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10.3390/cancers15020507
|
Characteristics and Clinical Outcomes of Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Receiving Ibrutinib for ≥5 Years in the RESONATE-2 Study
|
<jats:p>Primary results from the phase 3 RESONATE-2 study demonstrated superior efficacy and tolerability with ibrutinib versus chlorambucil in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Here, we describe characteristics and outcomes of patients who received ibrutinib treatment for ≥5 years in RESONATE-2. Patients aged ≥65 years with previously untreated CLL/SLL, without del(17p), were randomly assigned 1:1 to once-daily ibrutinib 420 mg until disease progression/unacceptable toxicity (n = 136) or chlorambucil 0.5–0.8 mg/kg for ≤12 cycles (n = 133). Baseline characteristics in ibrutinib-randomized patients (n = 136) were generally similar between patients on ibrutinib treatment for ≥5 years (n = 79) versus those on treatment for <5 years (n = 57). In patients on ibrutinib treatment for ≥5 years, complete response rates improved over time, reaching 42% by 5 years. Estimated 7-year progression-free survival and overall survival rates were 82% and 94%, respectively. Adverse events (AEs) led to dose reductions in 16/79 patients (20%); these AEs were resolved for 13/16 patients (81%). AEs led to dose holds (≥7 days) in 45/79 patients (57%); these AEs were resolved for 43/45 patients (96%). More than half (58%) of ibrutinib-randomized patients benefitted from ibrutinib treatment for ≥5 years regardless of baseline characteristics. Dose modification resolved AEs for most patients, thereby facilitating continued treatment.</jats:p>
|
[
{
"section_content": "Ibrutinib is a once-daily oral Bruton tyrosine kinase (BTK) inhibitor that is approved as first-line treatment for patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) in the United States, Europe, and other countries.Ibrutinib has the longest follow-up of any targeted therapy across multiple randomized phase 3 studies and is the only therapy to date that has demonstrated a significant overall survival (OS) benefit compared with chemotherapy/chemoimmunotherapy in patients with previously untreated CLL/SLL [1][2][3][4].Initial approval of ibrutinib in the first-line setting was supported by results from the primary analysis of the phase 3 RESONATE-2 study, which demonstrated that ibrutinib was superior to chlorambucil with respect to both efficacy and tolerability [5].With up to 8 years of follow-up (median: 82.7 months; range, 0.1-96.6months) in the RESONATE-2 study, the majority of ibrutinib-randomized patients remained progression-free; median progression-free survival (PFS) was not yet reached at the latest data cut [1]. Previous studies suggest that patients who continue treatment with single-agent ibrutinib experience better survival outcomes than patients who discontinue treatment within the first few years [6][7][8][9].Additionally, real-world evidence suggests that dose management (dose reduction or temporary dose holds for up to 1-2 weeks) results in improvement in or resolution of adverse events (AEs) [10] without impacting disease outcomes [6,[10][11][12][13][14][15][16][17][18].Therefore, active management of AEs by dose modification might facilitate continued ibrutinib treatment and maximize clinical outcomes [12].As of May 2022, the US prescribing information for ibrutinib includes updates to recommended dose modifications for AEs [19]. As it is the only BTK inhibitor with long-term follow-up for up to 8 years, we have the opportunity to examine efficacy and safety outcomes in patients with longer-term experience on ibrutinib treatment.Here, we describe characteristics and outcomes of patients who received treatment with ibrutinib for ≥5 years in the RESONATE-2 study. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "",
"section_name": "Study Design and Patients",
"section_num": "2.1."
},
{
"section_content": ") is a multicenter, international, randomized, open-label, phase 3 study designed to compare the efficacy and safety of first-line treatment with ibrutinib versus chlorambucil in patients aged ≥65 years with previously untreated CLL/SLL who required therapy per the 2008 International Workshop on CLL (iwCLL) criteria [20].Patients with del(17p) were excluded.Detailed methods were previously reported [5].Briefly, eligible patients were randomly assigned in a 1:1 ratio to receive oral ibrutinib 420 mg once daily until occurrence of progressive disease or unacceptable toxicity, or chlorambucil 0.5 mg/kg, escalated to a maximum of 0.8 mg/kg as tolerated, on days 1 and 15 of each 28-day cycle for up to 12 cycles.After confirmation of progressive disease per iwCLL criteria [20,21], patients who were randomly assigned to the chlorambucil arm could cross over to second-line treatment with ibrutinib.Per protocol, ibrutinib was temporarily held for any unmanageable grade ≥ 3 AE that was considered by the investigator to be potentially related to the study's treatment.Other AEs, including AEs of grade 2 in severity, could be managed with a one-level dose reduction of ibrutinib if the AE was considered to be potentially manageable by dose reduction as judged by the investigator. This study was performed in accordance with International Conference on Harmonisation Guidelines for Good Clinical Practice and the principles of the Declaration of Helsinki.The study protocol was approved by institutional review boards of each participating institution, and all patients provided written informed consent before participation in the study.This study was registered with ClinicalTrials.gov,numbers NCT01722487 and NCT01724346. ",
"section_name": "RESONATE-2 (PCYC-1115 [NCT01722487]/PCYC-1116 [NCT01724346]",
"section_num": null
},
{
"section_content": "The current exploratory analysis evaluated baseline demographics and clinical characteristics, overall response rates (per iwCLL criteria [20,21]), PFS, OS, prevalence of AEs over time, and AEs leading to dose modifications (per protocol) for ibrutinib-randomized patients who were on ibrutinib treatment for ≥5 years.Since the study protocol provided flexibility for dose reductions based on an investigator's judgment, additional analyses were performed to retrospectively determine the incidence of dose reductions due to AEs for which dose reductions are recommended in the recently updated US prescribing information (grade 2 cardiac failure, grade 3 cardiac arrhythmia, grade 3 or 4 nonhematologic AEs [excluding cardiac failure and cardiac arrhythmia], grade 3 or 4 neutropenia with infection or fever, and grade 4 hematologic AEs) [19]. Baseline characteristics were also evaluated as potential predictors for remaining on treatment for ≥5 years using a multivariate logistic regression model including the following baseline characteristics: age group, sex, Eastern Cooperative Oncology Group performance status, Cumulative Illness Rating Scale score, creatinine clearance, TP53 mutation status, IGHV mutation status, del(11q) status, disease histology, bulky disease, β-2 microglobulin, Rai stage, any history of cytopenia, lactate dehydrogenase level, and geographic region.Additionally, PFS and OS were analyzed in subgroups of patients with and without dose reductions in the overall population of all ibrutinib-treated patients; these exploratory post hoc analyses were not powered for significance, and comparative statistics are provided for descriptive purposes only.PFS and OS were estimated using the Kaplan-Meier method. ",
"section_name": "Analysis",
"section_num": "2.2."
},
{
"section_content": "In RESONATE-2, 269 patients were randomly assigned to receive ibrutinib (n = 136) or chlorambucil (n = 133).Of the 136 patients in the ibrutinib arm, 79 (58%) received ibrutinib treatment for ≥5 years.Median follow-up duration for patients who were on ibrutinib treatment for ≥5 years (n = 79) was 89.2 months (range: 61.3-96.6).Of these 79 patients, 22 subsequently discontinued ibrutinib in years 5-6 (n = 9), 6-7 (n = 10), or 7-8 (n = 3); reasons for discontinuation in these 22 patients were progressive disease (n = 10), death (n = 4), AEs (n = 3), physician decision (n = 3), and patient withdrawal (n = 2). ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Within ibrutinib-randomized patients in the intention-to-treat population (n = 136), baseline characteristics in the subset of patients who were on ibrutinib treatment for ≥5 years (n = 79) were generally similar to those in the subset of patients who were on ibrutinib treatment for <5 years (n = 57) (Table 1).Compared with the subset of patients who were on ibrutinib treatment for <5 years, the subset of patients who were on ibrutinib treatment for ≥5 years were more likely to be in the youngest age group (65-69 years; 37% vs. 19% of patients) and had a longer interval between initial diagnosis and initiation of study treatment (median 35 vs. 26 months). ",
"section_name": "Baseline Characteristics",
"section_num": "3.1."
},
{
"section_content": "In multivariate analysis, several baseline characteristics showed a trend toward continuation of ibrutinib treatment for ≥5 years (age ≤ 73 years, female sex, creatinine clearance ≥60 mL/min, TP53 mutated, del(11q), CLL histology, absence of bulky disease [<5 cm], β-2 microglobulin >3.5 mg/L, Rai stage III/IV, absence of cytopenia, and lactate dehydrogenase ≤250 U/L), but none reached statistical significance (Figure 1). ",
"section_name": "Predictors of Ibrutinib Treatment for ≥5 Years",
"section_num": "3.2."
},
{
"section_content": "Responses deepened over time, as indicated by the improvement of complete response (CR) rates from 10% (8/79 patients) at 1 year to 42% (33/79 patients) by 5 years and 46% (36/79 patients) by 7 years (Figure 2a).In patients who were on ibrutinib treatment for ≥5 years, 23/79 (29%) had a documented response of partial response with lymphocytosis (PR-L); of these patients, 9/23 (39%) achieved a best response of partial response (PR), 1/23 (4%) achieved nodular PR (nPR), and 13/23 (57%) achieved CR.In the overall population of ibrutinib-randomized patients, 30/136 (22%) had a documented response of PR-L; of these patients, 13/30 (43%) achieved a best response of PR, 1/30 (3%) achieved nPR, and 15/30 (50%) achieved CR.In patients who were on ibrutinib treatment for ≥5 years, the median time to PR was 4.6 months (95% CI: 3.8-7.4),whereas the median time to CR was 32.3 months (95% CI: 19.7-37.7)for those patients achieving CR.With up to 8 years of follow-up, complete response was achieved in 44 patients in the overall population, 36 of whom received ibrutinib treatment for ≥5 years. In patients who were on ibrutinib treatment for ≥5 years, median PFS and OS were not yet reached; 7-year PFS and OS rates were 82% (95% CI: 71-89) and 94% (95% CI: 86-97), respectively (Figure 2b,c). ",
"section_name": "Efficacy in Patients on Ibrutinib Treatment for ≥5 Years",
"section_num": "3.3."
},
{
"section_content": "In patients who were on ibrutinib treatment for ≥5 years, the median duration of ibrutinib treatment was 89.2 months (range: 60.4-96.6).Median relative dose intensity of ibrutinib for these patients was 98% (range: 47-100).The most frequent AEs of any grade across the entire study period were diarrhea (42/79 patients; 53%), cough (34/79; 43%), and upper respiratory tract infection (33/79; 42%).Prevalence of the most frequent AEs of any grade and of grade ≥ 3 were generally highest in years 0-1 and decreased over time thereafter (Figure 3a,b).Prevalence of AEs of clinical interest of any grade over time are shown in Supplementary Figure S1.AEs of any grade (occurring in ≥25% of patients overall) by yearly interval; (b) Most frequent grade ≥ 3 AEs (occurring in ≥5% of patients overall) by yearly interval.Prevalence was determined by the proportion of patients with a given AE (existing event or new onset of an event) during each yearly interval.Multiple onsets of the same AE term within a specific yearly interval were counted once, and the same AE term continuing across several yearly intervals was counted in each of the intervals.Abbreviations: AE, adverse event; UTI, urinary tract infection; URTI, upper respiratory tract infection. ",
"section_name": "Prevalence of AEs over Time",
"section_num": "3.4."
},
{
"section_content": "AEs led to dose reductions in 16/79 patients (20%) who were on ibrutinib treatment for ≥5 years and in 31/135 patients (23%) in the overall population of all ibrutinib-treated patients (Table 2).Most patients (12/16; 75%) experienced only one AE leading to dose reduction. a Denominator is patients with dose reductions because of any AE.b The same patient may be counted in more than one category because of multiple AE events leading to dose reduction.c Of 12 AEs that recurred at same/higher grade at any point during treatment, 3/13 were infections, 2/13 were hematologic, 2/13 were cardiac, 1/13 was gastrointestinal, and 4/13 were other.Abbreviations: AE, adverse event; NR, not reached; SOC, system organ class. Among patients who were on ibrutinib treatment for ≥5 years, the lowest ibrutinib dose for most patients with dose reductions was 280 mg once daily (10/16 patients).At data cutoff, 3/16 patients were receiving ibrutinib 420 mg once daily, 10/16 were receiving 280 mg once daily, and 3/16 were receiving 140 mg once daily.The median duration of treatment with ibrutinib at a reduced dose was not reached (range: 8.4-84.0+months) for patients who were on ibrutinib treatment for ≥5 years compared with 36.1 months (range: 0.0-84.0+) in all ibrutinib-treated patients with dose reductions (n = 31). Following dose reduction, 13/16 patients (81%) had a resolution of the initial AE.Three patients had AEs that were not resolved at data cutoff (grade 3 malignant lung neoplasm, grade 2 fatigue, and grade 1 contusion in 1 patient each).When considering the subset of AEs for which dose reductions are recommended in the updated ibrutinib US prescribing information (as of May 2022), such AEs led to dose reductions in 4/79 patients (5%) (Table 3).Among these patients, AEs did not recur or recurred at a lower grade in 3/4 patients; 1 patient had recurrence at the same grade AE 3 years after initial resolution (grade 3 atrial fibrillation), that resolved without further dose reduction.Patients who were on ibrutinib treatment for <5 years (n = 56) experienced similar rates of AEs leading to dose reduction (15/56; 27%).Most common reasons for dose reduction by system organ class in this subgroup were hematologic (n = 3), cardiac (n = 3), and dermatologic (n = 3).Dose reductions were more common in response to grade 3 AEs (n = 8); however, 100% of AEs (15/15) were initially resolved.Six patients (40%) experienced a recurrence of their AE at the same or higher grade. AEs led to dose holds of ≥7 days in 45/79 patients (57%) who were on ibrutinib treatment for ≥5 years and in 79/135 patients (59%) in the overall population of all ibrutinibtreated patients (Table 4). Among patients who were on ibrutinib treatment for ≥5 years, ibrutinib was restarted at 420 mg once daily after dose holds of ≥7 days for most patients (42/45 patients).Following a dose hold of ≥7 days, 43/45 patients (96%) had resolution of the initial AE. Among patients who were on ibrutinib for ≥5 years, the frequency of AEs leading to dose reductions was highest in years 0-2 and lower in subsequent years, whereas the frequency of AEs leading to dose holds of ≥7 days remained relatively consistent across the first 6 years of treatment (Supplementary Figure S2).a The same patient may be counted in more than one category because of multiple AE events leading to dose holds; b Denominator is patients with dose holds ≥7 days because of any AE.Abbreviations: AE, adverse event; SOC, system organ class. ",
"section_name": "Dose Management with Ibrutinib Treatment",
"section_num": "3.5."
},
{
"section_content": "In the overall population of all ibrutinib-treated patients, median PFS for patients who had dose reductions (n = 31) was 87.7 months (95% CI: 56.9-NE) and was not reached (95% CI: 81.9-NE) for those without dose reductions (n = 104) (hazard ratio 0.96 [95% CI: 0.50-1.84];p = 0.9011; Figure 4a).Estimated 7-year PFS rates were 59% (95% CI: 39-74) and 59% (95% CI: 48-68) for patients with and without dose reductions, respectively.With up to 8 years of follow-up, median OS was not reached in either group (hazard ratio 1.28 [95% CI: 0.58-2.83];p = 0.5363; Figure 4b); estimated 7-year OS rates were 74% (95% CI: 54-86) and 79% (95% CI: 69-86) in patients with and without dose reductions, respectively. ",
"section_name": "Exploratory Post Hoc Analysis of Outcomes in Patients with Dose Reductions",
"section_num": "3.6."
},
{
"section_content": "Concomitant medications of clinical interest in patients who were on ibrutinib treatment for ≥5 years are shown in Supplementary Table S1.Anticoagulants and antiplatelet agents were frequently used during the treatment period (33% and 65%, respectively), as were antihypertensive medications, including agents acting on the renin-angiotensin system (61%), beta-blocking agents (46%), calcium channel blockers (35%), and other antihypertensives (10%).Overall, 67% of patients received medications to treat acid-related disorders, including proton pump inhibitors in 58% of patients. ",
"section_name": "Concomitant Medications",
"section_num": "3.7."
},
{
"section_content": "Results of the current analysis demonstrate that more than half of patients with previously untreated CLL/SLL were able to receive treatment with single-agent ibrutinib for ≥5 years.While real-world studies have suggested an increased risk of discontinuation of targeted therapies in patients with older age, higher comorbidity burden, higher tumor burden, and/or worse performance status at baseline [13,22,23], no individual baseline characteristics were identified as significant predictors for continuation of long-term ibrutinib treatment in the current study. Among patients who were on ibrutinib treatment for ≥5 years, responses deepened over time.This subgroup of patients had a higher CR rate over the course of the study (46%) relative to the overall population of ibrutinib-randomized patients (34%) [1], suggesting that patients with favorable responses may be more likely to continue on ibrutinib treatment.In line with this, a higher PFS rate at 7 years was seen in patients who remained on long-term ibrutinib treatment for ≥5 years (82%) relative to the overall ibrutinib-randomized population (59%) [1].These findings are consistent with those of previous studies, suggesting that continuation of ibrutinib treatment is associated with improved efficacy outcomes [6][7][8]. Safety results in patients who were on ibrutinib treatment for ≥5 years were consistent with those seen in the overall population of ibrutinib-treated patients, including incidences of AEs of clinical interest (hypertension, atrial fibrillation, and major hemorrhage) [1].AEs generally decreased over time with continued ibrutinib treatment, and no new safety signals emerged in patients who received ibrutinib treatment for ≥5 years.Treatment with ibrutinib was well tolerated irrespective of the frequent use of concomitant antithrombotics, antihypertensives, and acid-reducing agents.Since AEs are the most common reason for discontinuation of ibrutinib in the first-line setting [1,[23][24][25][26][27][28], optimization of AE management is crucial to enabling patients to remain on long-term therapy.In the subgroup of patients who were on ibrutinib treatment for ≥5 years, active management of AEs with dose reductions or dose holds was associated with AE resolution in the majority (>80%) of patients.Additionally, dose reductions helped to prevent recurrence or worsening of AEs for most patients, facilitating continued benefit from ibrutinib treatment. In the current study, disease assessments were performed at regularly scheduled intervals based on iwCLL criteria [20,21].With up to 8 years of follow-up in the RESONATE-2 study, PFS and OS were similar between patients with and without dose reductions in the overall population of ibrutinib-randomized patients.Patients who had dose reductions received reduced doses of ibrutinib for extended periods of time (median of 3 years in all ibrutinib-treated patients with dose reductions).Together, these results suggest that patients experiencing AEs leading to dose reduction continue to benefit from ibrutinib at the reduced dose.While two real-world studies found significantly worse PFS in patients receiving ibrutinib at reduced doses (<420 mg once daily), this finding from RESONATE-2 is consistent with several other studies that have found no significant difference in efficacy outcomes between patients with dose reductions due to AEs compared to patients without dose reductions [10,[12][13][14][15][16]29,30]. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Regardless of demographic and disease characteristics at baseline, more than half (58%) of the patients randomly assigned to the ibrutinib arm in the RESONATE-2 study continued to benefit from ibrutinib treatment for ≥5 years.With up to 8 years of follow-up, the subset of patients who received ibrutinib treatment for ≥5 years experienced sustained efficacy benefits as evidenced by improved depth of response over time and high PFS rates. For patients who received ibrutinib treatment for ≥5 years, the safety profile was consistent with previous reports of long-term ibrutinib treatment and no new or unexpected AEs were observed.Dose modification (dose reduction or dose hold) was effective in resolving AEs for most patients, likely facilitating continuation of ibrutinib treatment. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "We thank the patients who participated in the study and their supportive families, as well as the investigators and clinical research staff from the study centers.Editorial support was provided by Melanie Sweetlove, and funded by Pharmacyclics LLC, an AbbVie Company. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by Pharmacyclics LLC, an AbbVie Company. The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the Institutional Review Boards or Independent Ethics Committees of each participating institution. Informed Consent Statement: Informed consent was obtained from all patients involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by Pharmacyclics LLC, an AbbVie Company. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the Institutional Review Boards or Independent Ethics Committees of each participating institution. Informed Consent Statement: Informed consent was obtained from all patients involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Data Availability Statement: Requests for access to individual participant data from clinical studies conducted by Pharmacyclics LLC, an AbbVie Company, can be submitted through Yale Open Data Access (YODA) Project site at http://yoda.yale.edu. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following are available online at https://www.mdpi.com/article/10.3390/cancers15020507/s1: Figure S1, Adverse events of clinical interest of any grade by yearly interval; Figure S2, AEs leading to dose modifications over time in patients on long-term ibrutinib treatment for ≥5 years; Table S1 honoraria from Gilead, Janssen, Novartis, TG Therapeutics, and Pharmacyclics LLC, an AbbVie Company; consulting/advisory role and speakers bureau for BeiGene, Gilead, Janssen, TG Therapeutics, and Pharmacyclics LLC, an AbbVie Company; research funding from AstraZeneca, BeiGene, and Pharmacyclics LLC; an AbbVie Company; travel/accommodations/expenses from Gilead, Janssen, Novartis, TG Therapeutics, and Pharmacyclics LLC; an AbbVie Company.This study was sponsored by Pharmacyclics LLC, an AbbVie Company.The sponsor was involved in study design, data analysis, data interpretation, writing/review of the manuscript, and the decision to publish the results.The sponsor had no role in data collection. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
10.1186/s40364-020-00220-5
|
The biological function and clinical significance of SF3B1 mutations in cancer
|
<jats:title>Abstract</jats:title><jats:p>Spliceosome mutations have become the most interesting mutations detected in human cancer in recent years. The spliceosome, a large, dynamic multimegadalton small nuclear ribonucleoprotein composed of small nuclear RNAs associated with proteins, is responsible for removing introns from precursor mRNA (premRNA) and generating mature, spliced mRNAs. SF3B1 is the largest subunit of the spliceosome factor 3b (SF3B) complex, which is a core component of spliceosomes. Recurrent somatic mutations in <jats:italic>SF3B1</jats:italic> have been detected in human cancers, including hematological malignancies and solid tumors, and indicated to be related to patient prognosis. This review summarizes the research progress of <jats:italic>SF3B1</jats:italic> mutations in cancer, including <jats:italic>SF3B1</jats:italic> mutations in the HEAT domain, the multiple roles and aberrant splicing events of <jats:italic>SF3B1</jats:italic> mutations in the pathogenesis of tumors, and changes in mutated cancer cells regarding sensitivity to SF3B small-molecule inhibitors. In addition, the potential of <jats:italic>SF3B1</jats:italic> or its mutations to serve as biomarkers or therapeutic targets in cancer is discussed. The accumulated knowledge about <jats:italic>SF3B1</jats:italic> mutations in cancer provides critical insight into the integral role the SF3B1 protein plays in mRNA splicing and suggests new targets for anticancer therapy.</jats:p>
|
[
{
"section_content": "Of the 3.3 billion base pairs of haploid DNA in the human genome, approximately 20,000 protein-coding genes have been identified by the Encyclopedia of DNA Elements (EN-CODE) project [1].However, the number of protein-coding genes is surprisingly low given the proteomic complexity, as the number of protein isoforms expressed from this gene set has been estimated to be at least 5-10-fold higher [2][3][4].The generation of protein diversity is primarily due to the process of precursor mRNA (premRNA) splicing, which is controlled by a complex regulatory system that consists of an enormous number of sequence elements and trans-acting splicing factors; therefore, it is not surprising that the mRNA splicing machinery is susceptible to mutations and that these mutations are implicated in many human diseases, including cancer [5,6].Indeed, genomewide studies have revealed more than 15,000 tumorassociated splice variants in a wide variety of cancers [7,8]. Recently, the most interesting mutations detected in human cancer were found to target components of the spliceosome involved in the mRNA-splicing process, as indicated by genomic DNA analysis of a variety of human tumors studied through the Cancer Genome Project.One of the most exciting discoveries has been recurring somatic mutations in genes encoding 3 splicesite recognition protein components and serine/arginine-rich (SR) splicing factors, which were initially discovered in myelodysplastic syndrome (MDS) in 2011 and later reported in other hematological malignancies, including solid tumors [9][10][11][12][13].Spliceosome mutations in cancers have highlighted the importance of the spliceosome pathway as a direct player in carcinogenesis and led to questions regarding the functional roles and molecular mechanisms of these mutations [14]. In this review, we describe spliceosome-associated transcript processing and its impact on disease.Moreover, we focus on one of the frequently mutated spliceosome proteins: cancer-related splicing factor 3b subunit 1 (SF3B1).We mainly summarize the distribution of mutations in SF3B1, mutant expression in tumors and its prognostic value.In particular, we discuss the functional consequences of SF3B1 mutation in tumors, with multiple roles in tumor pathogenesis, aberrant splicing events, and changes in sensitivity to SF3B smallmolecule inhibitors.The potential value of SF3B1 or its mutation as a novel cancer therapeutic target and marker that is more sensitive to spliceosome inhibitors is also described.Finally, we explore the options available for future research on the biological function and clinical significance of SF3B1 mutations in cancer. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Precursor mRNA splicing is an essential step in the posttranscriptional regulation of gene expression and is a process that involves the removal of noncoding sequences (introns) from premRNA and the ligation of coding sequences (exons) to form mRNA. PremRNA splicing is catalyzed by the spliceosome, a complex consisting of 5 small nuclear RNAs (snRNAs) that associate with proteins to form particles termed small nuclear ribonucleoproteins (snRNPs) [15][16][17].To date, two types of spliceosomes with unique compositions have been characterized: U2-dependent (major) spliceosomes and U12-dependent (minor) spliceosomes.The former spliceosome has been found in all eukaryotes and consists of the U1, U2, U5, and U4/U6 snRNPs and numerous proteins.Each U1, U2, and U5 snRNP has a single snRNA and several proteins; the U4 and U6 snRNPs have 2 snRNAs and several proteins.This spliceosome catalyzes the vast majority of transcript splicing events, removing the most commonly encountered class of introns (U2-type introns) (more than 99% in humans) [18,19].In contrast, the U12-dependent spliceosome is found only in a number of organisms, and acts on U12-type introns (less than 1% of introns in humans) [20,21].The difference between U2-type and U12-type introns is in the consensus splicesite sequence.The U12-dependent spliceosome also consists of 5 snRNPs, U11, U12, U5, and U4atac/U6atac snRNPs [22,23]. The stepwise interactions between premRNA and both U2-and U12-dependent spliceosome snRNPs are highly ordered, as shown in Fig. 1.Briefly, assembly of the U2dependent spliceosome is initiated by interaction of the U1 snRNP with the 5′ splice site, which includes a GU, leading to the formation of the E complex.Then, the U2 snRNP binds to the branch site (BS) to generate the A complex or the prespliceosome.When the U5 and U4/ U6 snRNPs interact with the A complex, the B complex is generated.U4/U6 base-pairing interaction is disrupted, and U6 displaces U1 snRNA, which then binds to the 5′ splice site.After B complex rearrangement, a catalytically active B* complex is produced through U1 and U4 snRNP dissociation.The mRNA is released after completion of the first (the C complex is formed) and second catalytic steps of splicing, in which the intron is removed, and the spliceosome dissociates to be recycled Fig. 1 SF3B1 functions in the stepwise assembly of the U2-and U12-dependent spliceosomes.There are two types of spliceosomes: U2dependent spliceosomes (left) and U12-dependent spliceosomes (right).Assembly of the U2-dependent spliceosome consists of 5 snRNPs, U1, U2, U5, and U4/U6 snRNPs; the U12-dependent spliceosome also consists of 5 snRNPs: U11, U12, U5, and U4atac/U6atac snRNPs.The difference between the two spliceosomes is in the consensus splice-site sequences, namely, U2-type or U12-type premRNA introns.SF3B1 is shared in the core components between the two spliceosomes and plays a key role in the recognition and selection of the branch site (BS) by interacting with premRNA in a sequence-independent manner, reinforcing stability during U2 (or U12) snRNA/BS interaction for new premRNA splicing [24,25].Assembly of the U12dependent spliceosome is similar to that of the U2dependent spliceosome because U11, U12 and U4atac/ U6atac snRNPs are functional analogs of the snRNPs U1, U2 and U4/U6.U12-dependent spliceosome assembly also involves sequential formation of the A, B, B*, and C complexes, but the earliest E complex formation step does not occur [21,23].The differences in the premRNA splicing mechanism mediated by the U2-and U12-dependent spliceosomes appear to involve only early intron recognition events and not the catalytic process. A large, diverse and dynamic protein has been found that interacts with snRNAs to form snRNPs within the spliceosome.Although the U2-and U12-dependent spliceosomes differ in their snRNA composition, they share many proteins [26][27][28][29].For example, all of the subunits of the protein complex SF3B, namely, SF3B155/SF3B1, SF3B145, SF3B130, SF3B49, SF3B14a/p14, SF3B14b and SF3B10, are the same [30].SF3B contributes a molecular mass of ~450 kDa to each snRNP, and it has been demonstrated to play a key role in the recognition and selection of the branch site (BS) during splicing by interacting with the premRNA at or near the BS in a sequence-independent manner, reinforcing stability during the U2 snRNA/BS interaction [31].In addition, numerous splicing factors, including an array of regulatory elements and proteins, participate in premRNA splicing events involving the two types of spliceosomes, such as exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE), intronic splicing silencer (ISS), SR proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and others [5,17,[32][33][34].Thus, although premRNA splicing is traditionally considered to involve separate and sequential processes, it is difficult for spliceosomes and associated proteins to detect specific splice sites in a vast RNA pool.This complexity makes the premRNA splicing machinery susceptible to sequence polymorphisms and deleterious mutations, some of which eventually lead to diseases, the number of which is growing [5].Therefore, some specific mutations or polymorphisms of premRNA splicing factors have become important diagnostic markers and therapeutic targets in human diseases. ",
"section_name": "Precursor mRNA (premRNA) splicing",
"section_num": null
},
{
"section_content": "Two different modes of splicing have been defined: constitutive splicing and alternative splicing.Constitutive splicing is the process of removing introns from pre-mRNA and joining the exons together to form a mature mRNA sequence.Alternative splicing is the process by which the exons are either retained or targeted for removal in different combinations to yield a diverse array of mRNAs from a single premRNA [35].There are several distinct patterns of alternative splicing, including cassette exons (in which one or more exons are either skipped or included), alternative 5′ splice sites, intron retention, mutually exclusive exons, alternative 3′ splice sites, and complex splicing patterns [36].More than 90% of human genes produce transcripts that are alternatively spliced, and 60% of the splice variants encode distinct protein isoforms with unique cellular functions or properties [37][38][39].Thus, alternative splicing plays important biological roles in the proliferation, differentiation and/or development of cells.In humans, the regulation of alternative splicing is tightly controlled during normal biological events [40].Misregulation of alternative splicing can lead to the production of aberrant protein isoforms, which may contribute to serious diseases, including cancers.Thus, an in-depth investigation of alternative splicing regulation has become the trend to understand the mechanisms of human diseases. ",
"section_name": "Alternative splicing",
"section_num": null
},
{
"section_content": "Disease-related mutations can affect splicing by altering splice site sequences, splicing regulatory sequences, or genes of the splicing machinery itself (i.e., spliceosome mutations) [41,42], and mutations of the splice-site sequences or of the splicing regulatory sequences have been documented in a variety of human diseases.For example, mutations in the splice-site sequences of the HBB (hemoglobin, beta) gene lead to abnormal splicing of HBB and defective synthesis of its protein β-globin in human β + -thalassemia [43][44][45].In human multisystem proteinopathy and amyotrophic lateral sclerosis (ALS), mutations in the prion-like domains of hnRNPA2B1 and hnRNPA1 occur [46].An array of mutations in splicing cis-acting sequences include those of LKB1 (liver kinase B1), KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog), CDH17 (cadherin 17), KLF6 (Kruppel-like factor 6) and BRCA1 (breast cancer gene 1) in many types of cancers [47,48]. In addition to mutations that alter precursor RNA sequence elements that regulate splicing, components of the spliceosome machinery have been shown to be dysregulated in human disease.An example discovered relatively early is that mutations in tri-snRNP (small ribonucleoprotein), a preassembled complex of U4 snRNA hybridized to U6 (U4/U6 or U4atac/U6atac) that also contains U5 (U4/U6 or U5) and associated proteins, can cause an autosomal dominant form of retinitis pigmentosa (adRP) [6,34].Since 2011, recurrent somatic mutations have been identified in a number of spliceosome components in human malignancies through the Cancer Genome Project, such as U2AF1 (U2AF35), SRSF2 (SC35), SF3B1 (SF3B155 or SAP155), and ZRSR2 (URP) [13].These spliceosome component mutations indicates somatic mutations are an important molecular mechanism underlying splicing deregulation in diseases. In this review, we discuss recently discovered examples of disease-linked spliceosome mutations in SF3B1 that are shared in the core comments between the two spliceosomes (Fig. 1). ",
"section_name": "Splicing mutations in human disease",
"section_num": null
},
{
"section_content": "Distribution of mutations in the molecular architecture of SF3B1 SF3B1 (splicing factor 3b subunit 1) is the largest subunit of the SF3B complex and functions by serving as a core component of the U2 snRNP, which is critical for branch site recognition and for the early stages of spliceosome assembly [49].Structurally, the N-terminal hydrophilic region of SF3B1 has multiple U2AF2 binding motifs [50,51], which may facilitate localization of the U2 snRNP to the vicinity of the branch site.Two-thirds of the C-terminus consists of 22 nonidentical HEAT (Huntingtin, elongation factor 3, subunit An of protein phosphatase 2A, and phosphatidylinositol 3-kinase (PI3K) target of rapamycin 1) repeats, which form rodlike helical structures, providing major scaffolding for the U2 snRNP to support interactions with other SF3B subunits, including p14 [52].The HEAT-repeat superhelix of SF3B1 defines a composite RNA-binding platform for BS recognition.In human diseases, almost all mutations in SF3B1 are located in the HEAT domain, particularly from H4-H12, as shown in Fig. 2.Among the mutated codons, 700 account for more than 50% of the variants observed, and additional codons (666, 662, 622, and 625) have been found to be hotspots for mutation [41,[53][54][55]. ",
"section_name": "SF3B1 mutations in cancer",
"section_num": null
},
{
"section_content": "Mutations in SF3B1 have been implicated as common drivers of hematologic malignancies.Somatic SF3B1 mutations are found in approximately 30% of patients with MDS and as many as 80% of patients with the MDS subtype characterized by ring sideroblasts (MDS-RARS) [41,56,57].These mutations are also present in 20% of patients with myelodysplastic/myeloproliferative neoplasm (MDS/MPN) [41] and in 15% of patients with chronic myeloid leukemia (CLL) [58][59][60].More recently, SF3B1 mutations have been identified at relatively high frequency in some solid tumors, such as various pigmented tumors, including uveal melanoma (UM) [61,62], mucosal melanoma [63], leptomeningeal melanoma [64] and blue nevus-like cutaneous melanoma [65], and neuroblastomas that arise following chromothripsis [66], estrogen receptor-positive breast cancers (BC) [67], pancreatic ductal adenocarcinoma [68], prostate cancer [69], prolactinomas [70], acute myeloid leukemia [71,72], and many others [73][74][75]. ",
"section_name": "Mutant SF3B1 expression in cancer",
"section_num": null
},
{
"section_content": "The prognostic value of the SF3B1 mutation in MDS remains controversial.Most studies have claimed that patients carrying an SF3B1 mutation have a significantly better overall survival and a lower likelihood of their disease transforming into acute leukemia compared with patients without SF3B1 mutations [11,41,76,77].In contrast, some studies found no significant effect of mutation on clinical outcomes [78,79].Regardless, SF3B1 appears to be the only gene for which somatic mutations are associated with a good prognosis in MDS [41,80].As with MDS, SF3B1 mutations were found to confer a favorable prognosis in uveal melanoma (UM), with a younger age of onset and concurrent disomy 3 [81,82].In addition, patients with SF3B1-mutated UM had better survival (at 5 years) than did SF3B1 wild-type patients.Nonetheless, evidence also shows that the survival differences between patients with SF3B1-mutant tumors and SF3B1 wild-type tumors are not significant over time, as indicated by follow-up data (at 10 years).Moreover, SF3B1-mutant UM is reported to cause late metastasis (median 8.2 years after initial diagnosis), suggesting that patients with SF3B1 mutations are also at risk for metastasis, particularly late-onset metastasis [83].It was inferred that the positive prognostic value of SF3B1 mutation may be partly or completely lost after the acquisition of other gene mutations associated with disease progression [41].In contrast to those in MDS or UM, SF3B1 mutations that are cancer-related occur more commonly in advanced disease and tend to be associated with poor prognosis in other malignancies, including CLL.Thus, the prognostic relevance of SF3B1 mutations in disease may be dependent on cellular contexts. ",
"section_name": "Prognostic value of SF3B1 mutation in cancer",
"section_num": null
},
{
"section_content": "",
"section_name": "Functional consequences of SF3B1 mutation in cancer",
"section_num": null
},
{
"section_content": "To date, it remains unclear what functional role SF3B1 mutations play in carcinogenesis, and it has not been well established whether deregulated SF3B1 activity is required for the maintenance of cancer [84].To address these questions, the role of SF3B1 mutations in malignant hematopoiesis has been investigated in vitro and in vivo.Regarding MDS, an SF3B1 K700E conditional knock-in mouse has been generated [85], and heterozygous expression of SF3B1 K700E caused progressive macrocytic anemia [85].Moreover, SF3B1 K700E expression was associated with aberrant 3′ splice-site selection as well as increased nonsense-mediated decay [85].In another study, conditional SF3B1 flox-K700E/+ mice were generated by targeted modification of the SF3B1 locus in JM8 mouse embryonic stem cells (ESCs).SF3B1 K700E/+ mice develop progressive normocytic anemia without ring sideroblasts [86].In addition to erythropoiesis, SF3B1 K700E/+ mice had reduced numbers of hematopoietic stem cells (HSCs) and exhibited a myeloid cell bias [86].Furthermore, the self-renewal potential of SF3B1 K700E/+ HSCs was determined by their repopulating ability in competitive transplantation assays into either young or old recipient mice.The results revealed a fitness disadvantage of mutant over wild-type HSCs [86], which contrasts with observations that mutant SF3B1 drives clonal hematopoiesis and may even be the sole identifiable driver mutation in human MDS [87,88].In addition, simultaneous expression of SF3B1 and SRSF2 mutations in mice resulted in increased apoptosis and reduced quiescence of hematopoietic stem progenitor cells (HSPCs).Moreover, combined expression of SF3B1 and SRSF2 mutations impaired expression of regulators of HSPC survival and increased sensitivity to inflammatory stimulation [89].In human SF3B1-mutated CLL cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was found to be increased at baseline and upon irradiation, demonstrating that mutations in SF3B1 are associated with increased DNA damage and/or an aberrant response to DNA damage [90].In many cancer cells, SF3B1 mutation was found to dysregulate multiple cellular functions, including heme biosynthesis, immune infiltration, DNA damage response, R-loop formation, telomere maintenance, and Notch signaling [74,[91][92][93], as well as many cellular pathways, such as the mitochondrial, Notch and NF-κB pathways [91].These results suggest that SF3B1 mutations play multiple roles in the pathogenesis of tumors. ",
"section_name": "Multiple roles in tumor pathogenesis",
"section_num": null
},
{
"section_content": "In terms of SF3B1 as a core component of splicing machinery, it has been clearly proven that common and tumor-specific splicing aberrations are induced by SF3B1 mutations, and aberrant 3′ ss selection has been established as the most frequent splicing defect [94], with increased alternative 3′ splice site usage [85] and usage of cryptic 3′ splice sites [86], as shown in Fig. 3. Strikingly, SF3B1 variants utilize a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3′ ss to enable aberrant splicing during the second step [94].SF3B1 mutations result in neomorphic activity, causing hundreds of alterations both through aberrant splicing and dysregulated gene expression in common alternative splicing signatures in different types of cancers [95].Furthermore, SF3B1 mutations are linked to various RNA processing mechanisms, such as alternative terminal exons, alternative 3′ acceptor splice sites, alternative cassette exons, alternative first exon, alternative branch point usage, and intron retention [96,97].Approximately 50% of aberrantly spliced mRNAs are subjected to nonsense-mediated decay, resulting in downregulation of gene and protein expression [94,98].However, few of these genes have been functionally implicated in driving the diseases known to be associated with SF3B1 mutations, including the genes shown in Fig. 4. Thus, the functional consequences and mechanisms of SF3B1 mutations in cancers need to be further investigated. ",
"section_name": "Aberrant splicing events",
"section_num": null
},
{
"section_content": "Fifteen aberrant splicing (PARVG, RPRD1A, DOM3Z, CXXC1, AP1G2, SNRPN, TCEA2, NICN1, ABCC5, ERCC3, SNRPN, PPOX, GPR108, PSTPIP1, NICN1) events have been correlated with clinical variables that showed a significant difference between SF3B1 mut and SF3B1 wt patients with MDS, including a lower percentage of bone marrow (BM) blasts and higher number of white blood cells, absolute neutrophil count (ANC), and platelet count (Plt) in the SF3B1 mut group [92].Moreover, isoform expression of extracellular exosome/focal adhesion genes (CRTC2, PPOX, AHSA2, DHP5) produced by aberrant splicing events in SF3B1 mut patients has been identified as a significant survival predictor in MDS [92,99].The functions of eight genes (BRD9, SUGP1, MAP3K7, TERC, KLF8, DVL2, SEPT2, and ABCB7) with deregulated expression due to SF3B1 mutations in tumors are discussed below. ",
"section_name": "Associations between aberrant splicing and clinical variables or patient survival",
"section_num": null
},
{
"section_content": "Homo sapiens bromodomain containing (BRD) 9 is a core component of the recently described mammalian BRG1-associated factor (BAF) chromatin remodeling complex that plays an important role in maintaining the transcriptional network of pluripotency [100].Previous studies have reported that BRD9 is required for the survival of some cancer types, particularly cancers with mutations that affect polybromo-associated BAF and canonical BAF6 [101][102][103].Recently, it was found that total levels of BRD9 mRNA were reduced in patients with CLL, MDS and UM carrying SF3B1 mutation.Further study found that mutant SF3B1 suppressed levels of full-length BRD9 protein without generating a truncated BRD9 protein in UM (MEL270) or myeloid leukemia (K562) cells that express SF3B1 K700E .Mutant SF3B1 recognizes an aberrant, deep intronic branch point within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element, causing subsequent degradation of BRD9 mRNA.Depletion of BRD9 in turn causes loss of noncanonical BAF at CTCF-associated loci, resulting in alteration of BAF localization to chromatin.BRD9 loss may also alter the expression of distinct genes involved in apoptosis and cell growth, adhesion and migration.In addition, it was also found that disruption of ncBAFdependent regulation of HTRA1 (HtrA serine peptidase 1) contributes to the protumorigenic effects of BRD9 loss.Correcting misspliced BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumor growth [100].These results implicate the disruption of noncanonical BAF in the diverse cancer types associated with SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies [104]. ",
"section_name": "BRD9",
"section_num": null
},
{
"section_content": "SUGP1 (SURP And G-patch domain-containing protein, a member of the SURP family of splicing factors that likely interact with SF1 and RNA helicases) is also greatly reduced in samples from MDS patient harboring SF3B1 mutation [53,[105][106][107].Furthermore, loss or weakening of the interaction of SUGP1 with SF3B1 in the spliceosome was found to be the sole cause of defects in BP recognition, which results in the use of cryptic 3′ ss typically located 10-30 nt upstream of canonical 3′ ss [53].That is, under normal conditions, the WT SF3B1 spliceosome uses a canonical BP and 30'ss for splicing; when the interaction between SF3B1 and SUGP1 is disrupted by SF3B1 mutations, the mutant SF3B1 spliceosome uses an upstream BP and a cryptic 30′ ss for splicing.Furthermore, SUGP1 can associate with the mutant spliceosome and partially rescue splicing defects [53].These data suggest that loss of SUGP1 is a common defect in spliceosomes with cancerassociated SF3B1 mutations and that the mutant spliceosome is \"repairable\" in principle via restoration of SUGP1 assembly [108]. ",
"section_name": "SUGP1",
"section_num": null
},
{
"section_content": "MAP3K7 (mitogen-activated protein kinase kinase kinase 7) encodes a kinase that mediates tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and Toll-like receptor signaling through the NF-κB, JNK, and MAPK pathways.Loss of MAP3K7 results in the attenuation or promotion of inflammation, depending on the cellular context [109][110][111].Reduced MAP3K7 mRNA and protein are both found in isogenic cell lines and primary myeloid and lymphoid cells from MDS and CLL patients carrying SF3B1 mutations [89].In contrast, hyperactive NF-κB signaling with increasing p-p65 levels is detected in SF3B1 K700E human myeloid and lymphoid leukemia cells stimulated with LPS.Re-expression of MAP3K7 in SF3B1 K700E cells leads to a significant decrease in p-p65 in both the resting state and following LPS exposure.Additionally, restoration of MAP3K7 expression in SF3B1 K700E cells results in partial rescue of cell clonogenicity [89].These data suggest that SF3B1 mutations affect the splicing of MAP3K7, which, at least in part, the results in hyperactivation of NF-κB signaling, which is associated with MDS pathogenesis.TERC TERC (telomerase RNA component), encoding an essential RNA component of telomeres, plays a role in cell proliferative potential, telomerase activity and telomere length, and TERC mutations have been found in autosomal dominant dyskeratosis congenita (DC), aplastic anemia, MDS, and cervical cancer [112][113][114][115]. TERC is a noteworthy target with significantly increased expression in SF3B1-mutated CLL samples, as based on amplification using total RNA [91].Upregulation of TERC and TERT (telomerase reverse transcriptase) was confirmed in Nalm6 isogenic cell lines with K700E and H622Q mutations.Not surprisingly, higher telomerase activity was detected in Nalm-6 SF3B1 K700E cells than in wild-type cell lines.These data suggest that mutant SF3B1 may affect telomerase activity through dysregulation of TERC and TERT expression [91]. ",
"section_name": "MAP3K7",
"section_num": null
},
{
"section_content": "KLF8 (Kruppel-like transcription factor 8) has been found to be involved in tumor cell proliferation, transformation, and progression and in DNA damage repair in several different tumors, including renal cell carcinoma, hepatocellular carcinoma and breast cancer [116].KLF8 is consistently upregulated in SF3B1-mutated CLL samples at both single-cell and bulk RNA levels.Overexpression of SF3B1 in cells induces phosphorylation of H2AX (γH2AX) [90,91] and CHK2 (checkpoint kinase 2) [91], two markers of DNA damage, following exposure to gamma irradiation.These data demonstrate that single mutations in SF3B1 are associated with increased DNA damage and/or an aberrant response to damage and that SF3B1 mutation-associated gene dysregulation is a contributor to altered DNA responses [90,91]. ",
"section_name": "KLF8",
"section_num": null
},
{
"section_content": "Dishevelled 2 (DVL2) has been previously reported to play a role in both canonical and noncanonical Wnt signaling by binding to the cytoplasmic C-terminus of frizzled family members and inducing transduction of the Wnt signal to downstream effectors [117,118].Relatively high expression of the DVL2 splice variant and the protein product of altered DVL2 have been confirmed in individual primary CLL cells with SF3B1 mutation [91].Interestingly, altering DVL2 does not influence Wnt signaling, but it does affect the Notch pathway: expression of altered DVL2 markedly abrogates the activation of Notch1 and expression of the Notch pathway target gene HES1 (hairy and enhancer of split-1).Moreover, combined expression of WT and altered DVL2 reverses these suppressive effects, suggesting a dominant impact of altered DVL2 on the WT isoform [91].These data implicate DVL2 as a target of mutated SF3B1 through which alternative splicing modulates Notch signaling activity. ",
"section_name": "DVL2",
"section_num": null
},
{
"section_content": "SEPT2 (septin 2), a member of the septin family of guanosine triphosphatases, plays a role in the biogenesis of polarized columnar-shaped epithelium by maintaining polyglutamylated microtubules and an important role in the regulation of mitosis and cell growth [119,120].SEPT2 is significantly downregulated in CD34 + cells with SF3B1 mutations in patients with MDS [92].In addition, the effect of SEPT2 silencing on erythroid cell growth and differentiation has been studied in human BM CD34 + cells.It was found that SEPT2 silencing leads to significantly impaired growth, G1/S transition arrest and a significant decrease in intermediate and late erythroid cell populations [92].These results suggest that aberrant splicing of SEPT2 may lead to impaired erythropoiesis in association with SF3B1 mutations in patients with MDS. ",
"section_name": "SEPT2",
"section_num": null
},
{
"section_content": "ABCB7 (ATP binding cassette subfamily B member 7) encodes a half-transporter involved in the transport of heme from the mitochondria to the cytosol and plays a role in mitochondrial iron accumulation, isodicentric chromosome formation (X)(q13) and sideroblastic anemia, which is involved in many hematologic malignancies [121][122][123].Aberrant splicing of ABCB7 occurs in MDS RARS and SF3B1 mut MDS patients and significant ABCB7 downregulation in SF3B1 mut cases [92,96].In normal bone marrow, ABCB7 downregulation reduces erythroid differentiation, growth and colony formation and results in a gene expression pattern similar to that observed in intermediate MDS-RARS erythroblasts and in the accumulation of FTMT.Moreover, silencing SF3B1 results in downregulation of ABCB7 in K562 cells undergoing erythroid differentiation, implicating ABCB7 in the acquisition of the RARS phenotype [124].Furthermore, an ABCB7 cryptic 3′ splice site event was detected in SF3B1-mutant HSCs carrying the SF3B1 K700E mutation; nonsense-mediated RNA decay (NMD) can target the aberrantly spliced ABCB7 transcript and underlies the downregulation of ABCB7 observed in MDS patients with SF3B1 mutation.Moreover, treatment of SF3B1 K700E -mutant cells with cycloheximide resulted in an increase in the aberrantly spliced form of the ABCB7 transcript [96].Interestingly, the sequence of the ABCB7 cryptic ss is not conserved in mice, and there is no aberrant splicing of ABCB7 in SF3B1-mutant murine cells [85,86], which indicates significant differences in the transcripts affected because of SF3B1 K700E between humans and mice.These data provide strong evidence that SF3B1 mutation leads to aberrant ABCB7 splicing and downregulation via NMD in human cancer cells and suggest an ABCB7-based therapeutic approach for treating these malignancies. In addition, the roles and mechanisms of many other key cancer-related genes that have been detected by RNA sequencing and validated by qRT-PCR in distinct types of cancer need to be further investigated, such as TMEM14C (transmembrane protein 14C), SEPT6 (septin 6), and ENOSF1 (enolase superfamily member 1), as shown in Fig. 4.More genes that can be categorized into one of two large groups, a cell-autonomous gene set and a set of genes in immune cells with signatures related to immune cell infiltration, need to be confirmed in vitro and in vivo [74]. ",
"section_name": "ABCB7",
"section_num": null
},
{
"section_content": "Because the spliceosome SF3B complex has emerged as a potential therapeutic target, SF3B small-molecule inhibitors are currently under development and have entered clinical trials [125][126][127].These inhibitors specifically target the SF3B protein complex, leading to the loss of spliceosome function with regard to 3 splice site recognition and resulting in aberrant alternative splicing/mRNA transport.Among them, three bacterial fermentation products (FR901464, herboxidiene, and pladienolide) were identified as natural compounds with antitumorigenic properties [126].In addition to natural drugs, meayamycin, E7107, and spliceostatin A (SSA) have been developed as synthetic analogs with improved stability and solubility [58].Among them, pladienolide (Pla) stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating the transition of SF3B to the closed conformation to form the adenosine-binding pocket and stably accommodate the BS/U2 duplex [128,129].Although Pla-B might be located in the vicinity of both SF3B1 and SF3B3, it only binds to SF3B3 [130].Additionally, E7107, a synthetic derivative of pladienolide D, and SSA, a methylated derivative antitumor natural product FR901464, mainly destabilize U2 snRNP assembly at 3′ splice sites by blocking SF3B from binding to RNA [126].E7107 targets SF3B1 to block ATP-dependent A complex formation as well as a conformational change in U2 that exposes the snRNA region responsible for base pairing to the branchpoint sequence [131], whereas SSA induces a conformational shift in the U2 snRNP to bind to \"decoy\" sequences that can occur upstream of the branchpoint sequence [58]. Interestingly, one disease-associated SF3B1 mutation has the opposite effects on sensitivity to SF3B smallmolecule inhibitors, as shown in Fig. 5.An R1074H mutation in SF3B1 confers resistance to pladienolide activity by impairing the ability of pladienolide B to bind to SF3B due to the physical interference caused by SF3B1 mutation [132], and it was speculated that SF3B1 mutations in other HEAT repeats might have the same effect [58].These data indicate that the mutant SF3B1 protein has an impaired response to pladienolide B. In contrast to pladienolide B, it was reported that the SF3B1 mutant cell lines Panc 05.04 (pancreatic, K700E) and ESS-1 (endometrial, K666N) were more sensitive to SAA, leading to inhibition of cell growth [133].In contrast to SF3B1 +/+ murine HSPCs, SF3B1 +/K700E HSPCs were more sensitive to the spliceosome modulator E7107E [85].Furthermore, treatment of SF3B1 +/K700E recipients with E7107 in vivo caused a significant decrease in CD45.2 chimerism in the peripheral blood, bone marrow, and spleen [85].These data demonstrate that SF3B1 mutation enhances the sensitivity of tumor cells to the spliceosome modulator E7107 and SAA but not pladienolide B. SF3B1 mutation therefore sensitizes cells to pharmacologic targeting of wild-type SF3B1, consistent with the observation that the growth of SF3B1-mutant endometrial cancer and uveal melanoma cell lines was impaired by deletion of wild-type, but not mutant, SF3B1 [84].Although the mechanism of the enhancement sensitivity to splicing inhibitors is unknown, probably because a mutated SF3B1 gene may be unable to tolerate further perturbations in splicing and therefore be preferentially sensitive to pharmacological splicing inhibition [134], these findings suggest that there may be a therapeutic window for the use of spliceosome modulators in the treatment of hematologic malignancies with SF3B1 mutation [85].Overall, mutated SF3B1 has been detected in hematological malignancies and tumors and has been proven to be related to patient prognosis, transcription, alternative splicing, and sensitivity to SF3B small-molecule inhibitors.However, only a few genes affected by SF3B1 mutation have been extensively studied to date, and the roles and mechanisms of these genes still need to be confirmed. ",
"section_name": "Changes in sensitivity to SF3B small-molecule inhibitors",
"section_num": null
},
{
"section_content": "Primary tumors with SF3B1 mutations display alternative splicing in select key genes in cancer, including CLL, MDS, and uveal melanoma; this signature is conserved between cancer sites and is independent of the mutant amino acid [95], which implies that SF3B1 mutations may represent an important clinical significance in tumors.Indeed, SF3B1 is the most commonly mutated spliceosomal component gene in breast cancer, and mutations affecting this gene are significantly associated with ER-positive disease [67].Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3B complex inhibitor spliceostatin A, and treatment resulted in perturbation of the splicing signature [67].Thus, given the multiple roles in the pathogenesis of tumors and splicing events induced by SF3B1 mutations in cancer, as well as the potentially increased sensitivity of cancers to some SF3B small-molecule inhibitors, SF3B1 or its mutation may represent a prognostic biomarker and therapeutic target for cancer, and pharmacological modulation of splicing may represent an important therapeutic strategy [134].For example, although SF3B1 mutations occur at a low frequency (1.1%) in prostate cancer (PCa) [69], SF3B1 mRNA and protein levels are higher in tumor glands than in nontumor adjacent regions [135].Notably, SF3B1 expression correlated positively with clinical and molecular features, including Gleason score and vascular and perineural invasion.Pharmacological blockade of SF3B1 with pladienolide-B reduced malignant features of PCa cells and modulated key signaling pathways, malignancy markers, and expression of oncogenic splicing variants AR-v7 and In1-ghrelin, spliceosome components and splicing factors as well as expression of EJC and SURF components and NMD factors [135].That is, dysregulation of SF3B1 expression may be involved in the development, progression, and aggressiveness of PCa, and SF3B1 might represent a new prognostic biomarker and therapeutic target in this devastating pathology.These results also indicate that SF3B1 inhibition leads to a decrease in the aggressiveness features of PCa cells through both direct and indirect mechanisms, possibly involving the modulation of different types of cellular stress processes.As SF3B1 variants exhibit an impaired response to pladienolide B [132], other inhibitors, such as E7107E and SSA, may have better antitumor activity for PCa.The prognostic and therapeutic potential of SF3B1 in other types of cancer needs to be further studied. ",
"section_name": "SF3B1 or its mutation as a novel therapeutic target in cancer",
"section_num": null
},
{
"section_content": "In eukaryotes, the RNA splicing system, as essential cellular machinery, is critical for successful transcription through constitutive splicing and guarantees the functional diversity of protein products through alternative splicing.Thus, deregulation of this machinery causes severe developmental abnormalities [42,92,136].Recent studies have shown that mutations in the RNA splicing machinery significantly affect the RNA splicing system by altering many splicing patterns associated with 3′ splice sites, suggesting that aberrant splicing patterns induced by spliceosome mutations are directly linked to disease phenotypes [13].SF3B1 is a shared core component of snRNPs both in major and minor spliceosomes and plays a critical role in the early and later stages of spliceosome assembly.SF3B1 mutations occur in many types of tumor and play an important role in the development and progression of cancer.However, the functional impact and mechanisms of the SF3B1 mutationderegulated splicing pattern on oncogenesis need to be better understood.In addition, although it has become clear that aberrant premRNA alternative splicing is a major contributor to cancer phenotypes, studies on the misregulation of alternative splicing induced by SF3B1 mutation in cancer have not kept pace with the latest data.Moreover, mutant SF3B1 may have a distinct function not only in the direct regulation of RNA splicing but also in the elongation and stability of DNA, which may be important for the acquisition of specific disease phenotypes [36].Further work is also required to evaluate the molecular mechanism by which mutations in SF3B1 HEAT domains may influence the base-pairing potential of U2 snRNA [14].Future clinical work is also needed to explore the relationship between SF3B1 mutations and mutations in other cancer-related genes, including those of other spliceosome-associated proteins, splicing regulatory factors, and transcriptional factors.Moreover, a valuable tool for dissecting the effects of SF3B1 mutations on the transformation, splicing, and functions of SF3B1 was established in a mouse model [87,88], and investigation of the role of the yeast ortholog Hsh155 supports a novel mechanism in which SF3B1 helps to define the BS during premRNA splicing [137].These findings are part of wide ongoing effort to generate genetically engineered model systems to study the biological and biochemical consequences of spliceosomal mutations in model systems as diverse as yeast, zebrafish, mouse, and human cells [138].Finally, as the effects of splicing-modulation strategies targeting SF3B1 mutations are currently unpredictable, laboratory, preclinical and clinical studies are required understand the biological and clinical significance of SF3B1 mutations in cancer. ",
"section_name": "Conclusions and future perspectives",
"section_num": null
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This study was supported by the Qingdao Source Innovation Program (18-2-2-76-jch), the National Natural Science Foundation of China (91849209, 31671447), and the Innovative Talent Program of Qingdao (18-1-2-6-zhc). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Not applicable, all information in this review can be found in the reference list. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Abbreviations ABCB7: ATP-binding cassette subfamily B member 7; adRP: Autosomal dominant retinitis pigmentosa; ALS: Amyotrophic lateral sclerosis; BC: Breast cancer; BRD9: Homo sapiens bromodomain containing 9; BS: Branch site; CLL: Chronic myeloid leukemia; ENOSF1: Enolase superfamily member 1; ESE: Exonic splicing enhancer; ESS: Exonic splicing silencer; HSC: Hematopoietic stem cells; HSPC: Hematopoietic stem progenitor cell; hnRNP: Heterogeneous nuclear ribonucleoprotein; ISE: Intronic splicing enhancer; ISS: Intronic splicing silencer; KLF8: Kruppel-like transcription factor 8; MAP 3 K7: Mitogen-activated protein kinase kinase kinase 7; MDS: Myelodysplastic syndrome; snRNA: Small nuclear RNA; snRNP: Small nuclear ribonucleoprotein; SRSF2: Serine/arginine-rich splicing factor 2; SF3B1: Splicing factor 3b subunit 1; SURP1: SURP and G-Patch domain containing 1; TERC: Telomerase RNA component; TERT: Telomerase reverse transcriptase; TMEM14C: Transmembrane protein 14C; U2AF1: U2 small nuclear RNA auxiliary factor 1; UM: Uveal melanoma; ZRSR2: CCCH type zinc finger, RNA binding motif and serine/arginine rich protein 2 All authors contributed to the literature search and writing of the manuscript.All authors read and approved the final manuscript. No ethics approval was required for this review that did not involve patients or patient data. All authors consent to publication. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Abbreviations ABCB7: ATP-binding cassette subfamily B member 7; adRP: Autosomal dominant retinitis pigmentosa; ALS: Amyotrophic lateral sclerosis; BC: Breast cancer; BRD9: Homo sapiens bromodomain containing 9; BS: Branch site; CLL: Chronic myeloid leukemia; ENOSF1: Enolase superfamily member 1; ESE: Exonic splicing enhancer; ESS: Exonic splicing silencer; HSC: Hematopoietic stem cells; HSPC: Hematopoietic stem progenitor cell; hnRNP: Heterogeneous nuclear ribonucleoprotein; ISE: Intronic splicing enhancer; ISS: Intronic splicing silencer; KLF8: Kruppel-like transcription factor 8; MAP 3 K7: Mitogen-activated protein kinase kinase kinase 7; MDS: Myelodysplastic syndrome; snRNA: Small nuclear RNA; snRNP: Small nuclear ribonucleoprotein; SRSF2: Serine/arginine-rich splicing factor 2; SF3B1: Splicing factor 3b subunit 1; SURP1: SURP and G-Patch domain containing 1; TERC: Telomerase RNA component; TERT: Telomerase reverse transcriptase; TMEM14C: Transmembrane protein 14C; U2AF1: U2 small nuclear RNA auxiliary factor 1; UM: Uveal melanoma; ZRSR2: CCCH type zinc finger, RNA binding motif and serine/arginine rich protein 2 ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All authors contributed to the literature search and writing of the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "No ethics approval was required for this review that did not involve patients or patient data. ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "All authors consent to publication. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.21320/2500-2139-2020-13-4-366-381
|
Mantle Cell Lymphoma: History, Current Principles of Diagnosis, and Treatment (Literature Review)
|
<jats:p>Mantle cell lymphoma (MCL) is a heterogeneous disease with a broad spectrum of clinical manifestations from rare indolent cases requiring no immediate treatment to aggressive fast-proliferating tumors. Differences in clinical behavior are rooted in molecular grounds which in the latest edition of WHO hematopoietic and lymphoid tissue tumor classification formed the basis for dividing MCL into two variants: classical (in most cases) and indolent. In last decades, our insight into biology and disease development mechanisms has been considerably enhanced. Further, it will help to risk stratify patients not only according to clinical factors (MIPI) but also taking into account molecular and biological properties of tumor (Ki-67 proliferation index, ТР53, NOTCH1, and NOTCH2 mutations, complex karyotype, and unmutated IGHV status). Treatment algorithms based on intensive chemotherapy with high-dose cytarabine and autologous hematopoietic stem cell transplantation with further rituxi-mab maintenance therapy ensure long-term monitoring of the disease in many MCL patients. The use of new “chemo-free” regimens and rational combinations (bortezomib, BTK inhibitors, lenalidomide, and venetoclax) offers the hope of a departure from conventional chemotherapy for a certain part of patients. Novel drugs with unique modes of action enabled, to some extent, to deconstruct the stigma of MCL fatality.</jats:p>
|
[
{
"section_content": "Для цитирования: Тумян Г.С.Мантийноклеточная лимфома: история, современные принципы диагностики, лечение (обзор литературы).Клиническая онкогематология.2020;13(4):366-81.DOI: 10.21320/2500-2139-2020-13-4-366-381 For citation: Tumyan GS.Mantle Cell Lymphoma: History, Current Principles of Diagnosis, and Treatment (Literature Review).Cl inical oncohematology.2020;13(4):366-81.(In Russ ).DOI: 10.21320/2500-2139-2020-13-4-366-381 ",
"section_name": "Мантийноклеточная лимфома",
"section_num": null
},
{
"section_content": "Современная история МКЛ начинается с классификации лимфом Генри Раппапорта, опубликованной в журнале «Cancer» в 1954 г. [1].Она основана на морфологическом описании клеточного состава и определении характера роста опухоли (нодулярный и диффузный).К этому времени отсутствовало понимание феномена «бласт-трансформации» и была принята концепция, что крупные клетки в опухолевой ткани имеют нелимфоидное происхождение и являются потомками стромальных (ретикулярных) клеток лимфатического узла.Было выделено пять типов лимфом: лимфоцитарная (хорошо дифференцированная), лимфоцитарная (мало дифференцированная, лимфобластная), смешанная (лимфоцитарная и ретикулярная), ретикулярная, типа Ходжкина.Эта классификация нашла широкое распространение в США. В течение последующего десятилетия в медицинской науке произошла настоящая революция, которая, безусловно, перевернула все ранее существующие понятия в иммунологии и онкогематологии.Стало очевидным, что лимфоциты представляют собой гетерогенную группу клеток, которые имеют разное линейное происхождение (Т-, В-, NK-) и выполняют разные функции.Лимфомы стали рассматриваться как опухоли иммунной системы, а лимфоцит перестал восприниматься как конечный продукт дифференцировки лимфоидных клеток.Было показано, что под влиянием различных факторов (митогенов или антигенов) лимфоидные клетки могут трансформироваться в крупные пролиферирующие клетки, ранее называвшиеся ретикулярными.Все эти знания легли в основу создания Кильской классификации, предложенной К. Леннертом и представителями его школы [2].В новой классификации наряду с морфологическими особенностями учитывали иммунофенотип клеток лимфоидной опухоли и их соответствие нормальным лимфоцитам разных этапов В-и Т-клеточной дифференцировки.Так появилось новое понятие «неопухолевого эквивалента» (postulated normal counterpart).Уже в Кильской классификации было определено, что большинство лимфом происходит из В-клеток зародышевого (герминативного) центра вторичного лимфоидного фолликула.В зависимости от морфологии и характера течения заболевания было предложено разделять лимфомы на опухоли низкой (мелкие клетки) и высокой (преимущественно бластные элементы) степени злокачественности. К этому времени возможности стадирования и лечения лимфом стали расширяться и возникла большая необходимость в проведении многоцентровых клинических исследований.Путаница в систематизации опухолей делала невозможным сопоставление полученных результатов и тормозила дальнейшие научные изыскания.В Европе повсеместно использовалась Кильская классификация, которая плохо стыковалась с классификацией Г. Раппапорта, принятой патологами и клиницистами в США.По инициативе Национального института рака в 1982 г. публикуется «Рабочая схема» для клинического применения (Working Formulation, WF).По сути это «рабочая платформа», целью которой было совместить уже существующие классификационные системы для совместной работы патологов и клиницистов [3].В этом руководстве учитывается степень злокачественности опухоли и характер ее роста, но не принимаются во внимание данные иммунофенотипа клеток.Несмотря на некоторую полезность WF, желаемая цель не была достигнута, поскольку в Европе продолжают использовать Кильскую классификацию, а отсутствие иммунологической составляющей в WF делает ее нежизнеспособной и бесперспективной. В 1991 г.Peter Isaacson и Harald Stein создали Международную группу по изучению лимфом (ILSG), которая объединила европейских и американских гематопатологов для попытки систематизации опухолей лимфоидной природы с учетом современных понятий.К тому времени уже были внедрены в практику иммуногистохимические методы обработки парафиновых блоков, разработаны новые методики (полимеразная цепная реакция [ПЦР] и флюоресцентная гибридизация in situ [FISH]), которые позволяли провести генетические анализы на криостатных срезах, что значительно облегчило тестирование образцов рутинной биопсии.В качестве маркера определения клональности и линейной принадлежности опухоли изучались перестройки генов, кодирующих синтез В-и Т-клеточных рецепторов.Стало возможным определение соматических гипермутаций в генах, кодирующих синтез вариабельных участков тяжелых цепей иммуноглобулинов (IGHV), что позволило не только говорить о линейной принадлежности опухоли, но и о стадии ее дифференцировки (герминальная, постгерминальная). Именно в это время Международная группа представила консенсусный отчет по определению новой самостоятельной нозологической единицы, которую назвали мантийноклеточной лимфомой (МКЛ) [4].Установлено, что внутри большой группы лимфом низкой степени злокачественности можно выделить особый вариант, который на тот момент определялся как лимфоцитарная лимфома с промежуточной дифференцировкой (lymphocytic lymphoma of intermediate differentiation), либо лимфома из клеток мантийной зоны (mantle zone lymphoma), либо центроцитарная лимфома (centrocytic lymphoma) согласно Кильской классификации.При этом В-клеточном варианте опу- Понимание молекулярных механизмов развития любой опухоли -это, как правило, более поздний этап изучения болезни, который следует после описания ее морфологических, иммунофенотипических и клинических особенностей.В этом контексте не является исключением и МКЛ, при которой подробные патоморфологические и клинические характеристики опухоли предшествовали распознаванию ключевого гена CCND1, критичного в патогенезе этой болезни.Как только стали понятны механизмы становления заболевания, был получен широкий доступ к новым диагностическим инструментам (например, определение циклина D1), которые помогают в дифференциальной диагностике лимфом.Амплификация или перестройка гена CCND1, расположенного на хромосоме 11q13, и как следствие, гиперэкспрессия белка циклин D1 приводят к укорочению фазы G1, уменьшению размера клеток и потере контроля клеточного цикла [10,11].Кроме того, избыточная экспрессия циклина D1 нарушает экспрессию и других генов, участвующих в пролиферации и делении клеток (c-myc, c-jun и Cyclin A).Elaine S. Jaffe в журнале «Blood» еще в 2008 г. [12] представила наглядную схему эволюции понимания и идентификации МКЛ (рис.2). ",
"section_name": "ИСТОРИЯ ВОПРОСА",
"section_num": null
},
{
"section_content": "МКЛ является гетерогенным заболеванием с широким спектром клинических проявлений от редких индолентных случаев, не требующих немедленного начала терапии, до агрессивных, быстро пролиферирующих типов опухоли.Разное клиническое поведение имеет серьезное молекулярное обоснование, которое позволило в новой классификации ВОЗ разделить опухоль на два варианта.Классическая МКЛ (80-90 % случаев) происходит из наивных В-клеток прегерминального этапа дифференцировки, отличается отсутствием мутаций генов IGHV, обычно SOX11-позитивная, проявляется нодальным/экстранодальным вовлечением и агрессивным течением (рис.3). Напротив, более редкий индолентный ненодальный тип МКЛ (10-20 %) характеризуется нали- Рис. 2. Схема эволюции понимания и идентификации МКЛ (цит.по [12]).МКЛ в Кильской классификации и модифицированной классификации Раппапорта рассматривалась в группах центроцитарной лимфомы и лимфоцитарной лимфомы промежуточной степени дифференцировки.В это время еще отсутствовали точные критерии идентификации морфологически сходных форм лимфоидных опухолей.Определение характерного иммунофенотипа МКЛ (CD5+, CD23-, CD10-) помогло в дифференциальной диагностике мелкоклеточных опухолей (ФЛ, ХЛЛ, ЛМЗ).Выявление в абсолютном большинстве случаев t(11;14)(q13;q32) позволило в рутинной практике использовать в диагностике МКЛ иммуногистохимическое определение белка циклин D1.Кроме того, вторичные генетические события, такие как делеция/мутация p53 и p16, были идентифицированы при ряде агрессивных подвариантов МКЛ, которые ранее гистологически обозначались как бластоидный подтип.Все вышеперечисленное позволило четко обозначить границы нозологически очерченного варианта лимфоидной злокачественной опухоли, обозначенного как мантийноклеточная лимфома ВДЛ -высокая степень дифференцировки, лимфоцитарная; ДГЛ -диффузная гистиоцитарная лимфома; ЛМЗ -лимфома маргинальной зоны; МКЛ -мантийноклеточная лимфома; НДЛ -низкая степень дифференцировки, лимфоцитарная; ПДЛ -промежуточная степень дифференцировки, лимфоцитарная; ФЛ -фолликулярная лимфома; ХЛЛ -хронический лимфолейкоз.[12]).The Kiel classifi cation and the modifi ed Rappaport classifi cation regarded MCL within centrocytic and lymphocytic lymphoma groups with intermediate grade of diff erentiation.At that time there were no precise criteria for identifying morphologically similar forms of lymphoid tumors.Determination of specifi c MCL immunophenotype (CD5+, CD23-, CD10-) helped in the diff erential diagnosis of small cell tumors (FL, CLL, MZL).Identifi cation of t(11;14)(q13;q32) in the vast majority of cases allowed to use immunohistochemical determination of Cyclin D1 protein in the routine practice for MCL diagnosis.Besides, secondary genetic events, such as p53 and p16 deletion/mutation, were identifi ed in several aggressive MCL subvariants which had been previously histologically designated as blastoid subtype.All said above allowed to mark the boundaries of nosologically distinct variant of lymphoid malignant tumor designated as mantle cell lymphoma ВДЛ -well-differentiated lymphocytic lymphoma; ДГЛ -diffuse histiocytic lymphoma; ЛМЗ -marginal zone lymphoma; МКЛ -mantle cell lymphoma; НДЛ -poorly differentiated lymphocytic lymphoma; ПДЛ -lymphocytic lymphoma of intermediate differentiation; ФЛ -follicular lymphoma; ХЛЛ -chronic lymphocytic leukemia.Этот комплекс способствует фосфорилированию главного гена-супрессора опухолевого роста -гена ретинобластомы (RB1).Инактивация RB1, опосредованная комплексом Cyclin D1-CDK4 или делецией гена RB1 (наблюдается в 30 % МКЛ), приводит к высвобождению фактора транскрипции E2F, ключевого триггера перехода клеток из фазы G1 в S-фазу клеточного деления [14].Активность E2F может быть дополнительно повышена за счет амплификации гена MYC, что ассоциируется с бластоидным вариантом и крайне агрессивным течением заболевания [15,16].Описаны редкие случаи циклин D1-негативной МКЛ, при которой определяются перестройки в генах СCND2 и CCND3.Эта крайне редкая подгруппа циклин D1-/D2-/D3-негативной МКЛ отличается агрессивным течением с признаками дисрегуляции циклина E [17]. ",
"section_name": "ПАТОГЕНЕЗ И МОЛЕКУЛЯРНАЯ БИОЛОГИЯ",
"section_num": null
},
{
"section_content": "",
"section_name": "Fig. 2. Evolution in the understanding and identifi cation of MCL (quoted from",
"section_num": null
},
{
"section_content": "Следующим важным звеном в патогенезе опухоли является гиперэкспрессия транскрипционного фактора SOX11, который не встречается в нормальных В-лимфоцитах.SOX11-опосредованная активация PAX5 блокирует программу дифференцировки В-лимфоцитов путем подавления белка BLIMP1 и тем самым предотвращает попадание клеток МКЛ в герминативный центр [18,19].В опухолевых клетках белок SOX11 регулирует клеточную миграцию, инвазию и рост, участвует в ангиогенезе и служит маркером агрессивного течения заболевания [20,21].Кроме того, экспрессия SOX11 -решающий маркер при установлении диагноза в тех крайне редких случаях, когда, несмотря на отсутствие t(11;14)(q13;q32) с гиперэкспрессией циклина D1, опухоль по другим параметрам, включая профиль экспрессии генов, демонстрирует соответствие МКЛ [22]. В зависимости от функции вовлеченного гена молекулярные нарушения при МКЛ можно объединить в несколько подгрупп.Это нарушения, связанные с генами контроля клеточного цикла (CCND1, RB1, CDK2, CDK4, CDKN2A, CDKN1B, TP53, MYC), участия в сигнальном пути генотоксического стресса (TP53, ATM, CDKN2A, MDM2), регуляции апоптоза (BCL2, MDM2, TP53, CDKN2A), выживания и пролиферации клеток (TRAF2, BIRC3, CARD11) и, наконец, генами эпигенетической модификации (NSD2/WHSC1, MLL2, MLL3) [23].Наиболее частыми являются мутации генов ATM (40-50 %), CCND1 (14-35 %), TP53 (14-31 %), MLL3 (16 %), MLL2 (12-20 %), TRAF2 (7-10 %), RB1 (10 %), NOTCH1 (5-14 %).В одних случаях, например при вовлечении гена АТМ, увеличивается генетическая нестабильность опухоли, однако нет данных о неблагоприятном влиянии этой генетической поломки на прогноз МКЛ [24].Другие, например мутация ТР53, относятся к маркерам крайне агрессивного течения заболевания [25].Изучение и понимание механизмов развития опухоли позволяют идентифицировать ключевые гены, вовлеченные в патогенез заболевания, и использовать их в дальнейшем в качестве мишеней для таргетной терапии. ",
"section_name": "Рис. 3. Последовательные этапы патогенеза классической (нодальной) и индолентной (ненодальной) мантийноклеточной лимфомы (МКЛ) (цит. по [13])",
"section_num": null
},
{
"section_content": "МКЛ составляет около 2-10 % всех неходжкинских лимфом.Медиана возраста составляет 60 лет, муж-чины болеют значительно чаще, чем женщины (2:1).Заболевание обычно протекает с генерализованной лимфаденопатией, гепатоспленомегалией и поражением костного мозга.Нередко диагностируется значимый лейкоцитарный лимфоцитоз, что требует дифференциальной диагностики с другими мелкоклеточными лимфомами.При бластоидном варианте с высоким лейкоцитозом необходимо исключить острый лейкоз.Из экстранодальных поражений часто наблюдается вовлечение ЖКТ, кольца Вальдейера, орбиты.Локальные стадии заболевания крайне редки. Европейская группа по изучению МКЛ провела большой ретроспективный анализ (1998-2015 гг.) данных пациентов, у которых опухоль проявлялась изолированным или преимущественно изолированным экстранодальным вовлечением по типу MALT (MALT МКЛ).В работу были включены клинические данные 127 больных (61 % мужчин) с медианой возраста 65 лет.Зоны поражения распределялись следующим образом: кольцо Вальдейера (32 %), ЖКТ (25 %), орбита (13 %), слюнные железы (13 %), другие локализации (1 %).При медиане наблюдения 80 мес.5-летняя выживаемость без прогрессирования (ВБП) составила 45 %, общая выживаемость (ОВ) -71 %.В сравнении со 128 больными с классической МКЛ за тот же период времени показатели отдаленной выживаемости при MALT MКЛ оказались статистически значимо лучше: медиана ВБП 4,5 vs 2,8 года (p = 0,001), медиана ОВ 9,8 vs 6,9 года (p = 0,018) соответственно.Таким образом, кроме описанного в классификации ВОЗ индолентного подварианта следует иметь в виду наличие отдельной клинической формы MALT МКЛ, которая также характеризуется относительно благоприятным прогнозом и не требует интенсивных режимов терапии [26]. Для определения индивидуального прогноза ОВ на примере 455 случаев классической МКЛ был разработан международный прогностический индекс (MIPI).Он позволяет стратифицировать пациентов на три группы: низкий (44 % больных, медиана ОВ не достигнута), промежуточный (35 %, медиана ОВ 51 мес.) и высокий риск (21 %, медиана ОВ 29 мес.) [27]. В качестве независимых неблагоприятных факторов были выбраны возраст, соматический статус по ECOG, уровень лактатдегидрогеназы и число лейкоцитов в крови (табл.1).Каждый прогностический признак оценивается от 0 до 3 баллов (сумма не должна превышать 11 баллов): низкий (0-3 балла), промежуточный (4-5 баллов), высокий риск (6-11 баллов). Позднее проведено другое крупное исследование, в котором наряду с клиническими параметрами 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Общая выживаемость 1 2 3 4 5 6 7 8 9 10 11 12 13 ",
"section_name": "КЛИНИЧЕСКИЕ ПРОЯВЛЕНИЯ, ФАКТОРЫ ПРОГНОЗА",
"section_num": null
},
{
"section_content": "Результаты молекулярно-генетических исследований при МКЛ позволили установить наличие большого числа повторяющихся нарушений генов, участвующих в регуляции клеточного цикла, репарации ДНК и эпигенетической модификации [28].Показано негативное влияние мутации генов TP53, NOTCH1 и NOTCH2 на прогноз заболевания [29][30][31].Скандинавская группа по изучению лимфом провела серьезный анализ прогностической ценности повторяющихся генетических аберраций в образцах костного мозга 183 первичных молодых пациентов с МКЛ, которые получали интенсивные режимы терапии в рамках протоколов MCL2 и MCL3 [32].Показано, что наибольшее независимое неблагоприятное влияние на ОВ оказывает наличие мутации ТР53, которая определялась у 11 % больных.В этой подгруппе у 50 % пациентов диагностированы рецидивы в течение 1-го года, а медиана ОВ составила 1,8 года по сравнению с 12,7 года у больных МКЛ с TP53 без мутаций (p < 0,0001).Мутации TP53 четко ассоциировались с индексом Ki-67 > 30 %, бластоидной морфологией, высоким риском по MIPI и плохим ответом на терапию (рис.6). Таким образом, наличие мутации гена TP53 позволяет идентифицировать фенотипически отличную и высокоагрессивную форму MКЛ, которую не удается контролировать содержащими цитарабин режимами и высокодозной химиотерапией (ВДХТ) с трансплантацией аутологичных гемопоэтических стволовых клеток (аутоТГСК).Определение статуса ТР53 ко времени диагностики позволяет выделить эту ",
"section_name": "Б",
"section_num": null
},
{
"section_content": "Рис. 6. Общая выживаемость больных МКЛ в зависимости от мутационного статуса гена ТР53 (цит.по [32]) ",
"section_name": "Мантийноклеточная лимфома",
"section_num": null
},
{
"section_content": "Современные подходы к лечению первичных больных МКЛ продолжают изменяться по мере накопления данных о молекулярных механизмах развития болезни и появления новых лекарственных средств.Выбор программы терапии зависит от биологических особенностей опухоли, возраста и соматического статуса пациента, наличия и тяжести сопутствующих заболеваний.В большинстве случаев МКЛ отличается агрессивным течением и требует начала противоопухолевой терапии.Вместе с тем примерно в 10-20 % случаев опухоль можно охарактеризовать как индолентную.Американские коллеги [33] предложили использовать термин «тлеющая» МКЛ, которая имеет ряд клинико-морфологических и других характеристик (табл.2).Для этой категории больных, а также при бессимптомном лейкемическом небластоидном варианте допустима тактика «наблюдай и жди». ",
"section_name": "ЛЕЧЕНИЕ",
"section_num": null
},
{
"section_content": "Перед началом терапии специалисту необходимо ответить на два основных вопроса: является ли больной кандидатом для интенсивных режимов, включая ВДХТ с аутоТГСК, и с учетом конкретной ситуации (доступность трансплантационных стационаров, предпочтения пациента) видит ли он ограничения в реальной возможности выполнения аутоТГСК.Ответы на эти два вопроса позволяют принять правильное решение по выбору адекватной терапии (табл.3). В том случае, если у больного нет противопоказаний для получения интенсивных режимов, но существуют опасения невозможности своевременного проведения аутоТГСК, программой выбора может служить режим R-HyperCVAD/R-MA, который не тре-бует высокодозной консолидации.Согласно данным американских ученых, которые лечили 97 больных МКЛ по этой программе без аутоТГСК, при медиане наблюдения более 1 3 лет общая эффективность (ОЭ) терапии составила 97 %, а медиана ВБП у молодых пациентов была 78 мес.[34].В дальнейших исследованиях было показано, что добавление бортезомиба к этому режиму не улучшило результаты лечения по сравнению с оригинальной программой R-Hyper-CVAD/R-МА [35].Несмотря на эффективность, данный вид терапии характеризуется высокой гематологической токсичностью и значительным числом вторых опухолей (6,2 %).Так, согласно данным итальянских авторов (n = 60) и группы SWOG (n = 49), только у 40 и 60 % больных соответственно удалось завершить полную программу в связи с высокой ее токсичностью [36,37]. В Европе более распространены интенсивные режимы индукции, включающие цитарабин в высоких дозах с последующей консолидацией ВДХТ и аутоТГСК [38,39].В качестве индукционных режимов рассматриваются альтернирующие схемы R-CHOP/R-DHAP (European MCL network) или R-maxi-CHOP/R-HD-araC (Nordic group).Результаты этих двух режимов вполне сопоставимы: медиана ВБП составляет 8,5-9 лет.Все перечисленные выше протоколы, с одной стороны, демонстрируют преимущества в добавлении цитарабина в высоких дозах, с другой -ставят под сомнение целесообразность сохранения R-CHOP в программе.В небольшом исследовании показана эффективность альтернирующего режима BR/R-HD-araC у 23 Таким образом, в настоящее время стандартом лечения первичных молодых (< 65 лет) больных с МКЛ являются интенсивные режимы иммунохимиотерапии с включением цитарабина в высоких дозах, консолидация ВДХТ с аутоТГСК и дальнейшая поддерживающая терапия ритуксимабом.Какие изменения в этой стратегии ожидают нас в будущем?С одной стороны, возможна интенсификация лечения с включением новых препаратов (ингибиторы тирозинкиназы Брутона [BTK]) и аллоТКМ в группе крайне высокого риска с мутацией ТР53.С другой стороны, это попытка деэскалации с отказом от высокодозной консолидации у больных, достигших МОБ-отрицательной ремиссии (исследование E4151, № NCT03267433), или применение «chemo-free window» (нехимиотерапевтическое окно) режима (ибрутиниб + ритуксимаб) с коротким курсом консолидации HyperCVAD без дальнейшего высокодозного этапа (исследование Window-1, № NCT02427620).Ответы на эти и другие вопросы ожидают нас в ближайшие 2-3 года. ",
"section_name": "Первая линия лечения молодых больных МКЛ",
"section_num": null
},
{
"section_content": "Медиана возраста больных составляет 60-65 лет, а значит, лечение именно этой популяции представляет основную проблему при МКЛ.В качестве первой линии терапии рассматриваются содержащие антрациклин или бендамустин режимы [42].В основе большого рандомизированного исследования M. Rummel и соавт.было как раз сравнение режимов R-CHOP и BR у первичных больных с индолентными лимфомами и МКЛ.Последняя группа была представлена 94 пациентами [43].Бендамустин продемонстрировал высокую эффективность и благоприятный профиль токсичности у всей анализируемой группы.При МКЛ медиана ВБП составила 35 и 22 мес.соответственно (p = 0,0044), различий в ОВ не получено.Эти данные были подтверждены в протоколе BRIGHT [44] Таким образом, в лечении пожилых отчетливо прослеживаются две сопоставимые по эффективности программы (BR и R-CHOP + Rmaint), которые, однако, позволяют получить длительные ремиссии только у половины больных.Безусловно, исследователями предпринимаются попытки улучшить эти результаты путем добавления новых препаратов.Первой попыткой были замена винкристина на бортезомиб и проведение рандомизированного сравнения режимов R-CHOP и VR-CAP у больных МКЛ, у которых не планируется ВДХТ [46].В протокол включено 487 первичных больных МКЛ.При медиане наблюдения 40 мес.медиана ВБП была статистически значимо выше в группе с бортезомибом (30 и 16 мес.соответственно; p = 0,0044).Однако это лечение оказалось более токсичным за счет большого числа серьезных тромбоцитопений (57 %).Интересен тот факт, что при более длительном сроке наблюдения (медиана 82 мес.) различия в ОВ после применения VR-CAP стали особенно очевидными (медиана ОВ 90 и 55 мес.соответственно; p = 0,001) [47]. Австралийские коллеги провели важный анализ результатов лечения 70 больных МКЛ (медиана возраста 69 лет) по содержащим антрациклин режимам с или без добавления цитарабина [48].Были проанализированы следующие режимы: R-CHOP-like (n = 39), R-CHOP/R-DHAC (n = 10), R-HyperCVAD/R-MA (n = 7), R-CHOP/Cytarabine (Nordic Protocol) (n = 10) и другие (n = 4).ВДХТ с аутоТГСК выполнена 16 больным.При медиане наблюдения 37 мес.содержащие цитарабин режимы в сравнении с R-CHOP показали лучшие результаты ОЭ (70 и 33 % соответственно; p < 0,001) и отдаленной выживаемости (p = 0,05).Однако они отличались более высокой токсичностью и были выполнимы у относительно молодых больных МКЛ.В такой ситуации итальянские коллеги [49,50] предложили добавлять цитарабин к менее токсичной схеме BR (сначала Ara-C по 800 мг/м 2 в 1-3-й день, затем доза из-за гематологической токсичности была снижена после первого анализа до 500 мг/м 2 в 1-3-й день).Режим R-BAC был протестирован у 57 больных (медиана возраста 71 год) и продемонстрировал удовлетворительные результаты: ОЭ 96 % (МОБ-отрицательные ремиссии 55 %), 2-летняя ВБП 83 %, 2-летняя ОВ 91 %.Следует отметить, что у 49 % пациентов отмечалась нейтропения II-IV степени и у 52 %тромбоцитопения; выполнить все 6 циклов удалось только у 63 % больных.Тем не менее режим R-BAC представляется перспективной опцией лечения МКЛ.Основные режимы первой линии лечения пожилых больных представлены в табл.4. В лечении пожилых больных МКЛ продолжается поиск баланса между эффективностью и токсичностью.В этом контексте чрезвычайно интересным представляется применение режимов «chemo-free».Наиболее изученной является схема R2, которая включает индукционный этап и поддерживающее лечение максимально до 3 лет: леналидомид 20 мг в 1-21-й день каждые 28 дней (всего 12 циклов), затем леналидомид по 15 мг в режиме поддержки + ритуксимаб еженедельно (4 введения), далее каждые 2 мес.[51].В протокол включено 38 пожилых больных МКЛ, причем 1 / 3 из них составляла группу высокого риска по MIPI.При медиане наблюдения 64 мес.5-летняя ВБП составила 64 %, 5-летняя ОВ -77 %. Продолжается большое число исследований с включением новых препаратов (ибрутиниб, акалабрутиниб) в комбинации с ритуксимабом или обинутузумабом, которые изучаются в первой линии терапии у пожилых больных МКЛ.Интересными представляются следующие протоколы лечения, результаты которых ожидаются в скором времени: Для контроля долгосрочной эффективности лечения больных МКЛ важна оценка МОБ как основного Г.С.Тумян КЛИНИЧЕСКАЯ ОНКОГЕМАТОЛОГИЯ маркера качества противоопухолевого ответа и риска развития рецидива [52].Вместе с тем в отличие от хронического лимфолейкоза МОБ при МКЛ изучена недостаточно, поскольку не у всех больных имеется поражение костного мозга ко времени диагностики или достаточно высокий опухолевый лимфоцитоз в крови, который можно оценить проточной цитофлюориметрией или аллель-специфической ПЦР.В этой связи интересным представляется использование новых методов обнаружения IgH-клона или количественный ПЦР-анализ t(11;14)(q13;q32) периферической крови больных МКЛ [53].Применение ритуксимаба, ингибиторов BTK или леналидомида в качестве поддерживающей терапии может служить инструментом для достижения МОБ-отрицательной ремиссии. ",
"section_name": "Первая линия лечения пожилых больных МКЛ",
"section_num": null
},
{
"section_content": "Несмотря на успехи в лечении больных МКЛ, заболевание считается неизлечимым.Американские ученые из Memorial Sloan Kettering Cancer Center (MSKCC) провели большой ретроспективный анализ своей базы данных за 15 лет (2000-2014 гг.).В исследование включено 404 больных МКЛ, которые получали лечение и наблюдались в этом центре на протяжении указанного периода времени [54].Этот анализ предоставляет уникальную возможность проследить за течением заболевания, оценить эффективность множественных линий терапии.Согласно их данным, каждая последующая ремиссия при МКЛ значительно короче предыдущей, а срок жизни больных неизбежно уменьшается.Так, медиана ОВ после первой линии составила 116,3 мес., а далее -41, 25, 14 и, наконец, 8,6 мес.соответственно линиям терапии; медиана ВБП -47, 14, 6, 5 и 3,2 мес.соответственно (рис.7).Тем не менее необходимо отметить, что медиана ОВ больных, у которых заболевание диагностировано после 2000 г., превышает 11 лет, в то время как до 1990 г. этот показатель не достигал 3 лет [55].Это связано с интенсификацией лечения и внедрением новых эффективных лекарственных средств.Особого внимания требуют больные МКЛ с ранним рецидивом или прогрессированием опухоли: медиана ВБП и ОВ после первого раннего рецидива составляет 9,6 и 29,9 мес.соответственно.Наиболее эффективны режимы первой и второй линий, все последующие линии терапии у больных МКЛ не различаются по длительности ремиссии и составляют в среднем менее 6 мес. Полученные результаты вновь указывают на то, что на ранних этапах течения болезни необходимо внедрять новые препараты в схемы терапии и комбинировать лекарственные средства с различным механизмом действия.Представляем перечень перспективных новых препаратов, которые уже успешно применяются или проходят испытания при рецидивах МКЛ.Венетоклакс -селективный ингибитор антиапоптотического белка Bcl-2 для приема внутрь.Гиперэкспрессия Bcl-2 обычно наблюдается при различных В-клеточных опухолях, в т. ч. и при МКЛ, и способствует выживанию клеток, блокируя в них апоптоз.В I фазу клинического исследования включено 28 больных с рецидивами МКЛ (медиана возраста 72 года, в среднем три линии предшествующей терапии).Препарат оказался эффективным в 75 % наблюдений с частотой ПР у 21 % больных и с медианой ВБП 14 мес.[64].Обнадеживающие результаты получены при использовании комбинации венетоклакса с ибрутинибом в рамках протокола AIM [65].В анализ включены данные 23 больных (75 % пациентов имели высокий риск по MIPI, у 50 % определялась мутация TP53).Эта комбинация оказалась эффективной в 71 % случаев (ПЭТ-отрицательные ремиссии у 62 % больных).Медиана ВБП не достигнута, 12-месячная ВБП составила 75 %.Особо следует отметить, что у половины больных с мутацией ТР53 (n = 12) достигнута ПР.Основным побочным эффектом III-IV степени была диарея (71 %) и нейтропения (33 %).Венетоклакс имеет большой потенциал в лечении больных МКЛ, особенно в случае прогрессирования после применения ингибиторов BTK. ",
"section_name": "РЕЦИДИВЫ",
"section_num": null
},
{
"section_content": "Ингибиторы mTOR-киназы и других сигнальных путей.Наиболее изученным при рецидивах МКЛ из группы ингибиторов mTOR представляется темсиролимус, который применялся в дозе 175 мг еженедельно в течение 3 нед., далее по 75 мг еженедельно до прогрессирования или непереносимой токсичности.Препарат вводился внутривенно, что затрудняло его длительное применение, при этом частота общего ответа составила 22 %, а медиана ВБП не превышала 4,8 мес.[66]. Ингибиторы PI3K являются перспективным классом препаратов в лечении больных с рецидивами МКЛ, особенно после терапии ибрутинибом.Первый в этом классе ингибитор PI3K-δ иделалисиб показал активность при различных В-клеточных опухолях, в т. ч. и при МКЛ [67].Парсациклиб (ингибитор PI3Kδ второго поколения), энтосплентиниб (ингибитор SYK-киназы) и энзастаурин (протеинкиназа С, PKC) сейчас проходят клиническое исследование при рецидивах МКЛ. ",
"section_name": "НОВЫЕ ПЕРСПЕКТИВНЫЕ ПРЕПАРАТЫ",
"section_num": null
},
{
"section_content": "Иммунотерапия.Ингибиторы контрольных иммунных точек (ипилимумаб, ниволумаб, пембролизу маб) и биспецифические моноклональные антитела (блинатумомаб) представляются определенным прорывом в лечении солидных опухолей и различных гематологических злокачественных опухолей (лимфома Ходжкина, лимфобластная лимфома).Однако при МКЛ отмечается очень низкая экспрессия PD-L1, PD-L2 на опухолевых клетках и не определяются РD1-экспрессирующие Т-лимфоциты в окружающей ткани при биопсии.Этим можно объяснить неэффективность перечисленных выше препаратов при МКЛ. Одним из примеров адоптивной иммунотерапии являются генетически модифицированные Т-лимфоциты, экспрессирующие химерные антигенные рецепторы (CAR T-клетки), которые широко применяются при агрессивных лимфомах.В исследовании I фазы ZUMA-2 изучается эффективность аутологичных анти-CD19 CAR T-лимфоцитов (KTE-C19) у больных с рецидивами МКЛ [33].Другим видом CAR T-клеточной терапии (JCAR-017) является СD19-направленный 4-1BB CAR T-клеточный продукт, применяемый при рецидивах МКЛ.Эти два протокола проходят первый этап изучения, но могут стать потенциально важным направлением в лечении рецидивов МКЛ после применения ингибиторов BTK. АллоТКМ рекомендуется больным из группы высокого риска с мутациями ТР53 при рецидивах МКЛ.Эта стратегия позволяет получить длительный контроль над болезнью примерно у 30 % больных [33]. ",
"section_name": "Мантийноклеточная лимфома",
"section_num": null
},
{
"section_content": "Последние десятилетия расширили наши знания о биологии и клональной эволюции МКЛ.Алгоритмы лечения, основанные на интенсивной химиоте-рапии, с включением цитарабина в высоких дозах, аутоТГСК с дальнейшей поддерживающей терапией ритуксимабом позволили длительно контролировать заболевание у значительного числа больных.С другой стороны, появление инновационных лекарственных средств (ингибиторы BTK, леналидомид, венетоклакс) с уникальным механизмом действия расширило возможности лечения рецидивов заболевания.Дальнейшее изучение различных молекулярных нарушений позволит в будущем стратифицировать больных МКЛ на группы риска и проводить адаптированную терапию с учетом МОБ-статуса (рис.9). Применение новых препаратов вселяет надежду на возможность ухода от традиционной химиотерапии у определенной части больных.Основной целью лечения МКЛ станет рациональная комбинация препаратов с эффективным уничтожением остаточного клона, что в итоге может привести к долгосрочному контролю заболевания. ",
"section_name": "ЗАКЛЮЧЕНИЕ",
"section_num": null
},
{
"section_content": "Автор заявляет об отсутствии конфликтов интересов.Г.С.Тумян, член редакционной коллегии журнала «Клиническая онкогематология.Фундаментальные исследования и клиническая практика», не участвовала в рецензировании рукописи. ",
"section_name": "КОНФЛИКТЫ ИНТЕРЕСОВ",
"section_num": null
},
{
"section_content": "Исследование не имело спонсорской поддержки.Рис. 9. Настоящее и будущее лечения МКЛ (цит.по [13]) BTK -тирозинкиназа Брутона; МКЛ -мантийноклеточная лимфома; МОБ -минимальная остаточная болезнь. ",
"section_name": "ИСТОЧНИКИ ФИНАНСИРОВАНИЯ",
"section_num": null
},
{
"section_content": "",
"section_name": "ЛИТЕРАТУРА/REFERENCES",
"section_num": null
}
] |
[] |
10.3390/onco4030013
|
A Systems Biology Analysis of Chronic Lymphocytic Leukemia
|
<jats:p>Whole-genome sequencing has revealed that TP53, NOTCH1, ATM, SF3B1, BIRC3, ABL, NXF1, BCR, and ZAP70 are often mutated in CLL, but not consistently across all CLL patients. This paper employs a statistical thermodynamics approach in combination with the systems biology of the CLL protein–protein interaction networks to identify the most significant participant proteins in the cancerous transformation. Betti number (a topology of complexity) estimates highlight a protein hierarchy, primarily in the Wnt pathway known for aberrant CLL activation. These individually identified proteins suggest a network-targeted strategy over single-target drug development. The findings advocate for a multi-target inhibition approach, limited to several key proteins to minimize side effects, thereby providing a foundation for designing therapies. This study emphasizes a shift towards a comprehensive, multi-scale analysis to enhance personalized treatment strategies for CLL, which could be experimentally validated using siRNA or small-molecule inhibitors. The result is not just the identification of these proteins but their rank-order, offering a potent signal amplification in the context of the 20,000 proteins produced by the human body, thus providing a strategic basis for therapeutic intervention in CLL, underscoring the necessity for a more holistic, cellular, chromosomal, and genome-wide study to develop tailored treatments for CLL patients.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a type of cancer that affects white blood cells and tends to progress slowly over many years.It is a chronic lymphoproliferative disorder characterized by an increased production of morphologically mature but immunologically dysfunctional B lymphocytes.As a result, these cells are unable to fight infections as well as normal white blood cells do [1]. The disease starts developing in the bone marrow, since here leukemia cells survive longer and eventually outnumber normal cells.Then, cells further grow and may spread to other parts of the body, including the spleen, the lymph nodes, and the liver [1].Since the growth of leukemia cells is slow, CLL may remain latent for many years before it causes symptoms, and it is usually harder to cure than acute leukemias [1]. From the genetic perspective, CLL is a unique disease with multiple gene signatures.One cohort of patients can exhibit a different gene-signature set than another cohort.Whole-genome sequencing has revealed that TP53, NOTCH1, ATM, SF3B1, BIRC3, ABL, NXF1, BCR, and ZAP70 are often mutated in CLL, but not consistently across all CLL patients [2][3][4].For example, NOTCH1 is mutated in about 10% of newly diagnosed patients and in about 15% to 20% of progressive ones.Similarly, SF3B1 is mutated in about 10% of newly diagnosed CLL patients and about 17% in late-stage disease [2].Just because a gene is mutated does not mean that it will be strongly expressed.One of the goals of our study is to show a molecular thermodynamics approach to determine the most energetically significant pathways supporting a given patient's CLL initiation and progression.This new molecular systems approach may shed light on optimal treatment for each patient-essentially personalized therapy.Before we present this new methodology, we provide an overview of the known biomarkers for CLL and then a survey of the current treatment options, as well as experimental drugs in development. It is of fundamental importance to obtain information about the patient's status and prognosis to define the therapeutic strategy.There exist several laboratory-based prognostic markers, such as high levels of serum beta-2 microglobulin (B2M) and the absolute lymphocyte count (ALC).However, chromosomal aberrations detected using Fluorescent In Situ Hybridization (FISH) serve as the main prognostic tools.The most common aberrations detected in CLL patients are as follows [5]: ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Deletions on the long arm of chromosome 13 (del(13q)): In patients with this aberration, the disease progresses slowly. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Deletions on the long arm of chromosome 11 (del(11q)): This usually occurs among young males and tends to manifest with bulky lymph nodes.It is associated with rapid disease progression and short survival.The 11q chromosome contains the Ataxia-Telangiectasia-mutated gene (ATM), and ATM kinase is responsible for inhibited cell cycle progression in the case of DNA damage.Furthermore, ATM kinase acts on p53 by phosphorylating it in order to induce apoptosis.Therefore, when 11q is deleted, this phosphorylation does not occur, and the cell damage cannot be repaired [6]. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Deletion on the short arm of chromosome 17 (del(17p)): This results in the loss of TP53, which is the most important prognostic marker in CLL.It is associated with rapid disease progression and resistance to fludarabine chemoimmunotherapy.In addition to the role of TP53 as a prognostic marker in CLL, it is also fundamentally a predictive marker for chemo-immunotherapy, guiding treatment decisions and potentially influencing the response to specific therapies, such as fludarabine chemoimmunotherapy [7]. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Immunoglobulin heavy-chain variable region gene (IGHV) mutational status: For prognosis and therapy choice, it is important to detect IGHV mutational status since the unmutated state is correlated with low survival. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Other markers, which are present in a low percentage of newly diagnosed CLL patients, but whose incidence increases in patients who are refractory to fludarabine chemotherapy, are mutations of NOTCH1, SF3B1, and BIRC3.Finally, combining genetics, clinical parameters, and biochemistry, the CLL International Prognostic Index (CLL-IPI) is a tool to predict the status of the disease [8]. Wnt signaling is a network of interacting protein pathways which control processes such as cell differentiation, cell cycle regulation, proliferation, apoptosis, cytoskeletal rearrangement, cell polarity, adhesion, motility, migration and invasion, and the interaction with the microenvironment [9].Wnt signaling is correlated with hematopoiesis and is linked with leukemogenesis of cancers such as CLL [9].Two Wnt signaling pathways are associated with CLL, namely the Wnt/β-catenin-dependent and -independent pathways.The Wnt/β-catenin is associated with cell proliferation, homeostasis, and cell cycle regulation, and thus its malfunction indicated a hallmark of many cancers.Regarding the Wnt/β-catenin independent pathway, the Wnt/PCP (Planar Cell Polarity) is the most important one, and it takes place in the regulation of cell polarity, migration, and invasion.Wnt pathways play a role in CLL pathogenesis and response to treatment.Moreover, the expression of Wnt signaling molecules from Wnt/β-catenin and Wnt/PCP pathways is defective in CLL.For example, ROR1 (receptor tyrosine kinase-like orphan receptor), a Wnt-5 (a Wnt protein)-dedicated receptor in the Wnt/PCP pathway, is expressed on the surface of CLL cells and not on the healthy B cells.Therefore, ROR1 is a sensitive marker of a possible relapse of patients with a more aggressive form of the disease. The scope of this study extends beyond the traditional single-target silver bullet approach in drug development, acknowledging the intricate network of proteins that drive the pathological transformation of CLL.A systems biology perspective indicates that targeting a manageable group of five or six network nodes could be more effective for combination therapy design, considering the potential for serious side effects due to overlapping off-target interactions.The statistical thermodynamics method applied here aims to identify and hierarchize such targets, which could be inhibited by existing approved or investigational drugs, setting the stage for a more nuanced and personalized treatment approach in CLL. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Unfortunately, currently available treatments may relieve CLL patients from their symptoms and extend their survival, but still CLL remains incurable [6].For patients without \"active disease\" and who are asymptomatic, or those with early-stage disease, the treatment consists of just a simple observation during which blood counts are performed every three months [6].For patients with \"active disease\", before choosing therapy, the clinical status must be evaluated in terms of general health, characteristics such as TP53 abnormalities or adverse cytogenetics, or relapsed disease [6].Standard treatment has been chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR).However, it has demonstrated a lack of efficacy, and it leads to numerous side effects, especially in patients with TP53 or NOTCH1 mutations, unmutated IGHV, and deletion of 17p or 11q [8]. Target agents are small molecules that have greater efficacy in patients harboring TP53 mutation or del(17p), whose examples include the following [6]: ",
"section_name": "Current Treatment Options and Experimental Drug Candidates",
"section_num": "2."
},
{
"section_content": "Bruton tyrosine kinase inhibitors (ibrutinib and acalabrutinib), • BCL-2 inhibitor (venetoclax), ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Purine analogs (fludarabine and pentostatin), • Alkylating agents (cyclophosphamide, chlorambucil, and bendamustine), ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Monoclonal antibodies (rituximab, ofatumumab, and obinutuzumab), The most common chemotherapy medications used are listed below, together with their main mode of action: ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Fludarabine: a purine analogue and an antineoplastic agent. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Cyclophosphamide: an alkylating agent. • Rituximab: a monoclonal antibody which targets the B-lymphocyte antigen CD20 expressed on the surface of B cells.• These three together (FCR) constitute a chemoimmunotherapy treatment: ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Bendamustine: an alkylating agent used along with Rituximab (BR) to form another combination chemoimmunotherapy treatment. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Chlorambucil: an alkylating agent. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Ibrutinib is a Bruton tyrosine kinase (BTK) inhibitor.BTK, an enzyme which works for B-cell survival and growth, helps delay the progression of cancer.It inhibits CLL cell migration, proliferation, and survival [10].Unfortunately, it presents some side effects, such as pneumonia, upper respiratory tract infection, atrial fibrillation, sinusitis, headaches, nausea, and many more [10]. • Acalabrutinib is a more selective irreversible BTK inhibitor since it acts just like ibrutinib, but without the side effects involving other kinases [10].Its most common side effects are headaches, tiredness, low red blood cells, low platelets, and low white blood cells [10]. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "PI3K (Phosphatidylinositol-3-kinase) inhibitors such as Idelalisib [11], which was FDA-approved in 2014 for use in combination with rituximab for treating relapsed CLL [12].However, Idelalisib is also toxic with nearly 40% of patients having had to interrupt the therapy due to rash or 3-4-grade transaminitis, and pulmonary infections.A PI3Kδ inhibitor called TGR 1202 has better selectivity compared to idelalisib.It was approved for medical use in the USA in February 2021.TGR 1202 reduces the phosphorylation of AKT in lymphoma and leukemia cells.• Venetoclax binds and inhibits the antiapoptotic protein B-cell lymphoma 2 (BCL-2) [9]. In CLL, the inhibition of this pathway has been considered an optimal therapeutic strategy [13].The use of Venetoclax was approved by the FDA in 2016 [13].Its side effects usually include low levels of white and red blood cells, respiratory infections, diarrhea, nausea, tiredness, and tumor lysis syndrome (TLS). ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Sotorasib (AMG510) is a highly selective and irreversible inhibitor which binds at an allosteric pocket, leading to the trapping of KRAS (Kirsten rat sarcoma virus) in an inactive GDP-bound state.Note that KRAS transmits signals for growth, division, and differentiation to the nucleus of the cell from the outside.KRAS mutations are among the most oncogenic events in carcinomas, including CLL, and a majority of them consist of missense mutation of the 12th codon (glycine).It was approved by the FDA in May 2021.Some of its side effects are diarrhea, nausea, and muscle or bone pain [14].• Adagrasib (MRTX849) is an irreversible covalent inhibitor of G12C KRAS mutation that makes a covalent bond to cysteine and binds in the switch-II pocket of KRAS in its inactive GDP state.It demonstrated improved antitumor activity when in combination with vistusertib (an mTOR inhibitor).In clinical trials, some patients experienced pneumonitis and heart failure, which led to the interruption of the treatment.Others experienced nausea, fatigue, and anemia.This inhibitor is still in clinical trials, together with numerous other experimental drugs under development [14]. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Experimental drug candidates also include AKT pathway allosteric inhibitors: ARQ092/ miransertib, BAY1125976, MK2206, and TAS-117 [15].• ATP-competitive AKT inhibitors: Capivasertib and ipatasertib showed a favorable safety profile, along with signs of activity in phase I monotherapy trials [14].Other AKT inhibitors include the following compounds: afuresertib (GSK2110183), uprosertib (GSK2141795 and GSK795), and ordidonin (NSC-250682) [16]. Additionally, various drug candidates are in development with MYC pathway-inhibition profiles [17]: • KI-MS2-008 [20], • L755507 [17]. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "The conceptual framework for understanding the thermodynamics and energetics of the molecular biology of human diseases from a network biology perspective has been developed over the past decade.Various studies were undertaken to quantify different signaling and metabolic pathways in various cancer types and other diseases, using metrics such as network entropy and the Gibbs free energy applied to each specific case [21][22][23][24][25][26].Here, we give only a brief summary of this approach.The transcriptome and other \"-omic\" (e.g., proteomic, genomic, etc.) measures can represent the energetic state of a cell.Here, we mean \"energetic\" from a thermodynamics perspective.There is a chemical potential between interacting molecules in a cell, and the chemical potential of all the proteins that interact with each other can be imagined forming a rugged landscape, not dissimilar to Waddington's epigenetic landscape [27,28]. The method we propose uses mRNA transcriptome data or RNA-seq data as a surrogate for protein concentration.This assumption is largely valid.Kim et al. [29] and Wihelm et al. [30] have shown an 83% correlation between mass spectrometry-generated proteomic information and transcriptomic information for multiple tissue types.Furthermore, Guo et al. [31] found a Spearman correlation of 0.8 in comparing RNAseq and mRNA transcriptome from TCGA human cancer data [32]. Given a set of transcriptome data, a representative of protein concentration, we overlay that on the human protein-protein interaction network from BioGrid [33].This means that we assign to each protein on the network the scaled (between 0 and 1) transcriptome value (or RNAseq value).From that, we can compute the Gibbs free energy of each protein-protein interaction using the mapping relation: where c i is the \"concentration\" of the protein i, normalized or rescaled to be between 0 and 1.The sum in the denominator is taken over all protein neighbors of i, including i. Therefore, the denominator can be considered a degree-entropy.Carrying out this mathematical operation essentially transforms the \"concentration\" value assigned to each protein to a Gibbs free energy.Thus, we replace the scalar value of transcriptome to a scalar function-the Gibbs free energy.The above equation is derived from a well-known concept in chemical thermodynamics [34].A biological cell or a group of cells (a tumor) exist in a complex chemical balance produced by a network of interacting molecular species, ranging from small molecules to some very large molecules on the order of hundreds to thousands of Daltons.The molecular concentration balance in this network is the Gibbs free energy, G.This thermodynamic quantity is typically expressed in the context of systems kept at a constant temperature and pressure, where the system can exchange molecules with the environment.For an arbitrary molecular system, the Gibbs function is given as a molar difference [35] in Equation (2): δG = µδn. ( where µ symbolizes the chemical potential, δG is the Gibbs energy, and δn is the molar difference (essentially concentration difference).Typically, one writes the chemical potential as follows: Equation ( 3) above assumes that the molar concentrations of other molecular components (other than i) are held constant, along with constant temperature and pressure.Using Equation ( 1) and given a network of interacting chemical species or proteins, and given their concentration, we can compute the Gibbs free energy for a single protein in the PPI. The Gibbs free energy is a negative number, so associated with each protein on the network is a negative energy well.This results in a rugged energy landscape represented schematically in Figure 1.If we use what is referred to as a topological filtration on this landscape, we essentially move a filtration plane up from the deepest energy well.As the filtration plane is moved up, larger-and-larger energetic subnetworks are captured.For convenience, we stop the filtration at energy threshold 32-meaning 32 nodes in the energetic subnetwork.We call these subnetworks Gibbs-homology networks. Onco 2024, 4, FOR PEER REVIEW 6 Equation ( 3) above assumes that the molar concentrations of other molecular components (other than i) are held constant, along with constant temperature and pressure.Using Equation ( 1) and given a network of interacting chemical species or proteins, and given their concentration, we can compute the Gibbs free energy for a single protein in the PPI. The Gibbs free energy is a negative number, so associated with each protein on the network is a negative energy well.This results in a rugged energy landscape represented schematically in Figure 1.If we use what is referred to as a topological filtration on this landscape, we essentially move a filtration plane up from the deepest energy well.As the filtration plane is moved up, larger-and-larger energetic subnetworks are captured.For convenience, we stop the filtration at energy threshold 32-meaning 32 nodes in the energetic subnetwork.We call these subnetworks Gibbs-homology networks.We now compute the Betti centrality, a topological measure, on the 32-node energetic networks, as described in Benzekry et al. [23].The concept is easily described.In networks, there are holes or rings of various sizes.In these energetic pathways, protein-protein interaction networks, the proteins form interaction rings.In densely connected, but not fully connected, networks, the rings or holes may consist of triangles and larger rings of interaction.To find the Betti centrality, we ask ourselves the following question: Which protein, when removed from the network, will change the overall total number of rings the most?The total number of rings is called the Betti number.Given a network G consisting of edges, e, and vertices, v, the Betti centrality is given by Equation (4): Hence, the difference from the total Betti number, B(G), and the Betti number of the network after removing node i gives the Betti centrality for node i.We compute this for all nodes in the threshold-32 energetic network.Often, there will be two or more proteins in the network that have equivalent Betti centrality. ",
"section_name": "Systems Biology Background",
"section_num": "3."
},
{
"section_content": "We report on a meta-analysis of 1001 samples from CLL patients and cancer cell lines.This study used online data from GEO [36]: GSE10139, GSE28654, GSE31048, GSE39671, GSE49896, GSE50006, and GSE69034.The data were mRNA expression numbers, all collected using Affymetrix Human Genome Array, HG-U133_2.We also used the human We now compute the Betti centrality, a topological measure, on the 32-node energetic networks, as described in Benzekry et al. [23].The concept is easily described.In networks, there are holes or rings of various sizes.In these energetic pathways, protein-protein interaction networks, the proteins form interaction rings.In densely connected, but not fully connected, networks, the rings or holes may consist of triangles and larger rings of interaction.To find the Betti centrality, we ask ourselves the following question: Which protein, when removed from the network, will change the overall total number of rings the most?The total number of rings is called the Betti number.Given a network G consisting of edges, e, and vertices, v, the Betti centrality is given by Equation (4): Hence, the difference from the total Betti number, B(G), and the Betti number of the network after removing node i gives the Betti centrality for node i.We compute this for all nodes in the threshold-32 energetic network.Often, there will be two or more proteins in the network that have equivalent Betti centrality. ",
"section_name": "Methodology and Datasets",
"section_num": "4."
},
{
"section_content": "We report on a meta-analysis of 1001 samples from CLL patients and cancer cell lines.This study used online data from GEO [36]: GSE10139, GSE28654, GSE31048, GSE39671, GSE49896, GSE50006, and GSE69034.The data were mRNA expression numbers, all collected using Affymetrix Human Genome Array, HG-U133_2.We also used the human protein-protein interaction network from Biogrid [33].In particular, we used the dataset downloaded from the BIOGRID-ORGANISM: homo_sapiens-3.5.172.2.An integrative analysis, including the t-SNE visualization of samples and subgroups, is detailed in Appendix C (Figure A1). Reiterating the method, we collected the GSE expression datasets, and then the expression value for each gene was overlaid on the human protein-protein interaction network for each protein or node in the network.For each node in the network, we then applied Equation (1), which resulted in the Gibbs energy for that node.This resulted in a rugged landscape similar to the one shown in Figure 1.Then, the procedure consisted in performing a filtration and computing the Betti number for zero nodes removed and then removing a node and recomputing the Betti number and replacing the node.This removalcomputation-replacement procedure resulted in a list of nodes that had the largest impact on complexity of the Gibbs homology network.We finally ranked significant nodes in a Pareto chart for each patient.Pareto charts were prepared at several filtration thresholds: 32, 48, 64, and 96. ",
"section_name": "Methodology and Datasets",
"section_num": "4."
},
{
"section_content": "Our discussion of the results is presented below, and it follows an analysis of the individual datasets and the research publication associated with it (if present) prior to presenting the meta-analysis Pareto chart and the network graphs.i. Ref. [37] (GSE10137) \"A genomic approach to improve prognosis and predict therapeutic response in chronic lymphocytic leukemia\", by Friedman et al., 2009.This was one of the papers with a large table in the Supplementary section.The table consisted of upregulated and downregulated probes indicative of progressive disease; upregulated and downregulated probes indicating chlorambucil resistance; upregulated and downregulated probes indicative of Pentostatin, Cyclophosphamide, and Rituximab signature.An important quote from the paper states that: \"Others have previously noted the prognostic significance of cytoskeletal genes and the tumor necrosis factor in CLL.Notably, probes for ZAP-70 did not constitute this genomic signature, although mean expression for ZAP-70 probes in samples from patients with progressive disease was higher than those from patients with stable disease\".The table of genes was parsed from the PDF document and used in our subsequent analysis (discussed below).ii.Ref. [3] (GSE28654) \"Gene expression profiling identifies ARSD as a new marker of disease progression and sphingolipid metabolism as a potential novel metabolism in chronic lymphocytic leukemia\" (Trojani et al., 2012) [3].A table in the manuscript lists about 65 genes that were selected as being differentially expressed in two cohorts of CLL patients.Of those genes, the authors selected 19 genes for PCR analysis because of their significance.Those genes are ZAP70, ARSD, LPL, ADAM29, AGPAT2, CRY1, MBOAT1, YPEL1, NRIP1, RIMKLB, P2RX1, EGR3, TGFBR3, APP, DCLK2, FGL2, ZNF667, CHPT1, and FUT8.An important quote from the paper is as follows: \"In the literature, lists of differentially expressed genes obtained using high-throughput microarray by different laboratories and research centers have often limited overlap [38,39].These differences are matters of important scientific discussions and are imputed, among other causes, to dataset dimensions: small number of subjects (some tens) with respect to the number of variables (tens of thousands of genomic probes in human).Notably and reassuringly the gene set list (65 genes) emerged from this study showed a substantial (but not quantitated) overlap with results from previously published microarray studies [40][41][42][43]\".The list of 65 genes was incorporated in our subsequent analysis.iii.Ref. [40] (GSE31048) \"Somatic mutation as a mechanism of Wnt/β-Catenin pathway activation in CLL\" [40].In the Supplementary Materials to this paper were two large tables listing genes.One table listed from their own study (Wnt pathway), and the other table listed Wnt genes from the literature and websites.Both tables were combined for the study.A quote from the paper: \". . .our data demonstrate that altered gene expression is indistinguishable between samples with and without mutations\".iv.Ref. [41] (GSE39671) \"Subnetwork-based analysis of chronic lymphocytic leukemia identifies pathways that associate with disease progression\" (Chuang et al., 2012) [41]. The Supplementary data included only figures and graphs.No table with a gene list.Of note is the following quote: \"Furthermore, the marker sets identified by different research groups often share few genes in common.Two landmark studies, Rosenwald and colleagues [42] and Klein and colleagues [43] each identify approximately 100 genes that were expressed differentially by CLL cells that use mutated versus unmutated IGHV genes.However, only 4 marker genes were identified in common between these studies\".v. Ref. [44] (GSE49896) \"miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1\" (Mraz, et al., 2014) [44]. The following is quoted from their paper: \"We identified miR-150 as being the most abundantly expressed miRNA in CLL.However, we observed significant heterogeneity in the expression levels of this miRNA among CLL cells of different patients.Lowlevel expression of miR-150 associated with unfavorable clinicobiological and prognostic markers, such as expression of ZAP-70 or use of unmutated IGHV (p < 0.005).Additionally, our data suggest that the levels of methylation of the upstream region of 1000 nt proximal to miR-150 associate with its expression.We demonstrated that GAB1 and FOXP1 genes represent newly defined direct targets of miR-150 in CLL cells.We also showed that high-level expression of GAB1 and FOXP1 associates with relatively high sensitivity of CLL cells to surface immunoglobulin ligation.High levels of GAB1/FOXP1 and low levels of miR-150 associate with a greater responsiveness to BCR ligation in CLL cells and more adverse clinical prognosis\".vi.GSE50006-no manuscript.vii.GSE69034-no manuscript. We created a master list of all genes cited and/or given in the tables associated with the above manuscripts.This list is in Appendix B. There were 515 genes in total.The list of genes was inputted into the DAVID platform for functional annotation analysis [45], and only 208 genes were found, which indicates that DAVID's database has annotations for only 208 of those genes.The missing genes might be due to them being less wellcharacterized, newer discoveries not yet integrated into DAVID's database, or they might be represented differently in the user's list compared to DAVID's nomenclature.To identify genes relevant for a generic condition like \"leukemia\", the KEGG [46] and OMIM [47] databases are used to filter and analyze the results such that both are integrated into DAVID.These databases contain curated information about genes related to specific pathways or diseases.By cross-referencing the 208 identified genes with \"leukemia\" in both KEGG and OMIM, genes whose expression or mutation is linked with the onset, progression, or other aspects of leukemia are pinpointed, aiming at narrowing down potential targets for research, therapeutic development, or further molecular study.Searching that file resulted in the following list: AKT1, CTBP1, CTBP2, CTBPA, SMAD4, HDAC1, LEF1, RARA, TCF3, TCF7, TCF7L1, TCF7L2, and MYC. Comparing the PublishedGeneList with our CLLnet96 list, only four were found: MYC, HDAC1, CTNNB1, and APP.Two of those, MYC and HDAC1, are known to participate in leukemia.The CLLnet96 list is assembled from all 1001 patients at Gibbs threshold 96.To reiterate the concept of threshold, for any given patient, the deepest well in the landscape is usually the same for all thresholds; but there may be differences based on the expression, and this gives rise to differences in the Gibbs homology network.An energy threshold of, say, 32 will result in a network of 32 nodes that are the largest negative energy values.This is called a topological filtration.Using this technique, we can produce 1 of these 32 threshold networks for each patient.If we do that, and then concatenate the entire list of nodes for each of the patients at this threshold, followed by sorting and discarding redundant nodes in the list, the result will be what we call the CLLnet32 list.By the nature of the filtration, CLLnet32 ⊂ CLLnet48 ⊂ CLLnet64 ⊂ CLLnet96, meaning that CLLnet32 is a proper subset of CLLnet48, etc. So, taking the list CLLnet96 will, by definition, incorporate all others.Comparing our CLLnet96 with the superset of published genes (i.e., PublishedGeneList in the Appendix B), we find only four that were both lists, MYC, HDAC1, CTNNB1, and APP. After comparing the PublishedGeneList and the CLLnet96 superset, we then used DAVID, an online bioinformatics resource that allows one to submit a list of genes (or other biological components, e.g., proteins), and it returns important information such as the KEGG pathway or OMIM associated with that gene.There are 98 genes in the superlist of CLL96net list.From that analysis, we find the following genes to be associated with leukemia (various types): KRAS, GRB2, HDAC1, NPM1, TP53, and MYC.In that superlist, CUL1, TP53, and CTNNB1 are associated with the Wnt signaling pathway. The published gene lists consisted of two parts.Keep in mind that, although the papers cited above included GSE expression data, most of them did not include tables of genes they identified from their analysis as being important.Instead, they were looking for prognostic markers for disease progression.So, Part 1 of the published gene list consisted of selections identified by the authors from GSE10137 and GSE28654.The combined list consisted of 320 genes.Of those 320 genes, 22 were found in DAVID.Only CEBPA and MYC were found to be associated with any form of leukemia.And CSNK2A1 and MYC were found to be associated with the Wnt signaling pathway.When we expand the published list to include GSE321048, which was a focused study on the Wnt pathway and CLL [40], the list expands to 515.Naturally, a huge number of genes were flagged by DAVID as being in the Wnt pathway (78 total).And a smaller subset was found to be associated with some form of leukemia: AKT1, CTBP1, CTBP2, CEBPA, SMAD4, HDAC1, LEF1, RARA, TCF3, TCF7, TCF7L1, TCFL2, and MYC.Looking for common genes between the expanded published and our larger list of 96 threshold, we find KRAS, GRB2, HDAC1, NPM1, TP53, MYC, APP, and CTNNB1. At threshold 32, CTNNB1 is the best Betti target once out of 1001 patients, but it is present in the threshold 32 networks 326 times.Keep in mind that anything found in the 32 threshold is energetically important.So, we find it in 32.5% of the population as a potentially good target for CLL (at 48 threshold 37.9%, at 64 threshold 44.1%, and at 96 threshold 58.9%).CTNNB1 is an important gene involved in CLL.It is also an important node in the Wnt pathway [48]. ",
"section_name": "Results",
"section_num": "5."
},
{
"section_content": "It is interesting that so many of the authors of the papers cited above did not find overlap among their gene list and other investigators.There was little overlap between those authors' lists until we included the dataset from GSE321048, the Wnt pathway.We speculate that the reason our Gibbs analysis of expression data did not overlap well with other expression data is that the Gibbs function includes a measure of network entropy (denominator in Equation ( 1)).Furthermore, many of the genes that are highly expressed, as reported in the literature, are not necessarily mutated based on whole-genome sequencing. Figure 2 shows the Wnt pathway from KEGG.After using the online R-script KEG-Graph at Bioconductor, it was converted to an edge-list of relevant protein-protein interactions [49]. The resulting edge-list was plotted using Cytoscape 3.7 [50].The PPI network is shown in Figure 3. Two nodes are highlighted.MYC is highlighted and connected to LEF1, TCF7, TCF7L1, and TCF7L2.MYC, as we will see, is an important player in the Wnt pathway.Also, CTNNB1 has 24 neighboring interactions and has a betweenness of 0.3155, the highest in this network.It also is an important player in the Wnt pathway.The resulting edge-list was plotted using Cytoscape 3.7 [50].The PPI network is shown in Figure 3. Two nodes are highlighted.MYC is highlighted and connected to LEF1, TCF7, TCF7L1, and TCF7L2.MYC, as we will see, is an important player in the Wnt pathway.Also, CTNNB1 has 24 neighboring interactions and has a betweenness of 0.3155, the highest in this network.It also is an important player in the Wnt pathway.As described above, we computed the Betti centrality for the Gibbs homology networks.Figure 4 shows a Pareto chart for the Betti centrality nodes at threshold-48.In our analysis of the 1001 expression samples, CTNNB1 was present as a key Betti centrality The resulting edge-list was plotted using Cytoscape 3.7 [50].The PPI network is shown in Figure 3. Two nodes are highlighted.MYC is highlighted and connected to LEF1, TCF7, TCF7L1, and TCF7L2.MYC, as we will see, is an important player in the Wnt pathway.Also, CTNNB1 has 24 neighboring interactions and has a betweenness of 0.3155, the highest in this network.It also is an important player in the Wnt pathway.As described above, we computed the Betti centrality for the Gibbs homology networks.Figure 4 shows a Pareto chart for the Betti centrality nodes at threshold-48.In our analysis of the 1001 expression samples, CTNNB1 was present as a key Betti centrality As described above, we computed the Betti centrality for the Gibbs homology networks.Figure 4 shows a Pareto chart for the Betti centrality nodes at threshold-48.In our analysis of the 1001 expression samples, CTNNB1 was present as a key Betti centrality node in three samples.Whereas MYC was not present as a key Betti centrality node at threshold-48, but at threshold-32, MYC was present 24 times; 12 times (50%), it was found in dataset GSE30671, which is associated with the manuscript by Chuang et al. [41].This again shows the inconsistency in gene expression values from samples of CLL patients.Of key importance is the fact that RPS15 is a Betti centrality node in three patients, and RPS15A is a Betti centrality node in five patients at threshold-48.These are shown in Figure 4. threshold-48, but at threshold-32, MYC was present 24 times; 12 times (50%), it was found in dataset GSE30671, which is associated with the manuscript by Chuang et al. [41].This again shows the inconsistency in gene expression values from samples of CLL patients.Of key importance is the fact that RPS15 is a Betti centrality node in three patients, and RPS15A is a Betti centrality node in five patients at threshold-48.These are shown in As shown in Figure 3, MYC is an important node in the Wnt pathway.It is directly connected to LEF1, TCF7, TCF7L1, and TCF7L2.Except for LEF1, which is a lymphoid enhancer-binding factor, the others are transcription factors.Figure 5 shows a Gibbs homology network at threshold-48 for a patient (GSM787065 part of GSE31048 [40]) whose RPS15 is the Betti centrality node.In the network diagram, the nodes are in a degree-sorted order, starting at the bottom, with MYC as the highest degree (48), and going around counterclockwise.RPS15 and MYC are pulled out of the network for easy locating, and MYC and all its first connections are highlighted in yellow. As we pointed out above, a gene can be mutated, and yet not overexpressed or underexpressed relative to normal.This is likely the main cause for differences in reported transcriptome data from various investigators.What is clear from the literature (e.g., Wang et al.) [40] is that the Wnt pathway is highly important, and overexpressed genes in that pathway are often indicative of cancer.MYC is a regulator of ribosome protein synthesis [51] and has been shown to be a key regulator in supporting and maintaining tumorigenesis [52].For example, Wu et al. [52] found that inactivation of MYC resulted in some tumors undergoing regression, and mutated RPS15 was identified in almost 20% of CLL patients who relapsed after FCR treatment.These mutations are associated with clinical aggressiveness in CLL, along with the mutant RPS15 displaying defective regulation of endogenous p53, which indicates a novel molecular mechanism underlying CLL pathobiology [52].RPS15 and RPS15A are often overexpressed in CLL [53], and our results confirm this with all (1001 patients) Gibbs-homology subnetworks at threshold-96, showing RPS15 or RPS15A as being an energetically important node.As shown in Figure 3, MYC is an important node in the Wnt pathway.It is directly connected to LEF1, TCF7, TCF7L1, and TCF7L2.Except for LEF1, which is a lymphoid enhancer-binding factor, the others are transcription factors.Figure 5 shows a Gibbs homology network at threshold-48 for a patient (GSM787065 part of GSE31048 [40]) whose RPS15 is the Betti centrality node.In the network diagram, the nodes are in a degree-sorted order, starting at the bottom, with MYC as the highest degree (48), and going around counterclockwise.RPS15 and MYC are pulled out of the network for easy locating, and MYC and all its first connections are highlighted in yellow. As we pointed out above, a gene can be mutated, and yet not overexpressed or underexpressed relative to normal.This is likely the main cause for differences in reported transcriptome data from various investigators.What is clear from the literature (e.g., Wang et al.) [40] is that the Wnt pathway is highly important, and overexpressed genes in that pathway are often indicative of cancer.MYC is a regulator of ribosome protein synthesis [51] and has been shown to be a key regulator in supporting and maintaining tumorigenesis [52].For example, Wu et al. [52] found that inactivation of MYC resulted in some tumors undergoing regression, and mutated RPS15 was identified in almost 20% of CLL patients who relapsed after FCR treatment.These mutations are associated with clinical aggressiveness in CLL, along with the mutant RPS15 displaying defective regulation of endogenous p53, which indicates a novel molecular mechanism underlying CLL pathobiology [52].RPS15 and RPS15A are often overexpressed in CLL [53], and our results confirm this with all (1001 patients) Gibbs-homology subnetworks at threshold-96, showing RPS15 or RPS15A as being an energetically important node. ",
"section_name": "Results and Discussion: Wnt Pathway",
"section_num": "6."
},
{
"section_content": "We showed in this study that the genes BTK, NFkB, JAK/STAT, NOTCH1, BCL2, and EEF2, among others, play a significant role in the support of CLL.Yet, some of them are only rarely studied in the literature because they are not strongly expressed; our research confirms this.Just because a gene or two are mutated does not mean that they will be strongly expressed.As pointed out above, gene expression found inconsistent sets of genes that were highly expressed and that had high Gibbs energy.We found the same inconsistent results when we looked at the Hi-C results for CLL patients.Speedy et al. [54] found that BCL2 was strongly implicated in the disease.They also found a disruption at the NFkB-binding site, but other genes, such as JAK/STAT, BTK, and EEF2, were not mentioned in their manuscript.Beekman et al. [55] found only NOTCH1, Puiggros et al. [56] found only NOTCH1 and SF3B1 as candidates for high risk of mutation, and Kiefer et al. [57] found NOTCH1 for trisomy 12. Though the actual causal agent of CLL is not well known, we can speculate that if there is some molecular agent (e.g., herbicide) or an energetic EM signal (e.g., X-ray), it will typically impact the cell only during a specific phase of the cell cycle [58].There are regions of the genome that are more sensitive to alterations due to some specific energy level in the overall molecular network we call a cell.These mutations are driven by the relevant chemical potential, stereochemistry, and Gibbs free energy.We argue that the locations of the relevant genes in the chromosome and the 4D dynamics of the nucleome may suggest a more holistic molecular and cellular approach to understanding CLL and, ",
"section_name": "Conclusions",
"section_num": "7."
},
{
"section_content": "We showed in this study that the genes BTK, NFkB, JAK/STAT, NOTCH1, BCL2, and EEF2, among others, play a significant role in the support of CLL.Yet, some of them are only rarely studied in the literature because they are not strongly expressed; our research confirms this.Just because a gene or two are mutated does not mean that they will be strongly expressed.As pointed out above, gene expression found inconsistent sets of genes that were highly expressed and that had high Gibbs energy.We found the same inconsistent results when we looked at the Hi-C results for CLL patients.Speedy et al. [54] found that BCL2 was strongly implicated in the disease.They also found a disruption at the NFkBbinding site, but other genes, such as JAK/STAT, BTK, and EEF2, were not mentioned in their manuscript.Beekman et al. [55] found only NOTCH1, Puiggros et al. [56] found only NOTCH1 and SF3B1 as candidates for high risk of mutation, and Kiefer et al. [57] found NOTCH1 for trisomy 12. Though the actual causal agent of CLL is not well known, we can speculate that if there is some molecular agent (e.g., herbicide) or an energetic EM signal (e.g., X-ray), it will typically impact the cell only during a specific phase of the cell cycle [58].There are regions of the genome that are more sensitive to alterations due to some specific energy level in the overall molecular network we call a cell.These mutations are driven by the relevant chemical potential, stereochemistry, and Gibbs free energy.We argue that the locations of the relevant genes in the chromosome and the 4D dynamics of the nucleome may suggest a more holistic molecular and cellular approach to understanding CLL and, therefore, new therapeutic strategies [59].Building on this notion, the insights from the 4D Nucleome Network [59] elucidate the intricacies of genome organization in space and time.The project underlines the critical role of the genome's three-dimensional organization in gene regulation.In the context of CLL, the spatial dynamics of chromatin can have a profound impact on gene expression patterns, emphasizing the importance of the genome's spatial and temporal dynamics in understanding and potentially treating the disease [60].Elucidating this idea further, the work conducted by Sawh et al. in 2022 revealed that the eukaryotic genome is a multilayered entity, exhibiting intricate organization levels that range from nucleosomes to larger chromosomal scales [61].These layers undergo significant remodeling across different tissues and developmental stages in C. elegans.It is noteworthy that advancements in C. elegans research, both imaging-based and sequencing-based, have unveiled the influence of histone modifications, regulatory elements, and broader chromosome configurations in this 4D organization.Specific revelations, such as the physiological implications of topologically associating domains and compartment variability during initial developmental phases, underscore the depth of genome dynamics.These insights provide compelling evidence that understanding such 4D genome organization nuances is crucial for decoding complex diseases like CLL.Interestingly enough, none of the genes described in Appendix A is in chromosome 13, which often has deletions in about 50% of CLL patients [62].In Appendix A, we support our argument for a larger view that CLL genes are widely spread throughout the whole genome and different chromosomes. In conclusion, our study challenges the conventional single-target paradigm in CLL therapy, advocating for a higher-level, network-oriented strategy.The identification and hierarchical ranking of 20-30 significant proteins, amidst the roughly 20,000 synthesized by the human organism, represent a leap in signal detection and amplification [63].This nuanced profiling, achieved via a statistical thermodynamics approach, underscores the potential of targeting a selective array of five or six network nodes.This selectivity is crucial to mitigate the risk of adverse effects caused by overlapping off-target interactions commonly seen with broader therapeutic targets.The proteins highlighted in our research, notably within the Wnt signaling pathway, are not merely isolated entities but components of a complex network that drives the CLL pathology.Therefore, our proposed method does not end at the identification of these proteins but extends to rank-ordering them in terms of therapeutic relevance.The next step for validating the findings involves experimental assays using siRNA [64] or small-molecule inhibitors, which will provide the empirical backbone for our theoretical model.Such an approach may revolutionize the current treatment regimens by transitioning from a one-size-fits-all model to a more customized, patient-specific strategy.This could be especially beneficial given the genetic variability among CLL patients, as indicated by the inconsistent mutation patterns observed in wholegenome sequencing.By incorporating the principles of systems biology and acknowledging the network dynamics of protein interactions, we can begin to envision a more effective, personalized therapeutic landscape for CLL.This, in turn, may pave the way for similar strategies in other cancers, marking a paradigm shift in oncological treatment towards precision medicine.genomic approach from GSE10139 to improve prognosis and therapeutic response predictions, and juxtaposes this with expression data from CLL tumors and healthy donor B cells from GSE50006.The inclusion of CLL-blood samples enriches the dataset, illustrating the heterogeneity within CLL and potentially reflecting different disease phases or subtypes.The blend of these datasets furnishes a more comprehensive understanding of the disease, highlighting the heterogeneity of CLL and reinforcing the necessity of personalized medicine approaches. ",
"section_name": "Conclusions",
"section_num": "7."
},
{
"section_content": "Cluster D presents a cohort of 152 patients, predominantly sick (144) with a small representation of normal B cells (8), combining data from GSE10139 and GSE50006.This distribution, primarily composed of CLL and CLL-blood samples, continues to emphasize the genetic and expression-level diversity found in CLL, supporting the need for an in-depth analysis to discern the nuances of the disease's progression and the potential response to treatments. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Cluster E is a homogeneous group consisting entirely of 100 sick patients from the GSE49896 dataset.This study spotlights the microRNA-150's influence on B-Cell Receptor signaling by modulating GAB1 and FOXP1 gene expressions, which are implicated in CLL.MicroRNAs are crucial post-transcriptional regulators, and their role in CLL adds an additional layer to our understanding of the disease's complexity and potential intervention points. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "In Cluster F, 130 CLL patients from the GSE39671 dataset were studied, all of whom had undergone treatment.The data represent a temporal progression, with sampling times to first treatment recorded, allowing for an exploration of the disease's evolution over time.The dataset's analysis provides prognostic subnetworks which can help predict disease progression and highlight the converging pathways in CLL, opening new avenues for tailored treatments. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Cluster G, comprising 75 CLL patients from the GSE69034 study, delves into the gene expression profiles linked with the MYD88 L265P mutations in conjunction with IGHV mutation status.The presence of the MYD88 L265P mutation, a notable variant found within the MYD88 gene that encodes a key adaptor protein in the Toll-like receptor and IL-1 receptor pathways, has been tied to specific prognostic outcomes in CLL.This mutation is known to activate downstream signaling pathways aberrantly, which can contribute to the uncontrolled proliferation of B cells characteristic of CLL.The dataset's inclusion in the study facilitates a detailed investigation into the mutation's role and its pathway associations in CLL, offering a potential explanation for the varying responses to treatment observed in patient populations.By analyzing the gene expression patterns influenced by the MYD88 L265P mutation, alongside the IGHV mutation status, a well-established prognostic marker in CLL, it unravels the complex interplay between genetic aberrations and their impact on the disease's clinical course.The correlation between MYD88 L265P mutations and factors such as treatment resistance, disease progression, and overall survival can be assessed.This is particularly crucial, as the mutation's impact on signaling pathways may suggest new therapeutic targets or strategies for intervention.Groundbreaking biomarkers are likely to be identified for early detection and prognosis by understanding the biological context in which these mutations operate, while also highlighting the therapeutic relevance of targeting the MYD88 pathway in certain subsets of CLL patients, thus implying the importance of precision medicine in the management of CLL.Based on the insights into the specific mutations driving the disease in individual patients, therapies can be customized to target these genetic abnormalities more effectively.In the case of MYD88 L265P, its presence could signify a need for targeted inhibitors that can mitigate its downstream effects, thereby introducing a new dimension to personalized CLL treatment paradigms. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Cluster H is a cohort of 84 CLL patients from GSE28654, all carrying the IgVHMT mutation and exhibiting negative ZAP-70 expression.The absence of ZAP-70 expression, a kinase linked to CLL, together with the mutational profile, provides a critical connection for investigation.This relationship implicates the substantial impact of the mutation on CLL's clinical progression and pinpoints the need for a detailed genetic analysis in crafting specialized treatments. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "In Cluster I, 28 sick patients from GSE28654 were categorized by the presence of the IgVHUM mutation and positive ZAP-70 expression, helping us to understand the disease's heterogeneity, since ZAP-70 positivity is often linked with a more aggressive CLL form.The combination of mutational status and ZAP-70 expression levels provides valuable prognostic information. The expression of ZAP-70 in CLL and its relevance as a molecular marker is particularly illuminating.For Cluster H, the collective profile of CLL patients characterized by the IgVHMT mutation yet displaying an absence of ZAP-70 expression represents a subset where traditional prognostic markers may predict a more favorable clinical course.In the broader landscape of our findings, this cluster could suggest that ZAP-70's negativity may reflect a less aggressive form of CLL, where the malignant B cells might not engage in the same signaling pathways that are characteristic of more virulent variants.Consequently, these insights bolster the argument for personalized therapeutic approaches, enabling clinicians to tailor treatments to the specific molecular makeup presented by individual CLL cases.Conversely, patients in Cluster I, characterized by the IgVHUM mutation concomitant with positive ZAP-70 expression, suggest a more aggressive manifestation of the disease.This association aligns with the understanding that ZAP-70 positivity mirrors the behavior of unmutated IgVH status, commonly linked to a robust disease progression and a less favorable response to conventional therapies.Here, ZAP-70 serves not just as a prognostic marker but potentially as a therapeutic target, whereby modulation of its expression or function could impact CLL cell survival.This reiterates the substantial role that ZAP-70 plays in CLL.It acts as a bifurcation point in the disease's prognostic roadmap, where its expression could either denote a need for more aggressive treatment or suggest a less intensive therapeutic course.The interplay of ZAP-70 with IgVH mutation status, as demonstrated in our clusters, provides a clearer understanding of disease heterogeneity and patient stratification.The overall results of the study thus advocate for the integration of ZAP-70 status into prognostic models and therapeutic decision-making algorithms, emphasizing its contribution not only to prognostication but potentially to the development of targeted CLL therapies. • Cluster J, mirroring Cluster G, includes another set of 75 CLL patients from the GSE69034 dataset, indicating the significant role of MYD88 L265P mutations in CLL, providing a robust dataset for the exploration of mutation-associated gene expression patterns and their prognostic significance. The heterogeneity of gene mutations across CLL patients underscores the intricate complexity of this malignancy, accentuating the necessity for individualized therapeutic strategies.The disparities unearthed by t-SNE analysis manifest in the distinct molecular signatures differentiating normal B cells from CLL-B cells, which reflect divergent evolutionary trajectories within the disease's progression.Notably, the aberrant expression of Wnt-pathway genes in CLL cells, as revealed by our cluster analysis, pinpoints this pathway's pivotal role in CLL pathobiology.The presence of specific gene expressions within clusters, particularly those highlighted by the t-SNE method (Clusters A and B), points to the pathway's disrupted regulation, which is suggestive of patient-specific disease mechanisms that contribute to CLL's heterogeneity.Simultaneously, the funding emphasizes that alterations in the Wnt signaling pathway are not universally present but vary among patients, reinforcing the pathway's contribution to the disease complexity.The therapeutic potential of targeting Wnt-pathway proteins is corroborated by their identified roles in vital cellular functions, with their significance accentuated by Betti number estimates, which propose these proteins as central players in CLL's pathogenesis rather than inconsequential elements.Such insights solidify the imperative for a more comprehensive, multi-scalar study from cellular to genomic dimensions to forge ahead with personalized treatments for CLL. ",
"section_name": "•",
"section_num": null
}
] |
[
{
"section_content": "Funding: J.A.T. acknowledges the funding support received from NSERC (Canada) for this project.J .Z., H.H., E.A.R., and H.T.S. acknowledge no external funding for this work. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The data for the CLL study are from the GEO Website and, more specifically, are the following datasets: GSE10139, GSE28654, GSE31048, GSE39671, GSE49896, GSE50006, and GSE69034. The data for the CLL study are from the GEO Website and, more specifically, are the following datasets: GSE10139, GSE28654, GSE31048, GSE39671, GSE49896, GSE50006, and GSE69034. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "The data for the CLL study are from the GEO Website and, more specifically, are the following datasets: GSE10139, GSE28654, GSE31048, GSE39671, GSE49896, GSE50006, and GSE69034. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Author Contributions: G.L.K. suggested the study; E.A.R. and G.L.K. selected the datasets; E.A.R. conducted the computational study and wrote the first draft with input from G.L.K.; H.T.S. supported E.A.R. and provided consulting on the project; G.P. contributed the study on drugs and rewrote the paper with the assistance of J.Z.; H.H. performed the clustering analysis; J.A.T. contributed to the cancer drug analysis and wrote the paper with G.P. and E.A.R.All authors have read and agreed to the published version of the manuscript.Funding: J.A.T. acknowledges the funding support received from NSERC (Canada) for this project.J.Z., H.H., E.A.R., and H.T.S. acknowledge no external funding for this work. Informed Consent Statement: Not applicable. Author Giannoula Lakka Klement is employed by CSTS Healthcare.Author Edward A. Rietman has been involved as a consultant in Applied Physics. Notes on the genomic location of key genes \"involved\" in CLL (from GeneCards.org)BTK is found in the X chromosome. Genes around BTK on UCSC Golden Path with GeneCards custom track. Xq22.1 by HGNC Xq22.1 by Entrez Gene Xq22.1 by Ensembl BTK Gene in genomic location: bands according to Ensembl, locations according to GeneLoc (and/or Entrez Gene and/or Ensembl if different) Onco 2024, 4, FOR PEER REVIEW 14 Conflicts of Interest: We declare no conflicts of interest. Notes on the genomic location of key genes \"involved\" in CLL (from GeneCards.org)BTK is found in the X chromosome.• Cluster A encapsulates a significant cohort focusing on the Wnt signaling pathway, a key player in CLL pathogenesis, with a total of 179 patients.Within this cluster, 21 are wild type, while 158 are CLL-patient samples, all derived from the GSE31048 dataset.This dataset offers a unique look at both normal and CLL-affected B cells, allowing for a direct comparison of Wnt pathway gene expression and Wnt-regulated gene expression.The marked difference in numbers between the normal (12 and 9, respectively) and sick (149 and 9, respectively) groups underscores the aberrant expression within CLL-affected B cells, highlighting the pathway's prominence in these cellular states.The study's in-depth focus on the Wnt pathway is well-founded, as it is pivotal in cellular processes that are often disrupted in CLL, thus potentially illuminating new therapeutic avenues. Cluster B includes a smaller, yet focused subset of 42 patients, of which 12 are wild-type and 30 are CLL patients.This cluster continues the examination of the Wnt pathway's role in CLL as part of the GSE31048 study, indicative of a unique or divergent role of Wnt signaling in this subset.It is particularly noteworthy that this dataset includes expression data from CLL B cells with and without Wnt3a treatment, providing insights into the pathway's functionality and potential for targeted therapies.The comparative analysis of Wnt gene expression between the normal and CLL B cells offers additional evidence for the pathway's critical role in the disease process. Cluster C, with 136 patients, 24 normal and 112 sick, merges data from two distinct studies, GSE10139 and GSE50006, providing a broader scope by incorporating a ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Author Contributions: G.L.K. suggested the study; E.A.R. and G.L.K. selected the datasets; E.A.R. conducted the computational study and wrote the first draft with input from G.L.K.; H.T.S. supported E.A.R. and provided consulting on the project; G.P. contributed the study on drugs and rewrote the paper with the assistance of J.Z.; H.H. performed the clustering analysis; J.A.T. contributed to the cancer drug analysis and wrote the paper with G.P. and E.A.R.All authors have read and agreed to the published version of the manuscript.Funding: J.A.T. acknowledges the funding support received from NSERC (Canada) for this project.J.Z., H.H., E.A.R., and H.T.S. acknowledge no external funding for this work. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Author Giannoula Lakka Klement is employed by CSTS Healthcare.Author Edward A. Rietman has been involved as a consultant in Applied Physics. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Notes on the genomic location of key genes \"involved\" in CLL (from GeneCards.org)BTK is found in the X chromosome. ",
"section_name": "Appendix A",
"section_num": null
},
{
"section_content": "Genes around BTK on UCSC Golden Path with GeneCards custom track. ",
"section_name": "Genomic View for BTK Gene",
"section_num": null
},
{
"section_content": "Xq22.1 by HGNC Xq22.1 by Entrez Gene Xq22.1 by Ensembl BTK Gene in genomic location: bands according to Ensembl, locations according to GeneLoc (and/or Entrez Gene and/or Ensembl if different) Onco 2024, 4, FOR PEER REVIEW 14 Conflicts of Interest: We declare no conflicts of interest. ",
"section_name": "Cytogenetic band:",
"section_num": null
},
{
"section_content": "Notes on the genomic location of key genes \"involved\" in CLL (from GeneCards.org)BTK is found in the X chromosome.• Cluster A encapsulates a significant cohort focusing on the Wnt signaling pathway, a key player in CLL pathogenesis, with a total of 179 patients.Within this cluster, 21 are wild type, while 158 are CLL-patient samples, all derived from the GSE31048 dataset.This dataset offers a unique look at both normal and CLL-affected B cells, allowing for a direct comparison of Wnt pathway gene expression and Wnt-regulated gene expression.The marked difference in numbers between the normal (12 and 9, respectively) and sick (149 and 9, respectively) groups underscores the aberrant expression within CLL-affected B cells, highlighting the pathway's prominence in these cellular states.The study's in-depth focus on the Wnt pathway is well-founded, as it is pivotal in cellular processes that are often disrupted in CLL, thus potentially illuminating new therapeutic avenues. ",
"section_name": "Appendix A",
"section_num": null
},
{
"section_content": "Cluster B includes a smaller, yet focused subset of 42 patients, of which 12 are wild-type and 30 are CLL patients.This cluster continues the examination of the Wnt pathway's role in CLL as part of the GSE31048 study, indicative of a unique or divergent role of Wnt signaling in this subset.It is particularly noteworthy that this dataset includes expression data from CLL B cells with and without Wnt3a treatment, providing insights into the pathway's functionality and potential for targeted therapies.The comparative analysis of Wnt gene expression between the normal and CLL B cells offers additional evidence for the pathway's critical role in the disease process. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "Cluster C, with 136 patients, 24 normal and 112 sick, merges data from two distinct studies, GSE10139 and GSE50006, providing a broader scope by incorporating a ",
"section_name": "•",
"section_num": null
}
] |
10.18632/oncotarget.27080
|
Methylation alteration of <i>SHANK1</i> as a predictive, diagnostic and prognostic biomarker for chronic lymphocytic leukemia
|
Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the clonal expansion of malignant B cells. To predict the clinical course of the disease, the identification of diagnostic biomarkers is urgently needed. Aberrant methylation patterns may predict CLL development and its course, being very early changes during carcinogenesis. Our aim was to identify CLL specific methylation patterns and to evaluate whether methylation aberrations in selected genes are associated with changes in gene expression. Here, by performing a genome-wide methylation analysis, we identified several CLL-specific methylation alterations. We focused on the most altered one, at a CpG island located in the body of SHANK1 gene, in our CLL cases compared to healthy controls. This methylation alteration was successfully validated in a larger cohort including 139 CLL and 20 control in silico samples. We also found a positive correlation between SHANK1 methylation level and absolute lymphocyte count, in particular CD19+ B cells, in CLL patients. Moreover, we were able to detect gains of methylation at SHANK1 in blood samples collected years prior to diagnosis. Overall, our results suggest methylation alteration at this SHANK1 CpG island as a biomarker for risk and diagnosis of CLL, and also in the personalized quantification of tumor aggressiveness.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL), one of the most common mature B cell neoplasm subtypes, is characterized by the monoclonal expansion of malignant B cells.CLL is a clinically and biologically heterogeneous disease.In fact, while some patients exhibit slow progression of the disease, others experience a more aggressive form and require adequate therapy to be initiated soon after diagnosis.No screening tests are currently recommended for this disease, and CLL is often diagnosed when undergoing blood tests for other reasons.The two staging systems currently used, Rai and Binet, remain good indicators of survival, but do not take into account other biological factors implied in the disease course, and consequently do not allow early recognition of the aggressive forms of the disease [1].To date, several recurrent genomic aberrations, such as del(17p), del(11q), trisomy 12, and del(13q), and genetic alterations, such as TP53, BIRC3, NOTCH1, SF3B1, have been proposed as prognostic biomarkers of CLL cases [2], and their characterization is recommended before initiating therapeutic treatment.However, since genetic aberrations might not be present at diagnosis, and they might occur later during the disease course, their characterization is not always sufficient to predict the clinical outcome in the initial stage.The mutational status of IGHV gene is one of the most commonly used prognostic biomarkers but, alone, it is inadequate to explain the clinical course heterogeneity of this disease.Therefore, diagnostic biomarkers and prognostic biomarkers characterizing different clinical courses in the early stages of the disease are urgently needed. Aberrant methylation patterns, which represent a molecular hallmark of cancer, are early events in tumor development, and they might represent early diagnostic biomarkers.It has been shown that methylome alterations would predict the diagnosis several years prior to the clinical appearance of the disease, thus proving to be potential markers for carrying out large-scale screening tests [3,4].Besides, aberrant methylation changes might represent a specific signature of different types of leukemia, depending on the originating cell [5].Our aim was to identify specific-CLL methylation patterns by conducting a genome-wide methylation analysis, and to evaluate whether methylation aberrations in selected genes are associated with changes in gene expression. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We conducted a first differential methylation analysis between 18 CLL cases and 6 population controls (Figure 1A), identifying 5001 CpG sites differentially methylated (adjusted p-value <0.05).As expected, the CLL methylome was characterized by a widespread hypomethylation, but by restricting the analysis to the regulatory regions, a switch towards hypermethylation, especially in the CpG islands (CGIs), was evident (Figure 2).Since the genome-wide methylation analysis was conducted on whole blood samples, which are characterized by a high level of cellular heterogeneity, we were not able to detect CpG islands significantly (adjusted p-value <0.05) hypermethylated in CLL samples compared to normal control samples.For this reason, we selected the 100 top-ranked differentially methylated CGIs (Supplementary Table 1). As the total number of lymphocytes is much higher in tumor samples than in control samples, it must be taken into account in the differential methylation analysis.For this reason, we repeated the differential methylation analysis using the absolute lymphocyte count data as a covariate in the limma model.This analysis identified 7886 CpG sites differentially methylated (adjusted p-value <0.05) between CLL and control samples.There was no substantial change in the list of the 100-top ranked differentially methylated CGIs (Supplementary Table 1) detected in this second analysis, which indicates that the differences in the DNA methylation pattern we observed between CLL cases and controls were not due to the different number of circulating lymphocytes. ",
"section_name": "Differential methylation analyses",
"section_num": null
},
{
"section_content": "In order to be able to subsequently carry out a functional study, and having little material available, we focused our attention on the most altered CGI, located in the gene body of SHANK1 (chr19:51198143-51198460, referred to hg19 assembly).This CGI was hypermethylated in the CLL samples, and it showed the highest mean differential methylation value (Δβ = 0.29) between CLL and control samples (Table 1 and Supplementary Table 1).We conducted a correlation analysis between the methylation percentage and the absolute lymphocyte count revealing a significant positive correlation (r = 0.78, p-value = 0.0045) (Figure 3A) between the two variables. Since this methylation alteration was detected analyzing whole blood samples and a differential cell type content has an effect on DNA methylation profile, we investigated cell type heterogeneity in our samples.The relative contributions of each principal immune components of whole blood (B cells, granulocytes, monocytes, NK cells, and T cells subsets) in each sample were estimated by applying the algorithm of Houseman et al. [6] based on the distinctive methylation profiles of each cell type. As expected, granulocytes, the most abundant leukocytes in peripheral blood in a healthy condition [7], contributed for most of cell type composition in normal blood control samples (data not shown). On the other hand, tumor samples with a high lymphocyte count, showed a high CD19+ B-cells contribution and higher SHANK1 methylation values, compared to samples with low CD19+ B-cells contribution (Table 2).Correlation analysis confirmed that there was a strong positive correlation between CD19+ B-cells contribution and SHANK1 methylation values (r = 0.91, p-value = 6,00e-08) (Figure 3B).Individual characteristics such as smoking status, age and BMI can affect DNA methylation and should be used for adjustment in differential methylation analysis.Since our sample size was small, we did not perform an adjustment of SHANK1 methylation values using these data.However, it can be observed that differences in SHANK1 methylation values in cases and between cases and controls are not associated to differences in smoking status, age or BMI (Table 3). To validate and increase the robustness of our data, we analyzed the methylation data for this CGI, obtained by Kulis et al. [8], of 139 CLLs (≥95% neoplastic cells) and 20 non-tumoral samples (normal B-cells from peripheral blood including total B cells and various subtypes of B-cells) (Figure 1B).Methylation data were retrieved from the ICGC Data Portal (https://dcc.icgc.org),DCC Data Release 27, DCC Project Code: CLLE-ES.We detected a Δβ value of 0.26 (p-value = 8.66e-12, adjusted p-value = 2.43e-10), confirming SHANK1 hypermethylation in CLL (Table 1). In addition, Kulis et al. identified the same CGI as hypermethylated in a subgroup of 59 CLL with a low or absent IGHV mutational load (U-CLLs) compared to 6 naïve B cells (CD5+NBC/NBC) (false discovery rate <0.05) [8] (Figure 1B, Table 1). To evaluate the potential role of SHANK1-associated CGI as a potential predictive biomarker, we conducted a differential methylation analysis between cases and matched controls in blood collected at baseline entry into the prospective cohort study (Figure 1A), finding a gain of methylation in the SHANK1 CGI (chr19:51198143-51198460) detectable in blood samples collected years before diagnosis of 82 CLL and small lymphocytic lymphoma (SLL) (Δβ = 0.047, p = 0.00863, adjusted p-value = 0,0921) (Table 1).Moreover, by extending the methylation analysis to the larger series of 438 mature B-cell neoplasms (MBCN) cases, including the 82 CLL/SLL, we confirmed significant differential methylation of the same CGI (Δβ = 0.03, p-value < 10 -7 , adjusted p-value = 7.47e-05) between cases and controls (Table 1).Figure 4 shows the methylation values observed in the extended cohort (Figure 4A) and in the CLL/ SLL subgroup (Figure 4B). ",
"section_name": "SHANK1 methylation alteration as a potential tumor biomarker",
"section_num": null
},
{
"section_content": "To investigate the impact of SHANK1-associated CGI hypermethylation on gene expression, we tested the gene expression level of SHANK1 in 27 CLL cases and 16 non-tumor subjects by qRT-PCR (Figure 1A).SHANK1 showed a significant almost 8-fold down-regulation (p-value <0.0001) in CLL cases compared to controls (Figure 5). SHANK1 downregulation was also confirmed using GUSB as reference gene in a subgroup of samples (data not shown). To validate SHANK1 downregulation observed in our CLL samples, we analyzed RNA-seq data publicly available.Differential gene expression data of ten CLL specimens versus five normal peripheral blood CD19+ B cells were retrieved from GEO database under the accession number GSE70830.In this dataset, SHANK1 gene showed a non-significant differential expression between CLL and control samples. Gene expression data of a large dataset [9] including 98 CLL and three subtypes of normal B cells (naïve, memory IgM/IgD, and memory IgG/IgA), from three different healthy individuals revealed that only three CLL samples show more than 10 reads and most CLL and normal samples have zero reads, therefore making these data unsuitable for a validation of SHANK1 downregulation. Whole blood gene expression data from The Genotype-Tissue Expression (GTEx) Portal (https:// gtexportal.org)revealed that SHANK1 is weakly expressed in whole blood (median Transcripts Per Kilobase Million (TPM): 0.030, number of samples: 407).RT-qPCR represents the most precise and sensitive method to detect differences in gene expression for weakly expressed genes.On the other hand, the accurate quantification of gene expression by RNA seq depends on the sequencing depth and it has been suggested that sequencing up to 100 million reads can be necessary to quantify precisely genes and transcripts that have low expression levels [10]. ",
"section_name": "SHANK1 gene expression analysis",
"section_num": null
},
{
"section_content": "Aberrant methylation patterns, one of the most striking features of cancer, might represent useful biomarkers for prediction of cancer risk, early diagnosis, prognosis, and for prediction of response to treatment and cancer relapse.Furthermore, as DNA methylation is an early epigenetic reversible modification, specific drugs can be developed to restore the DNA methylation pattern of normal cells at the initial stages of carcinogenesis. A substantial body of evidence suggests methylation changes as innovative biomarkers for both early cancer diagnosis and prognosis [11].Some such changes have already shown clinical relevance, such as BRCA1 in breast cancer, MGMT in glioblastoma multiform, and SEPT9, which has been approved by the Food and Drug Administration (FDA) for the diagnosis of colon cancer.Since global DNA methylation is similar in resting and proliferative compartments [12], aberrant methylation patterns may be an early event in CLL, as suggested by its occurrence in blood samples collected years before diagnosis of mature B-cells neoplasm [3] and CLL [4]. In addition, methylation alterations in CLL, such as ZAP70 [13] and HOXA4 [14], have been proposed as prognostic biomarker, and differential methylation profiles are used to classify CLL patients in three molecular CLL subtypes having different clinical features and deriving from B-cell subpopulations at different stages of differentiation [5,15,16].To our knowledge, biomarkers for detecting CLL at an early stage, which would permit the initiation of therapy in the first phases of the disease, are still lacking.In our study, we identified several CGIs differentially methylated (Supplementary Table 1), and we confirmed a genome-wide hypomethylation in CLL (Figure 2), as previously observed in other studies [8]. Although three CGIs resulted significantly hypomethylated in CLL samples (Supplementary Table 1), we do not suggest these alterations as diagnostic biomarkers since the difficulty of setting cut-off values to consider a sample as hypomethylated, especially when we want to look for an alteration that correlates, as in our case, with the number of tumor cells, then a biomarker that increases with the increase of tumor cells number.On the other hand, hypermethylated CGI showing high methylation values in tumoral samples could be more easily employed as tumoral biomarker. Our most striking finding was the hypermethylation of a CGI located in the gene body of SHANK1 in CLL samples.Although this differential methylation was not statistically significant after correction for multiple testing probably due to the fact that the sample size in our study was small, we were able to replicate our finding by in silico analysis of a larger CLL series [8].This type of approach has proved to be successful in the identification of other highly specific and sensitive tumor biomarkers [17][18][19][20].Moreover, the robustness of our data was verified in different validation and exploratory sets (Table 1).For these reasons, it is important to mention that, although we are aware of the importance of significance threshold for epigenome-wide studies (EWAS) [21], the methylation alterations detected are somatic epimutations, and even at the onset of the disease, thus the heterogeneity of the tumor, and in particular, as in this case, a blood tumor, needs to be taken into account.In our previous work [19], we have shown that in a heterogeneous tissue, such as adenoma, the methylation alterations detected were not statistically significant after multiple testing adjustment.In contrast, the same methylation alteration detected in colorectal carcinomas resist to any correction for multiple testing.In fact, adenomas include a mixture of cells showing methylation alterations and cells not showing the alterations compared to colorectal carcinomas where many tumor cells present the epimutations. In silico replication of our result analyzing data including CLL samples with ≥95% neoplastic cells and control normal B cells [8], confirmed that the detected SHANK1 methylation alteration belongs to neoplastic cells.The fact that we were able to detect this methylation alteration in CLL whole blood samples supported its potential use as a diagnostic biomarker to be introduced in clinical practice without the need of performing an expensive method such as cell sorting.Interestingly, Kulis et al [8] have shown that, in a differential methylation analysis between U-CLL samples and naïve B cells of control samples, the same SHANK1 CGI, identified as altered in our study, was significantly hypermethylated in U-CLL.IGHV mutational status is a prognostic biomarker in CLL: while patients with a high level of IGHV mutation (called as M-CLL) have a favorable prognosis, U-CLL is usually associated with poor outcomes.In our case series, patients with IGHV mutations had an average SHANK1 CGI methylation value of 35.0%, while it was 49.6 % in IGHV non-mutated patients (Table 3).Also, removing an outlier (69.0%methylation), probably due to the fact that this patient was first diagnosed with Follicular lymphoma, resulted in the methylation average dropping to 23.7%.Thus, SHANK1 methylation might be correlated with clinical outcomes in CLL.In this regard, it is interesting to note that we also observed that the β value for the altered CGI in SHANK1 correlates positively with the peripheral lymphocyte count at diagnosis (Figure 3), which also prompted us to repeat the differential methylation analysis by including the lymphocyte count as a covariate; this second analysis confirmed the same CGIs as the most affected by methylation changes (Supplementary Table 1).Inference of cell type contributions on tumor whole blood samples revealed that there was a strong positive correlation between CD19+ B-cells contributions and SHANK1 methylation values (Table 2).It is estimated that the increased number of lymphocytes, that is observed in the lymphocyte count at diagnosis, is mainly due (> 80%) to the proliferation of neoplastic cells.In fact, in an analysis of 110 patients with an absolute lymphocyte count of at least 5 x 10 9 /L, Shanafelt et al. have shown that monoclonal B-cells were more than 86% of B-cells, while polyclonal B-cells represented only a small fraction of total B-cells [22].Therefore, SHANK1 methylation might be a useful molecular biomarker in the personalized quantification of tumor aggression in CLL. Aberrant methylation of genes implicated in MBCN is detectable in blood samples collected many years before diagnosis [3,4].We found that within a prospective cohort, there was a significant gain of methylation (differential methylation of 3%) in the same SHANK1 CGI detectable in peripheral blood collected many years prior to diagnosis with MBCN (Figure 4A).In a subgroup of CLL/SLL cases within the prospective MBCN cohort, there was an even more pronounced gain of methylation (differential methylation of 4.7%) compared with matched unaffected controls (Figure 4B).Since there is an overlap between SHANK1 methylation levels in cases and controls, SHANK1 cannot be suggested as a predictive biomarker at individual levels.However, 210/438 (48%) MBCN samples showed a differential methylation greater than the average Δβ (0.03), ranging from 0.031 to 0.36, and 38/82 (46%) CLL/ SLL samples showed a differential methylation greater than the average Δβ (0.047), ranging from 0.06 to 0.33.It is important to mention that each case is matched to one control.Since we found that SHANK1 methylation values are positively correlated to lymphocyte counts at diagnosis and patients with poor prognosis according to IGHV mutational status showed higher methylation values, we can speculate that SHANK1 might be able to predict aggressive forms of disease years before diagnosis.Further studies including SHANK1 methylation status before diagnosis, at diagnosis and follow-up data are needed to investigate this hypothesis. This difference of methylation is in line with, and even greater than that observed in other genes years before cancer diagnosis [3].Thus, SHANK1 might be a potential predictive biomarker of CLL risk.Although SHANK1 methylation data at diagnosis in other MBCNs were not available in our study, we can speculate that methylation of this gene is an early event in leukemogenesis. The use of an epigenetic biomarker proving to be so traceable and informative without cells sorting, is particularly useful for preventive purposes.Extensive screening aimed to identify individuals at risk for CLL would not make much sense using detectors requiring cells sorting, while it is possible to find alterations in the methylation pattern of this specific CGI already years before the onset, starting from whole blood. SHANK1 is one of the three members of the SHANK (SH3 And Multiple Ankyrin Repeat Domains) gene family.Their respective proteins, SHANK1, SHANK2 and SHANK3 act as scaffold proteins and have a fundamental role in the formation, development and function of neuronal synapses.Mutations of the SHANK gene family are associated to developmental disorders, such as autism and schizophrenia.Investigating the association between SHANK genes methylation and their expression, Beri et al [23] showed that, although all these genes present several methylated CpG sites, only SHANK3 was highly methylated in tissues where its expression was low or absent, suggesting that methylation might regulate tissuespecific SHANK3 expression. In our work, the gene expression analysis revealed that SHANK1 was significantly down-regulated in CLL.The inability to validate this data, in silico, may be due to the fact that a gene already normally repressed, if further down-regulated, is hardly detectable by means of whole exome / whole transcriptome NGS / microarrays techniques.It will therefore be important to verify our observation on a larger database, by means of a targeted expression study and an ultrasensitive technique such as ddPCR.The association between methylation and gene expression is very complex and, while hypermethylation of CGIs located in gene promoters is usually associated with gene down-regulation, hypermethylation of intragenic CGIs has been correlated either positively and negatively with gene expression [8,[24][25][26][27].Intragenic DNA methylation might have a role in several molecular processes, such as regulation of cell-context specific alternative promoter in gene bodies [28], expression of intragenic non-coding RNA [29][30][31][32] and transposable elements [33], alternative splicing [34], alternative polyadenylation sites [35], and enhancer activation [36,37].It has also been shown that intragenic nucleosomes with H3K36 trimethylation (H3K36me3), a histone modification associated with transcript elongation, recruit DNA methyltransferases [38], suggesting that DNA methylation is unable to block transcript elongation.In CLL, Kulis et al [8], have shown that, in absence of CGIpromoter methylation, methylation of CpGs located in the gene bodies of around 900 genes shows a significant correlation (either positive or negative) with gene expression. Since methylation of intragenic CGIs might be inversely correlated with the expression of an alternative transcript and positively regulated with the expression of the main transcript [39], it would be interesting to investigate the association between the methylation of the intragenic CGI of SHANK1 and the expression of alternative transcripts.SHANK1, like the other two SHANK genes, SHANK2 and SHANK3, presents a complex transcriptional structure.This gene contains two different promoters, which generate the longest protein isoform (called SHANK1A) and the shortest one (called SHANK1B), and several splicing sites generating alterative transcripts (Figure 6).SHANK1 is mainly expressed in the brain (https://www.gtexportal.org/home/gene/SHANK1).The CGI found to be hypermethylated in our CLL cases is located in the gene body of the main transcript (uc002psx.1 or ENST00000293441), but it is upstream of the shorter transcript (uc002psw.1 or ENST00000391813) (Figure 6).Thus, we speculate that the hypermethylation of the intragenic CGI might be associated with a downregulation of this shorter transcript.Clearly, a gene expression study to accurately quantify each SHANK1 alternative transcript in a large number of CLL samples and controls is warranted to elucidate the impact of this intragenic CGI hypermethylation in gene expression regulation. To date, it is difficult to hypothesize whether the identified methylation alterations are really associated with a different gene expression profile and what this may possibly influence from a functional point of view.The putative activation or deactivation of isoforms that do not respect the normal cell differentiation program could obviously affect the cellular functionality, so as to induce it to a neoplastic iter.What is certain is that at the moment these alterations can provide useful biomarkers for the early diagnosis of a neoplasia, as well as, in the case of the marker we identified, associate with a different prognosis and even giving the possibility of predicting a risk of disease onset. Other SHANK1-associated CGIs are reportedly differentially methylated in other cancers, and they have been proposed as tumor biomarkers [19,40].The most plausible scenario would be that methylation changes in different SHANK1-associated CGIs would be related to different types of cancer, thus serving as specific signatures for different types of tumors. In summary, our results suggest SHANK1 as a promising tumor biomarker for CLL early diagnosis.Since no screening tests are recommended for CLL and patients can be asymptomatic, it is difficult to diagnose CLL in the first phases of the disease.The introduction of a methylation-based diagnostic biomarker in clinical practice could allow an early detection of CLL cases and the initiation of adequate therapeutic treatments in the first phases of the disease.SHANK1 hypermethylation can be easily found analyzing whole blood samples without cell sorting, supporting its utility in clinical practice.Moreover, since SHANK1 methylation levels correlated with lymphocyte count at diagnosis it can be particularly useful in the recognition of the most aggressive forms of the disease showing a fast progression.Finally, the detection of SHANK1associated CGI gain of methylation many years before diagnosis suggests that SHANK1 methylation levels may be predictive of CLL development.Thus, a screening test based on SHANK1 methylation levels assessment could be developed to monitor people at risk, such as people over age 50, or subjects presenting familiarity for the disease.Further studies on sequential samples before diagnosis, at diagnosis and during disease course are needed to monitor SHANK1 methylation levels and ascertain its involvement in CLL progression. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Eighteen Italian patients (10 men and 8 women, mean age at diagnosis: 65.3±12.3)with a diagnosis of CLL donated a blood sample during their first visit at the outpatient ambulatory of the Hematology department of the A. Businco Oncology Hospital, Cagliari (Sardinia, Italy).All patients were diagnosed using the 2008 WHO Classification of lymphoid neoplasms criteria.[41] Blood samples from six controls (mean age: 51.3±20.2),selected from the 151 participating in a population-based case-control study in the same area of CLL patients, were included in the genome-wide methylation analysis.Demographic characteristics (age, sex, smoking status and BMI) of CLL patients and controls and clinical and immunophenotypic characteristics of CLL samples are reported in Table 3. Mean age (p-value = 0,0711; Student t test), sex (p-value = 1,0000; Fisher's exact test), smoking status (p-value = 1,0000; Fisher's exact test) and mean BMI (p-value = 0,9613; Student t test) were not statistically significant different between cases and controls. In addition, blood samples were available for 27 out of the 29 CLL incident cases (mean age at diagnosis: 65.1±9.3)recruited for the same study at participating Italian hospitals in Novara, Florence, Perugia and Cagliari, and for a random sample of 16 out of the 455 population controls from the same areas, who accepted taking part to the full study protocol.We conducted the gene expression analysis for these 27 CLL cases and 16 controls. We also tested the hypothesis that the selected SHANK1 CpG island (CGI) might undergo methylation changes prior to the clinical manifestation of the disease using a case control study nested within a prospective cohort study of 438 samples of incident mature B-cell neoplasms (MBCN), including 82 CLL and small lymphocytic lymphoma (SLL) cases, considered two forms of the same disease only differing by the location where the cancer primarily occurs, and matched (individually matched to cases at 1:1 ratio based on age at enrollment, gender, ethnicity and DNA source) controls.Additional information about cases and controls demographic characteristics and matching criteria can be found in Woong Doo et al. [3].In this cohort, the mean time between blood collection and diagnosis was 9.5 years (range 0.6-17.8years) for CLL cases and 10.6 years (range 0.2-20 years) for the entire MBCN cohort.DNA was collected predominantly from whole blood samples and analyzed as previously described.In particular, of the 976 samples, 632 were processed from whole blood, 234 from Ficoll-separated mononuclear cells and 10 from buffy coat.[3] All the biological samples analyzed were obtained with written informed consent signed by patients/study participants and ethical approval granted by the \"Comitato Etico Azienda Ospedaliero Universitaria di Cagliari\" (269/09/CE, 26/05/2009). ",
"section_name": "Tumor specimens",
"section_num": null
},
{
"section_content": "DNA was isolated from peripheral whole blood lymphocytes using the DNA extraction 500 arrow® Kit (DiaSorin Ireland Ltd) kit.DNA was quantified with NanoDrop (NanoDrop Products Thermo Scientific Wilmington, DE) and by fluorometric reading (Quant-iT™ PicoGreen® dsDNA Assay Kit). RNA was extracted from PBMCs (Mononuclear cell fractions, isolated over a Ficoll-Hypaque density gradient) using Qiagen RNAeasy mini kit (Qiagen, Hilden, Germany) and quantified using NanoPhotometer (NanoPhotometer™Pearl, Denville®, Denville Scientific, Holliston, MA). ",
"section_name": "DNA and RNA extraction",
"section_num": null
},
{
"section_content": "The DNA extracted was bisulfite converted using the EZ DNA Methylation gold Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.Bisulfite converted DNA was hybridized to Illumina Infinium HumanMethylation450 BeadChips (450K), following the Illumina Infinium HD Methylation protocol.Hybridization fluorescent data were read on an Illumina HiScan SQ scanner. Illumina Methylation 450K raw data were analyzed using the RnBeads analysis software package as previously described.[42,43] Methylation levels [beta values (β)] were estimated as the ratio of signal intensity of the methylated alleles to the sum of methylated and unmethylated intensity signals of the alleles.The β values ranges from 0 (no methylation) to 1 (100% methylation). Differential methylation analysis was conducted on the CpG sites and other 4 genomic regions (tailing, genes, promoters and CpG Islands, so called CGIs).CpG-level p-values were corrected for multiple testing using the false discovery rate (FDR) method.CpGspecific uncorrected p-values within a given genomic region were combined to obtain aggregate p-values for each genomic region and then corrected for multiple testing.[42,43] Based on the absolute and relative effect size of the differences between the study groups, RnBeads combines statistical testing with a priority ranking scheme to assign a combined rank score for differential DNA methylation to each analyzed CpG site and genomic region.[42] The generated priority-ranked list was used to select the top-100 ranked differentially methylated CGIs.The selected CGIs were annotated to nearest genes by using R annotation package FDb.InfiniumMethylation.hg19.[44] An additional methylome analysis was performed adding absolute lymphocyte count data as a covariate in the limma analysis of differential DNA methylation.[42,45] DNA methylation analysis for the samples in the nested case-control study were also performed on the Illumina HM450K Infinium array as previously described.[3] Inference of cell type contributions was conducted using RnBeads applying the method of Houseman et al. [6].This method estimated the cell type contributions of whole blood samples based on methylation profiles of a sorted blood cell types reference.[46] The validity of this method for estimating whole blood cells composition in CLL samples has been recently demonstrated.[4,47] qRT-PCR 1μg RNA/sample was retro-transcribed using the High Capacity Kit (Applied Biosystems, Carlsbad, CA, USA).Gene expression analysis was performed by qRT-PCR, conducted on a DNA Engine Opticon 2 Real-Time Cycler (Bio-Rad, Hercules, CA, USA), using iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) for each gene tested and for the reference genes.Actin Beta (ACTB) gene, which is one of the most often used reference genes in B-CLL gene expression studies [48,49], was used as reference gene.A subgroup of tumoral and non-tumoral samples was also analyzed using Glucuronidase Beta (GUSB) as reference gene. Primers used in this study to conduct qRT-PCR are the following: -5'-AGACCATCAGTGCAAGCGAA-3' (SHANK1 forward) -5'-GGGATCGAAGCTCGACTCAG-3' (SHANK1 reverse) -5'-AAATCTGGCACCACACCTTC-3' (ACTB forward) -5'-AGCACAGCCTGGATAGCAAC-3' (ACTB reverse) -5'-CACCTAGAATCTGCTGGCTACT-3' (GUSB forward) -5'-AGAGTTGCTCACAAAGGTCACA-3' (GUSB reverse) Gene expression data were analyzed using the ΔΔCT method.We used a t-test for independent series to compare the average ΔCT of CLL cases and controls. ",
"section_name": "DNA methylation analysis",
"section_num": null
},
{
"section_content": "The power was estimated using a two-sample t test power calculation.A dataset of 18 samples, those available to us as discovery set, would guarantee a statistical power of 0.8 to detect a differential methylation level of at least 25%, using a type I error of 10e-8 (which takes into account the need to correct for multiple test).PC: PI of the multicenter study, participation in the study design, manuscript review. ",
"section_name": "Power calculation",
"section_num": null
},
{
"section_content": "PZ: study concept and design, supervision on methylation and gene expression analysis, supervision on statistical analysis and bioinformatic analysis, analysis and interpretation of data, drafting of the manuscript. All Authors discussed the results and commented the manuscript. ",
"section_name": "Author contributions",
"section_num": null
}
] |
[
{
"section_content": "www.oncotarget.comThe Melbourne Collaborative Cohort Study (MCCS) recruitment was funded by VicHealth and Cancer Council Victoria.The MCCS was further supported by Australian National Health and Medical Research Council grants 209057, 251553 and 1087683 and by infrastructure provided by Cancer Council Victoria.MCS is a National Health and Medical Research Council of Australia Senior Research Fellow. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "This work was supported by grants from the Italian Ministry for Education, University and Research (PRIN 2007 prot.2007WEJLZB and PRIN 2009 prot.20092ZELR2), and the Italian Association for Cancer Research (IG 2011/11855) to PC. Partly supported also by grants from Fondazione Banco di Sardegna (2012), Fondo per la Ricerca Locale (ex 60%), Università di Cagliari, Regione Autonoma della Sardegna (CRP-79303) to PZ. ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.1371/journal.pone.0013262
|
Tumor Necrosis Factor-α Regulates Distinct Molecular Pathways and Gene Networks in Cultured Skeletal Muscle Cells
|
Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood.We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes.TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells.
|
[
{
"section_content": "Skeletal muscle atrophy or wasting is a common phenomenon in a large number of systemic diseases including sepsis, diabetes, chronic obstructive pulmonary disease, heart failure, and cancer [1,2,3,4].Accumulating evidence suggests that inflammatory cytokines especially TNF-a play a major role in the development of muscular abnormalities resulting in loss of skeletal muscle mass and function [5].Increased levels of TNF-a have been observed under conditions that lead to skeletal muscle atrophy such as chronic heart failure, cancer, AIDS, and cachexia induced by bacteria [6].TNF-a transduces its biological activities by binding to two 55-and 75-kDa receptors [7].Trimeric occupation of TNF receptors by the ligand results in the recruitment of receptor-specific proteins leading to the activation of a cascade of protein kinases such as IkB kinase (IKK), transforming growth factor-b activated kinase 1 (TAK1), mitogen-activated protein kinases (MAPKs), and Akt and several downstream transcription factors [7,8,9]. Nuclear factor-kappa B (NF-kB) is a major proinflammatory transcription factor that regulates the expression of a plethora of genes especially those involved in inflammatory and immune responses [10,11].Depending on the type of stimuli, the activation of NF-kB can occur via either canonical or alternative pathway [10].The canonical NF-kB signaling pathway involves the upstream activation of inhibitors of kB (IkB) kinase-b (IKKb) and subsequent phosphorylation and degradation of IkB proteins.On the other hand, activation of the alternative NF-kB pathway requires the upstream activation of NF-kB-inducing kinase (NIK) and IKKa and the proteolytic processing of p100 subunit into p52 [10,12].Several recent studies have provided strong evidence that constitutive activation of NF-kB leads to skeletal muscle wasting and its inhibition prevents the loss of skeletal muscle mass in response to various catabolic stimuli including TNF-a [10,13,14,15,16,17].Li et al [17] showed that TNF-a-induced activation of NF-kB is responsible for the up-regulation of ubiquitin-conjugating E2 enzyme UbcH2 resulting in increased activity of ubiquitin-proteasome system and degradation of myofibril proteins.Furthermore, the inhibitory effect of TNF-a on myogenesis is mediated through the activation of NF-kB which downregulates the levels of myogenic regulatory factor MyoD in myoblasts through distinct mechanisms [18,19,20]. It is also noteworthy that the catabolic action of TNF-a in skeletal muscle may require the presence of other proinflammatory cytokines, such as TNF-related weak-inducer of apoptosis (TWEAK), interleukin-1b (IL-1b), interleukin-6 (IL-6), and interferon c (IFN-c) [16,21,22,23].A combination of TNF-a and IFN-c has been reported to cause a strong down-regulation of muscle specific gene products including MyoD in cultured muscle cells [18].However, it is not yet clear whether TNF-a augments the expression of other inflammatory cytokines or they are expressed by independent mechanisms in skeletal muscle cells. Recent investigations involving genome-wide gene expression profiling in skeletal muscle has helped in identifying several novel genes which mediate the loss of skeletal muscle mass in different muscle-wasting conditions [24,25,26,27,28].However, the effects of TNF-a on the global gene expression and intracellular pathways that it affects in skeletal muscle remain poorly understood.To attain a better molecular insight into the mechanisms of action of TNF-a in skeletal muscle, we focused the present investigation on identification of TNF-regulated gene expression, gene networks, and molecular pathways in skeletal muscle.Microarray analyses of control and TNF-treated myotubes revealed that TNF-a regulates the expression of several genes and pathways which may be related to its catabolic action in skeletal muscle.Furthermore, our study provides the initial evidence that TNF-a activates both canonical and alternative NF-kB signaling pathways in skeletal muscle cells. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "We have used microarray approach to identify the set of genes which TNF-a regulates in cultured C2C12 myotubes.To detect the expression of both early and late responsive genes, we have performed mRNA profiling after 18h of TNF-a treatment.Analysis using MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] dye showed no significant difference in cell viability between control and TNF-treated C2C12 myotube cultures after 18h (not depicted).Raw and normalized data of this microarray experiment has been submitted to ArrayExpress database (http://www.ebi.ac.uk/microarray-as/ae/) with accession number E-MEXP-2592. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "C2C12 myotubes were treated with TNF-a (10ng/ml) for 18h and the mRNA levels of different genes were monitored by cDNA microarray technique.The microarray gene expression profile appeared normally distributed for TNF-treated samples (Figure 1A) indicating that our analyses of differentially expressed genes is not biased due to skewed distribution of certain genes.Out of approximately 25,000 genes present on our microarray chips, TNF-a significantly (p,0.05)affected the expression of a total of 5,939 genes, out of which 3,349 genes were down regulated and 2,590 genes were up regulated.We have also filtered the genes with the fold change $1.2 or #1.2, which yielded 1,822 differentially regulated genes.In particular, 723 genes were significantly down regulated whereas 1,099 genes were significantly up regulated by TNF treatment.The volcano plot of differentially expressed genes with these cut-off p-values and fold changes is presented in the Figure 1B.Further analysis of differentially regulated genes showed that about 51 genes were differentially regulated by TNF with p-values #0.0001 (28 upregulated and 23 down-regulated with fold values $1.2), 181 genes with p-value #0.001 (91 up-regulated and 90 downregulated), 751 genes with p-values #0.01 (406 up-regulated and 345 down-regulated), and 839 genes with p-values #0.05, of which 574 genes were up-regulated and 265 genes were downregulated (Table 1).The functional annotations of important genes differentially expressed in TNF-treated myotubes are presented in the Table S1.We next sought to determine whether the expression levels of some of the genes which showed significant up-or downregulation in microarray experiment can be recapitulated in independent quantitative real time-PCR (QRT-PCR) assays.QRT-PCR assays were performed for the genes which showed high fold change and/or have a direct or indirect relation with skeletal muscle wasting.As shown in Figure 2A, the expression of Nfkbia (also known as IkBa), Nfkb1, Nfkb2, IL-6, Vcam1, Ccl5, Cxcl5, and Ccl2 was found to be significantly increased in TNFtreated samples in QRT-PCR assays.Similarly, the reduced expression of Notch1, TIMP2, and MyoD in TNF-treated samples was confirmed by independent QRT-PCR assays (Figure 2B) suggesting a direct correlation between microarray and QRT-PCR analysis for almost all the genes tested. C2C12 myoblasts differentiate into myotubes in low serum conditions.However, the differentiation of C2C12 myoblasts into myotubes is never complete [29].C2C12 cultures incubated in differentiation medium still contain a significant number of undifferentiated myoblasts [29].We investigated whether the observed changes in gene expression in response to TNF-a occur in undifferentiated myoblasts or myotubes or both in C2C12 cultures.To answer this question, we first studied whether TNF-a can affect the gene expression in undifferentiated C2C12 myoblasts incubated in growth medium.Treatment with TNF-a significantly increased the mRNA levels of NFKB1, NFKB2, and VCAM-1 (Figure 3A) and reduced the levels of MyoD and Notch1 (Figure 3B) in C2C12 myoblasts. To evaluate whether TNF-a affects gene expression in myotubes, we performed QRT-PCR assays for a few select genes using myosin heavy chain 4 (MHC4, expressed only in differentiated muscles) as the normalizing gene.The mRNA levels of NFKB1, NFKB2, and VCAM-1 were significantly higher (Figure 4A) and that of MyoD were significantly lower (Figure 4B) in TNF-treated cultures compared to untreated cultures.Taken together, these results indicate that TNF-a modulates gene expression in both myotubes and myoblasts in a similar fashion. In addition to TNF-a, several other proinflammatory cytokines such as IL-1b and IL-6 have been postulated to be the mediators of muscle-wasting in various chronic diseases [6].We investigated whether IL-1b and IL-6 can also modulate gene expression in C2C12 cultures similar to TNF-a.Interestingly, we observed that IL-1b but not IL-6 significantly augmented the expression of NFKB1, NFKB2, and VCAM-1 in C2C12 cultures.However, the fold increase in their mRNA levels in response to IL-1b was significantly lower compared to TNF-a (Figure 4A).Moreover, we found that while TNF-a significantly reduced mRNA level of MyoD in C2C12 myotubes, neither IL-1b nor IL-6 had any significant effect on the expression of MyoD (Figure 4B).The data suggest that TNF-a may be the most potent stimulus whereas other cytokines may also contribute to muscle wastage to some extent. To further confirm our microarray findings, we also performed Western blots for a few select proteins affected by TNF-a.Consistent with their mRNA levels, the protein levels of NFKB1, NFKB2, and MMP-9 were significantly increased in TNF-treated myotubes compared to untreated myotubes (Figures 5A and5B).It was interesting to note that while TNF-a increased the mRNA levels of NFKB1 by ,15 fold, the increase in NFKB1 protein level was only ,1.6 fold (Figures 5A and5B).Although the exact reasons remain unknown, it is possible that in addition to increasing the expression, TNF-a also increases the turnover of NFKB1 protein.Furthermore, there is also a possibility that NFKB1 mRNA is subjected to post-transcriptional modifications (including those involving micro RNAs) which may limit its translation into protein.In contrast to mRNA levels, the protein levels of NF-kB inhibitor IkBa was found to be significantly reduced (Figures 5A and5B).The reduced levels of IkBa in TNF -treated myotubes could be attributed to its enhanced degradation in response to NF-kB activation stimuli [10,12].In agreement with microarray data, we found reduced protein levels of Notch1 and TIMP-2 in TNF-treated myotubes (Figures 5C, and5D). ",
"section_name": "Identification of differentially expressed genes in TNFtreated C2C12 myotubes by microarray technique",
"section_num": null
},
{
"section_content": "TNF-a is a well-known activator of canonical NF-kB signaling pathway which involves the upstream activation of IkB kinase-b (IKKb) and subsequent phosphorylation and degradation of IkBa protein [7].Although it has been reported that TNF-a activates NF-kB in skeletal muscle cells [7,10,17,30], there has been no report whether TNF-a can augment the activation of alternative NF-kB signaling pathway which involves the activation of IKKa and proteolytic processing of p100 subunit into p52.Surprisingly, our microarray and subsequent QRT-PCR and Western blot revealed increased expression of both NFKB1 (e.g.p105/p50) and NFKB2 (e.g.p100/p52) in TNF-treated myotubes (Figures 2, 4, and5).These results prompted us to investigate whether TNF-a activates both canonical and alternative NF-kB pathways and the time points at which these two pathways are up-regulated after treatment with TNF-a.C2C12 myotubes were treated with TNFa for different time periods ranging from 0-24h and the activation of NF-kB was measured by electrophoretic mobility shift assays (EMSA).As shown in Figure 6A, treatment with TNF-a led to sustained activation of NF-kB in C2C12 myotubes though it peaked at 1h, 6h and 18h.These results are consistent with a previously published report also demonstrating biphasic activation of NF-kB in C2C12 myotubes upon TNF-a-treatment [31].To investigate whether TNF-a-induced activation involves canonical, alternative, or both pathways, we performed Western blot using TNF-a-treated myotubes.As shown in Figure 6B, treatment with TNF-a increased the phosphorylation and reduced the levels of IkBa protein.IkBa protein levels remained lower compared to untreated cultures indicating that TNF-a stimulates the canonical NF-kB signaling pathway in myotubes.Interestingly, we found that TNF-a did not affect the expression or proteolytic processing of p100 subunit into p52 up to 6h.However, after 6h, a significant increase was noticed in protein levels of both p100 and p52 suggesting the activation of alternative NF-kB pathway.IKKa is the kinase which phosphorylates p100 protein leading to its proteolytic processing into p52 subunit [10].To further investigate whether the activation of IKKa is increased in response to TNF-a treatment, we performed Western blot using antibody which recognizes the phosphorylated (activated) IKKa protein.As shown in Figure 6B, TNF-a-induced the activation of IKKa in a timedependent manner.Consistent with proteolytic degradation of p100 protein, the activation of IKKa was noticeable at 12h and later time points (Figure 6B). To further confirm that TNF-a activates both canonical and alternative NF-kB signaling pathways, we also performed supershift assays using nuclear extracts from TNF-treated myotubes.As shown in Figure 6C, incubation of nuclear-extracts from 1h and 18h TNF-treated myotubes with anti-p50 and anti-p65 shifted the NF-kB/DNA complex to higher molecular weight indicating that activated NF-kB complex contains p50 and p65 (the components of canonical NF-kB pathway) proteins.Although a slight shift was observed with antibodies against RelB and p52 at 18h, a remarkable increase in shifted bands was evident with both the antibodies at 24h and 36h after treatment with TNF-a.These results suggest that TNF-a initially activates canonical NF-kB pathway followed by the activation of alternative pathway. We have recently reported that TWEAK, a member of TNF super family, is a potent activator of alternative NF-kB signaling pathway in skeletal muscle cells [32].By performing QRT-PCR, we investigated whether TNF-a augments the expression of TWEAK or its receptor Fn14 in C2C12 myotubes.Our results showed that the mRNA levels of TWEAK were comparable between control and TNF-treated myotubes (Figure 6D).Furthermore, we could not detect TWEAK protein in culture supernatants of control or TNF-treated myotubes by ELISA.However, QRT-PCR assays showed a moderate increase (,1.7 fold) in the mRNA levels of Fn14 in TNF-treated myotubes (Figure 6D). Although TNF-a was found to stimulate alternative NF-kB pathway, it was not clear whether TNF-a by itself is sufficient or other cytokines, chemokines, and growth factors produced in culture supernatants are responsible for the activation of alternative NF-kB pathway in TNF-treated myotubes.To address this issue, C2C12 myotubes were initially incubated with TNF-a for 9h, the medium of the cells was replaced with fresh differentiation medium (without TNF-a), and the cultures were incubated for additional 9h.In a parallel culture, C2C12 myotubes were continuously treated with TNF-a for 18h.At the end of the incubation period, the cells were collected and Western blot was performed using p100/p52 antibody.As shown in Figure 7A, TNF-a increased the levels of p100 and p52 protein in both the cultures (i.e.those incubated with TNF-a for 9h or 18h).Furthermore, we found that IL-1b (another inflammatory cytokine) activated classical NF-kB pathway evident by degradation of IkBa protein (at 30 min and 1h) but did not affect the protein levels of p100/p52 in C2C12 cultures (Figure 7B).Collectively, these results suggest that TNF-a alone may be sufficient to activate both classical and alternative pathways in C2C12 myotubes. ",
"section_name": "TNF-a causes activation of both canonical and alternative NF-kB signaling pathways in myotubes",
"section_num": null
},
{
"section_content": "Although TNF-a was found to differentially regulate the expression of a large number of genes, it was not clear how the expression of these genes affect the activity of various cellular and molecular pathways in skeletal muscle cells.To understand the effects of TNF on various canonical pathways, we used Ingenuity Pathway Analysis (IPA) software.We first used a set of differentially regulated genes with fold values $1.5 and p-value of #0.05 from microarray analyses as an input for IPA software.However, this set of genes was not sufficient to generate pathways affected by TNF-a.We then reduced the stringency and used the set of genes with fold change (both up-and down-regulated genes) values $1.2 and p-value of #0.05 in the microarray experiment.We found that TNF-a affected the expression of genes involved in specific molecular pathways in myotubes (Figure 8).The major pathways affected by TNF-a in myotubes were those that regulate hepatic fibrosis, LXR/RXR activity, oxidative stress, mitochondrial dysfunction, and TGF-b and NF-kB signaling (Figure 8).Interestingly, this bioinformatics analysis of pathways using differentially regulated genes is consistent with the experimental evidence that skeletal muscle-wasting and other muscular disorders such as muscular dystrophy, involve the activation of many of these molecular pathways [6,10,11,33,34]. ",
"section_name": "Effect of TNF-a on various canonical pathways in C2C12 myotubes",
"section_num": null
},
{
"section_content": "In order to understand the interaction between different genes, we generated common networks using Ingenuity Pathway Analysis (IPA) software.The dataset of differentially expressed genes by TNF in C2C12 myotubes with selected stringency (p value #0.05 and fold $1.2) was uploaded into the IPA software tool.Networks of these genes were then algorithmically generated based on their connectivity.The graphical representation of the molecular relationships between genes developed by IPA is presented in Figure 9 and Figure 10.Based on the input information, the genes that are down-regulated are shown in green and the upregulated genes are shown in red (Figure 9 and Figure 10).Cytokines, growth factors and oxidative stress enzymes such as TWEAK, IL-6, IGF binding protein (IGFBP), and SOD were found to be involved in the network related to drug metabolism, neurological disease, organismal injury and abnormalities (Figure 9).This network also showed that many of these genes are regulated by each other either directly or indirectly. Furthermore, the networks related to cell cycle, cancer and nervous system development and function showed that several genes such as Nfkb2, NFkBia, BRCA1, Vegf, Jag2, Notch1, EGR1, FoxS1 and collagen type I were regulated by TNF (Figure 10). ",
"section_name": "TNF-a regulates distinct gene networks in myotubes",
"section_num": null
},
{
"section_content": "Although there is significant amount of literature suggesting the role of TNF-a in skeletal muscle wasting [3,35,36,37,38], the molecular mechanisms by which TNF-a induces muscle loss remain poorly understood.In this study, we have employed combination of microarray, bioinformatics tools, and biochemical techniques to identify potential mechanisms by which TNF-a might be regulating skeletal muscle mass. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The microarray analysis of TNF-treated C2C12 myotubes revealed that a total of 723 genes were significantly down regulated and 1,099 genes were significantly up regulated by TNF treatment with a p-value #0.05 and fold values of $1.2.Among the significantly up-regulated genes CXCL5 (8-fold), (1.27-fold) and Creb5 (1.24-fold) are the genes having either direct or indirect role in many pathways induced by TNF-a.Infiltration of leukocytes is a characteristic feature of acute inflammatory condition which is found in many secondary myopathies.A study on large selection of alpha/beta-chemokines and their receptors in normal controls and in the inflammatory myopathies showed a general increase of specific chemokines and chemokine receptors [39].An increase in expression levels of these chemokines and induction of neutrophil influx have been previously reported upon injection of TNF-a [40].A recent study by Vieira et al [41] showed that intraperitoneal injection of KC/CXCL1 and LIX/ CXCL5 induces dose and time-dependent neutrophil recruitment and TNF-a production.Consistent with these published reports, our microarray data show increased expression of many chemokine ligands and their receptors, especially, Cxcl5 which is up-regulated by 8-fold.The validation by QRT-PCR further confirmed the up-regulation of these chemokine networks by TNF-a (Figure 2A).These observations suggest that TNF-a induces the expression of chemokines and their receptors, leading to acute inflammation and myopathy. Vascular cell adhesion molecule-1 (VCAM1), also known as CD106, is expressed in a number of tissues including skeletal muscle and its expression is increased in response to proinflammatory cytokines.It is one of the important target genes of NF-kB transcription factor [12].The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to different tissues.It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis.Recent studies of Do et al. [42] on microarray analysis of TNF treated human SGBS adipocytes also reveal the up-regulation of this inflammatory gene.Consistent with these studies, our microarray analysis of TNFtreated myotubes showed increased expression of VCAM1, which was further validated by QRT-PCR (Figures 2A and4A). Interleukin IL-6 is an important cytokine which acts as both pro-inflammatory and anti-inflammatory molecule.This cytokine is also called as 'myokine' because it is also expressed by skeletal muscle and its levels are elevated during muscle contraction [43].In one study, enhanced gene expression of IL-6 and decreased levels of insulin signaling pathway and Akt pathway was observed when enterocytes were treated with TNF-a [44].Similar findings were observed in an independent study, where the infusion of angiotenisn II (AngII) increased circulating IL-6 in mice and triggered protein degradation via suppression of insulin signaling [45].Our experiments demonstrate that TNF-a induces the expression of IL-6 in myotubes (Figure 2A) suggesting that TNFa may also function through augmenting IL-6 expression to further accelerate inflammation and protein degradation in skeletal muscle.Recent studies have demonstrated that myogenic transcription factors such as serum response factor (SRF), MEF2c, and MyoD control the expression of myomiRs in skeletal and cardiac muscles [46].Our microarray experiment showed that TNF-a inhibits the expression of MEF2c and MyoD transcription factors in cultured myotubes (Table S1).We have previously demonstrated that TWEAK reduces the levels of MyoD and myogenin in differentiating C2C12 cultures [47].We have also shown that TWEAK down-regulates the expression levels of MEF2C in both microarray as well as QRT-PCR in C2C12 myotubes [28].In correlation with these observations, we have also shown that TWEAK down-regulates the myomiRs regulated by MEF2c including miR-1, 133 and 206 [28].The microarray analysis of TNF-treated myotubes in the present study showed a significant reduction in MEF2c and MyoD.The down-regulation of MyoD was further confirmed by QRT-PCR (Figures 2A and4B).From these observations, we can speculate that similar to C2C12 myotubes were incubated with or without TNF-a (10 ng/ml) continuously for 18h or for only 9h followed by changing the medium with fresh differentiation medium without TNF-a and incubation for additional 9h.The cell lysate made were analyzed by Western blotting for p100/p52 protein.A representative immunoblot presented here demonstrate that removal of TNF-a after 9h did not affect the TNF-induced expression/processing of p100/ p52 proteins.B).C2C12 myoblasts were treated with IL-1b (10 ng/ml) for indicated time periods and the levels of p100/p52 and IkBa protein were measured by Western blot.Data presented here show that IL-1b induced the degradation of IkBa protein (after 30 min and 1h) but did not affect the levels of either p100 or p52 proteins.TNF-a or IL-1b did not affect the levels of an unrelated protein tubulin in C2C12 cultures.doi:10.1371/journal.pone.0013262.g007TWEAK, TNF-a might also block myogenic differentiation by down-regulation of specific myogenic regulatory factors and thereby inhibiting the expression of various MyomiRs (miR-1, 133 and 206).These possibilities will be investigated in future studies. ",
"section_name": "TNF-a induces the expression of several cytokines, chemokines, protein kinases, and transcriptional factors in cultured myotubes",
"section_num": null
},
{
"section_content": "We also observed down-regulation of an antioxidant enzyme, superoxide dismutase 1 (SOD1).SOD1 is a well-known player of the anti-oxidative defense.The direct evidence of SOD1 in muscular atrophy was recently reported by Muller et al. [48], where they showed a dramatic increase in mitochondrial reactive oxygen species (ROS) in three conditions of muscular atrophy in animals lacking Sod1.In another study, mutations in Sod1gene (SOD1 G93A ) selectively in skeletal muscle showed progressive muscle atrophy with concomitant reduction in muscle strength, alterations in contractile apparatus, and mitochondrial dysfunction [49].In this study they also analyzed the molecular pathways associated with muscle atrophy by Sod1 mutation and found that accumulation of oxidative stress initiated autophagy and thereby degradation of muscles.This suggests that skeletal muscle is the primary target of Sod1 mutation-mediated toxicity whereby oxidative stress triggers muscle atrophy.The down-regulation of this important antioxidant in response to TNF-a treatment in our microarray data suggests a possible link between the regulation of Sod1 and TNF-induced muscular atrophy. Another important gene that was significantly down-regulated by TNF and is worth mentioning is Notch1.Notch-1 receptors are transmembrane proteins which are expressed in a broad range of tissues and function in diverse developmental and cell maturation processes [50,51].The intracellular regions of Notch receptors contain several functional motifs: ankyrin/CDC10 repeats, RAM, nuclear localization signals (NLS), PEST sequences, and a glutamine-rich domain [50].Ligands such as Delta or Jagged/ Serrate induce a second cleavage that requires presenilins at a site within the transmembrane region of Notch [52,53] leading to release of the intracellular fragment of Notch, which binds to numerous nuclear and cytoplasmic proteins.Besides its role in regulation of the activity of other transcription factors, recent studies have shown that constitutively expressed Notch-1 functions as a novel IkB-like molecule and regulates NF-kB-mediated gene expression through a direct interaction with the NFkB1 (i.e.p50) subunit [54,55].This interaction prevents NF-kB from binding to NF-kB recognition sites in DNA to regulate NF-kB-dependent Figure 8. Differentially expressed genes associated with canonical pathway in the Ingenuity Pathway Analysis (IPA).Top canonical pathways affected by TNF-a treatment were identified by using IPA analysis.Here the differentially regulated genes with p-value #0.05 and fold change $1.2 were considered for IPA analysis.Bars represent 2log p-value and percentage of genes present in the data set compared to the total number of genes present in each selected pathway in IPA data base.The yellow line represents 2log p-value of affected genes to the total number of genes in a pathway.Each bar was represented in two different colors in which red correspond to the up-regulated genes and green corresponds to down-regulated genes.The percentage of up-regulated and down-regulated genes in each selected canonical pathway can be measured in percentage scale given on y-axis (left side).doi:10.1371/journal.pone.0013262.g008gene expression [54,55].Therefore, the reduced levels of Notch1 (Figures 2B and5C) may be responsible, at least in part, for sustained activation of NF-kB in skeletal muscle cells in response to TNF-a. ",
"section_name": "TNF suppresses the expression of genes involved in myogenesis, regeneration and ubiquitin proteasomal degradation",
"section_num": null
},
{
"section_content": "Accumulating evidence strongly suggests that NF-kB is one the most important signaling pathways, the activation of which leads to skeletal muscle wastage [10].NF-kB activation can occur through two parallel pathways.The canonical NF-kB signaling pathway involves the upstream activation of IkB kinase-b (IKKb) and subsequent phosphorylation and degradation of IkB proteins [56,57,58].In contrast, the activation of the alternative NF-kB pathway requires the upstream activation of NF-kB-inducing kinase (NIK or MAP3K14) and IKKa and the proteolytic processing of NFkB2 (p100 subunit) into p52 protein [57,58].Previous studies from our group and other laboratories have demonstrated that TWEAK increases the activation of both canonical and alternative NF-kB pathways [32,59].However, there has been no report on the activation of alternative NF-kB pathway by TNF-a in skeletal muscle.The microarray analysis in the present study showed that TNF-a increases the expression levels of both NFkB1 and NFkB2 in skeletal muscle cells (Figures 2A, 3A, 4A, 5A, and6B).We also performed Western blots and EMSA and super-shift assays with radio-labeled NF-kB at different time points after the addition of TNF-a in cultured myotubes.These experiments demonstrated that TNF-a induces NF-kB activity via canonical pathway initially (Figure 6B).However a time-dependent increase in the components of alternative pathway was observed at later time points.These include the phosphorylation of IKKa, proteolytic degradation of p100 into p52 (Figure 6B), and presence of p52 and RelB subunits in NF-kB/DNA complex in super shift assays (Figure 6C).Since the activation of alternative pathway persisted even upon removal of TNF-a from culture medium after 9h (Figure 7A), these observations suggest that TNF-a alone might provide sufficient signal to activate alternative NF-kB pathway in skeletal muscle.It is also noteworthy that the activation of alternative NF-kB pathway in myotubes may be specific to TNF-a and TWEAK.This inference is supported by our findings that while IL-1b augmented the degradation of IkBa protein (an important event in the activation of classical pathway), it did not affect the levels of p100 and p52 proteins (Figure 7B).In future studies, it will be interesting to investigate which pathway mediates the loss of skeletal muscle mass in response to TNF-a. ",
"section_name": "TNF-a induces the activation of both canonical and alternative NF-kB signaling pathways in myotubes",
"section_num": null
},
{
"section_content": "The Ingenuity pathway analysis (IPA) of the selected genes that are differentially regulated by TNF treatment in C2C12 myotubes showed that it affects the activation of multiple canonical pathways in skeletal muscle cells.The major pathways affected by TNF-a are those involved in initiation and manifestation of fibrosis, oxidative stress, and mitochondrial dysfunction (Figure 8).Interestingly, it has now become increasingly clear that skeletal muscle wasting in both chronic diseases and disuse conditions leads to the up-regulation of these pathways in skeletal muscle [10,23,60,61] suggesting that TNF-a may be one of the important stimuli for their activation in catabolic conditions.Our analysis also showed that TNF-a affects the pathway involved in neurological disease and organismal injury (Figure 9) and nervous system development, cell cycle and cancer (Figure 10).Though TNF and TWEAK are two cytokines that share similar functions, it is of considerable importance that they affect distinct gene networks.Our previous studies showed the up-regulation of proteasomal pathway by TWEAK [28] but not by TNF-a.These studies further indicate that different inflammatory cytokines may regulate different set of molecular pathways in skeletal muscle and coordinated activation of these pathways may be responsible for the loss of skeletal muscle mass and myopathy in a particular disease state. Conclusions: The data presented in this study suggest that TNF-a affects the expression of specific set of genes which are involved in the regulation of various molecular pathways/ processes including extracellular matrix degradation, NF-kB signaling, Notch1 signaling, chemokine network, apoptosis, and muscle cell proliferation and differentiation.The study has also identified the activation of alternative NF-kB signaling pathway in C2C12 myotubes in response to TNF-a.The present study will provide strong basis for further delineating the molecular mechanisms of TNF-induced muscle wasting in different disease states. ",
"section_name": "TNF-a affects multiple molecular pathways and gene networks in C2C12 myotubes",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Horse serum was purchased from Sigma Chemical Company (St. Louis, MO).Recombinant mouse TNF-a protein and antibodies against MMP-9 and TIMP2 were purchased from R&D Systems (Minneapolis, MN).Antibodies against IkBa, p50, p65, c-Jun, phospho-p52, phospho-IkB, phospho-IKKa and Notch1 were purchased from Santa Cruz Biotechnology (San Diego, CA).Tubulin, p52/100, RelB, IKKa, IKKb and NFkB2 antibodies were obtained from Cell Signaling Technology (Beverly, MA).NF-kB consensus oligonucleotides and Dual-Luciferase assay kits were purchased from Promega (Madison, WI).Primers for PCR were synthesized by Integrated DNA Technologies (Coralville, IA) or Sigma-Genosys (Woodlands, TX). 32P-c-ATP was obtained from MP Biomedicals (Solon, OH). Cell Culture C2C12 myoblastic cell line was obtained from American Type Culture Collection (Rockville, MD).These cells were grown in Dulbecco's modified Eagle's Medium (DMEM) containing 20% fetal bovine serum.C2C12 myoblasts were differentiated into myotubes by incubation in differentiation medium (DM, 2% horse serum in DMEM) for 96h as described [47,62].Myotubes were maintained in DM and medium was changed every 48h. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Total RNA was isolated from control and TNF-treated C2C12 myotubes using the Agilent total RNA isolation kit (Agilent Technologies, Palo Alto, CA).Any contaminating DNA was removed using DNA-free TM kit from Ambion (Ambion, Austin, TX).The total RNA concentration was determined by NanoDrop spectrophotometer, and RNA quality was determined by 18S/28S ribosomal peak intensity on an Agilent Bioanalyzer.RNA samples from five wells per condition were separately subjected to microarray analyses.. Custom cDNA slides were spotted with Oligator ''MEEBO'' mouse genome set with 38,467 cDNA probes (Illumina, Inc., San Diego, CA), which allows interrogation of 25,000 genes.A Q-Array2 robot (Genetix) was used for spotting.The array includes positive controls, doped sequences, and random sequences to insure correct gene expression values were obtained from each array.A total of 250 ng RNA was used to synthesize ",
"section_name": "cDNA Microarray",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by National Institutes of Health grant RO1 AG029623 to AK.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": "double stranded cDNA using the Low RNA Input Fluorescent Linear Application Kit (Agilent).The microarray slides were scanned using a GSI Lumonics ScanArray 4200A Genepix scanner (Axon).The image intensities were analyzed using the ImaGene 5.6 software (Biodiscovery, Inc., El Segundo, CA).Expression analysis of microarray experiments was performed with GeneSpring 7.1 (Silicon Genetics, Palo Alto, CA) using the raw intensity data generated by the ImaGene software.Local background was subtracted from total signal intensities and was used as intensity measures.The data were normalized using per spot and per chip LOWESS normalization.Data analysis was performed using SAS (SAS Institute, Cary, NC), R and Q value software.The probe sets with absent calls across all samples were removed to reduce the multiple-testing problem.The expression levels were normalized to the chip median and log transformed.Two-way ANOVA tests were carried out to identify differentially expressed genes.For each probe set, the model y ijk ~mza i zb j zc ij ze ijk was fit, where y ijk is the log-transformed expression level of the k th chip in the i th treatment and the j th replicate.The variable m represents the grand mean expression, a i is the effect due to the treatment, b j is the effect due to the replicate, c ij is the interaction effect between treatment and replicate, and e ijk is an error term, which is assumed to be normally distributed with mean 0 and variance s 2 .Q values computed using Q value software indicates the false detection rate for each probe set.Ratio comparison was performed by dividing expression levels in TNF-treated myotubes with the expression levels in untreated myotubes.Functional classification of select probe sets was performed at NIH DAVID server (http://apps1.niaid.nih.gov/david/upload.asp).Volcano plots were prepared using the R program.The complete raw and normalized microarray data have been submitted in MIAME compliant ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/) database with accession number E-MEXP-2592. The expression of the differentially regulated genes from the microarray data set was validated using QRT-PCR as previously described [47,62].Briefly, purified RNA (1 mg) from myotubes was used to synthesize first strand cDNA by reverse transcription system using Ambion's oligo-dT primer and Qiagen's Omniscript reverse transcriptase according to the manufacturer's instructions.The first strand cDNA reaction (0.5 ml) was subjected to real time PCR amplification using gene specific primers.The primers were designed using Vector NTI Xi software (Invitrogen). Quantification of mRNA was done using the SYBR Green method on ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA).Approximately 25ml of reaction volume was used for the real time PCR assay that consisted of 26 (12.5ml)Brilliant SYBR Green QPCR Master Mix (Applied Biosystems), 400nM of primers (0.5 ml each from the stock), 11ml of water, and 0.5 ml of template.The thermal conditions consisted of an initial denaturation at 95uC for 10 minutes followed by 40 cycles of denaturation at 95uC for 15 sec, annealing and extension at 60uC for 1 minute, and a final step melting curve of 95uC for 15 sec, 60uC for 15 sec, and 95uC for 15 sec.All reactions were carried out in triplicate to reduce variation.The data was analyzed using SDS software version 2.0, and the results were exported to Microsoft Excel for further analysis.Data normalization was accomplished using the endogenous control b-actin and the normalized values were subjected to a 2 2DDCt formula to calculate the fold change between the control and experimental groups.The formula and its derivations were obtained from the ABI Prism 7900 Sequence Detection System user guide. Relative levels of gene expression were first computed with GeneSpring 7.1 to obtain data sets of differentially regulated genes based on cut-off values of 5% error rate (p,0.05,determined by ttest with Benjamini and Hochberg Multiple Testing Correction).These data sets included up and down regulated genes when C2C12 myotubes were treated with TNF.The second step of analysis consisted of identifying canonical pathways.Tab separated (txt) files containing Accession IDs and symbols derived from MEEBO genome set and the normalized expression ratios were then uploaded to Ingenuity Pathways Analysis.Ingenuity Pathways Analysis is a web-delivered bioinformatics tool (IPA 5.0, http://www.ingenuity.com) to identify pathways and functional networks.IPA knowledge database is generated from the peerreviewed scientific publications that enables discovery.The Accession IDs and symbols in each data set were queried against all genes stored in the IPA knowledge database for pathway analysis.Canonical pathways analysis identified the pathways from IPA library of canonical pathways that were most significant to the data set.The significance of the association between the data set and the canonical pathways was measured in 2 ways: 1) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed.2) Fisher's exact test was used to calculate a p-value determining the probability that the association between the genes in the data set and the canonical pathway is explained by chance alone. Western blotting was performed to measure the levels of different proteins using a standard protocol in our laboratory as described [32,47,62].The dilution of primary antibody was as follows: anti-IkB (1:1000), anti-NFkB2 (1:500), anti-MMP-9 (1:2000), anti-p52/100 (1:1000), anti-p50 (1:1000), anti-RelB (1:1000), anti-phospho IKKa (1:500) anti-Notch1 (1:1000), anti-TIMP2 (1:500), and anti-tubulin (1:3000).Immunoblots were quantified using ImageQuant TL software (GE Healthcare). The activation of NF-kB transcription factors was measured by EMSA.A detailed procedure for the preparation of nuclear and cytoplasmic extracts and EMSA has been described previously [47]. Methods used for statistical analysis of the cDNA microarray has been described above.For all other studies, results were expressed as mean 6 SD.The Student's t test was used to compare quantitative data populations with normal distributions and equal variance.A value of P,0.05 was considered statistically significant unless otherwise specified. ",
"section_name": "Quantitative Real-Time-PCR (QRT-PCR)",
"section_num": null
},
{
"section_content": "double stranded cDNA using the Low RNA Input Fluorescent Linear Application Kit (Agilent).The microarray slides were scanned using a GSI Lumonics ScanArray 4200A Genepix scanner (Axon).The image intensities were analyzed using the ImaGene 5.6 software (Biodiscovery, Inc., El Segundo, CA).Expression analysis of microarray experiments was performed with GeneSpring 7.1 (Silicon Genetics, Palo Alto, CA) using the raw intensity data generated by the ImaGene software.Local background was subtracted from total signal intensities and was used as intensity measures.The data were normalized using per spot and per chip LOWESS normalization.Data analysis was performed using SAS (SAS Institute, Cary, NC), R and Q value software.The probe sets with absent calls across all samples were removed to reduce the multiple-testing problem.The expression levels were normalized to the chip median and log transformed.Two-way ANOVA tests were carried out to identify differentially expressed genes.For each probe set, the model y ijk ~mza i zb j zc ij ze ijk was fit, where y ijk is the log-transformed expression level of the k th chip in the i th treatment and the j th replicate.The variable m represents the grand mean expression, a i is the effect due to the treatment, b j is the effect due to the replicate, c ij is the interaction effect between treatment and replicate, and e ijk is an error term, which is assumed to be normally distributed with mean 0 and variance s 2 .Q values computed using Q value software indicates the false detection rate for each probe set.Ratio comparison was performed by dividing expression levels in TNF-treated myotubes with the expression levels in untreated myotubes.Functional classification of select probe sets was performed at NIH DAVID server (http://apps1.niaid.nih.gov/david/upload.asp).Volcano plots were prepared using the R program.The complete raw and normalized microarray data have been submitted in MIAME compliant ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/) database with accession number E-MEXP-2592. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The expression of the differentially regulated genes from the microarray data set was validated using QRT-PCR as previously described [47,62].Briefly, purified RNA (1 mg) from myotubes was used to synthesize first strand cDNA by reverse transcription system using Ambion's oligo-dT primer and Qiagen's Omniscript reverse transcriptase according to the manufacturer's instructions.The first strand cDNA reaction (0.5 ml) was subjected to real time PCR amplification using gene specific primers.The primers were designed using Vector NTI Xi software (Invitrogen). Quantification of mRNA was done using the SYBR Green method on ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA).Approximately 25ml of reaction volume was used for the real time PCR assay that consisted of 26 (12.5ml)Brilliant SYBR Green QPCR Master Mix (Applied Biosystems), 400nM of primers (0.5 ml each from the stock), 11ml of water, and 0.5 ml of template.The thermal conditions consisted of an initial denaturation at 95uC for 10 minutes followed by 40 cycles of denaturation at 95uC for 15 sec, annealing and extension at 60uC for 1 minute, and a final step melting curve of 95uC for 15 sec, 60uC for 15 sec, and 95uC for 15 sec.All reactions were carried out in triplicate to reduce variation.The data was analyzed using SDS software version 2.0, and the results were exported to Microsoft Excel for further analysis.Data normalization was accomplished using the endogenous control b-actin and the normalized values were subjected to a 2 2DDCt formula to calculate the fold change between the control and experimental groups.The formula and its derivations were obtained from the ABI Prism 7900 Sequence Detection System user guide. ",
"section_name": "Quantitative Real-Time-PCR (QRT-PCR)",
"section_num": null
},
{
"section_content": "Relative levels of gene expression were first computed with GeneSpring 7.1 to obtain data sets of differentially regulated genes based on cut-off values of 5% error rate (p,0.05,determined by ttest with Benjamini and Hochberg Multiple Testing Correction).These data sets included up and down regulated genes when C2C12 myotubes were treated with TNF.The second step of analysis consisted of identifying canonical pathways.Tab separated (txt) files containing Accession IDs and symbols derived from MEEBO genome set and the normalized expression ratios were then uploaded to Ingenuity Pathways Analysis.Ingenuity Pathways Analysis is a web-delivered bioinformatics tool (IPA 5.0, http://www.ingenuity.com) to identify pathways and functional networks.IPA knowledge database is generated from the peerreviewed scientific publications that enables discovery.The Accession IDs and symbols in each data set were queried against all genes stored in the IPA knowledge database for pathway analysis.Canonical pathways analysis identified the pathways from IPA library of canonical pathways that were most significant to the data set.The significance of the association between the data set and the canonical pathways was measured in 2 ways: 1) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed.2) Fisher's exact test was used to calculate a p-value determining the probability that the association between the genes in the data set and the canonical pathway is explained by chance alone. ",
"section_name": "Pathways and Networks Analyses",
"section_num": null
},
{
"section_content": "Western blotting was performed to measure the levels of different proteins using a standard protocol in our laboratory as described [32,47,62].The dilution of primary antibody was as follows: anti-IkB (1:1000), anti-NFkB2 (1:500), anti-MMP-9 (1:2000), anti-p52/100 (1:1000), anti-p50 (1:1000), anti-RelB (1:1000), anti-phospho IKKa (1:500) anti-Notch1 (1:1000), anti-TIMP2 (1:500), and anti-tubulin (1:3000).Immunoblots were quantified using ImageQuant TL software (GE Healthcare). ",
"section_name": "Western Blot",
"section_num": null
},
{
"section_content": "The activation of NF-kB transcription factors was measured by EMSA.A detailed procedure for the preparation of nuclear and cytoplasmic extracts and EMSA has been described previously [47]. ",
"section_name": "Electrophoretic mobility shift assay (EMSA)",
"section_num": null
},
{
"section_content": "Methods used for statistical analysis of the cDNA microarray has been described above.For all other studies, results were expressed as mean 6 SD.The Student's t test was used to compare quantitative data populations with normal distributions and equal variance.A value of P,0.05 was considered statistically significant unless otherwise specified. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
10.3390/cardiogenetics13030010
|
GMDS Intragenic Deletions Associate with Congenital Heart Disease including Ebstein Anomaly
|
<jats:p>Ebstein anomaly is a rare heterogeneous congenital heart defect (CHD) with a largely unknown etiology. We present a 6-year-old girl with Ebstein anomaly, atrial septum defect, hypoplastic right ventricle, and persistent left superior vena cava who has a de novo intragenic ~403 kb deletion of the GDP-mannose 4,6-dehydratase (GMDS) gene. GMDS is located on chromosome 6p25.3 and encodes the rate limiting enzyme in GDP-fucose synthesis, which is used to fucosylate many proteins, including Notch1, which plays a critical role during mammalian cardiac development. The GMDS locus has sporadically been associated with Ebstein anomaly (large deletion) and tetralogy of Fallot (small deletion). Given its function and the association with CHD, we hypothesized that loss-of-function of, or alterations in, GMDS could play a role in the development of Ebstein anomaly. We collected a further 134 cases with Ebstein anomaly and screened them for genomic aberrations of the GMDS locus. No additional GMDS genomic aberrations were identified. In conclusion, we describe a de novo intragenic GMDS deletion associated with Ebstein anomaly. Together with previous reports, this second case suggests that GMDS deletions could be a rare cause for congenital heart disease, in particular Ebstein anomaly.</jats:p>
|
[
{
"section_content": "Ebstein anomaly is a rare congenital heart defect in which the tricuspid valve is malformed and displaced downward into the right ventricle. It accounts for less than 1% of all congenital heart diseases.Older studies reported a live birth prevalence of Ebstein anomaly of around 1:200,000 [1], but more recent population studies have reported a lower birth prevalence of around 1:20,000 [2][3][4].This has been attributed to different factors, i.e., earlier and better diagnoses [5], better technology, and differences in ascertainment methods and classifications [5,6]. The clinical presentation of Ebstein anomaly varies widely and depends on the degree of anatomic abnormalities.Additional cardiac anomalies, such as patent foramen ovale, atrial septum defect, pulmonary stenosis or atresia, and ventricular septum defect, are often present in cases with Ebstein anomaly [7].Approximately 20% of cases with Ebstein anomaly have extra-cardiac malformations suggesting that the cardiac defect could be part of a genetic syndrome [2].The etiology of Ebstein anomaly is largely unknown.Epidemiologic studies have reported associations between Ebstein anomaly and environmental factors, such as peri-conceptional exposure to pesticides and varnishes as well as maternal infections and maternal health conditions [2, 3,7].In addition, familial recurrence and identification of genetic variants segregating with this congenital disorder also suggest a genetic component [8,9].Although Ebstein anomaly has been sporadically described in genetic syndromes, including Down, CHARGE, Noonan, and Cornelia de Lange syndrome [10][11][12][13], a consistent association between Ebstein anomaly and a distinct genetic condition has not yet been identified [10].Pathogenic variants in three genes (MYH7, NKX2-5, and GATA4) and different copy number variants have been described in cases with Ebstein anomaly but only account for a small proportion of cases [6,[13][14][15].In 2016, Sicko et al. performed a genome-wide investigation of copy number variants in a cohort of 47 cases with isolated Ebstein anomaly [6].They identified several (new) candidate copy number variants, including one intragenic GMDS deletion of approximately 345 kb on chromosome 6p25.3. Here, we report a second case of Ebstein anomaly associated with a de novo 403 kb intragenic GMDS deletion.Additionally, we analyzed a further 134 cases with isolated Ebstein anomaly for abnormalities in the GMDS genomic locus, specifically copy number variants. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "The patient was enrolled at Amsterdam UMC in Amsterdam, the Netherlands.The parents provided written consent for research use of their data, and the study was conducted in accordance with the Declaration of Helsinki.Clinical data were retrieved from the patient's records.A total of 134 additional patients were evaluated from the Netherlands and the patient's island of origin.We obtained genomic DNA from 129 cases with the main clinical diagnosis of either Ebstein malformation, congenital pulmonary valve stenosis, or tricuspid atresia.These cases were collected through the Dutch national biobank of adult cases with congenital heart defects (CONCOR) [16].Subsequently, we screened these cases for copy number variants (CNVs) in GMDS using digital droplet PCR for three locations across the whole GMDS locus, including the previously identified deletion.Using this method, we could positively identify the deletion in the proband, validating the array-CGH results.From the same Caribbean island, genomic DNA from five cases with Ebstein anomaly was analyzed.Under Dutch law, assessment of the study protocol by our ethics committee was not required since only genetic and clinical data collected during regular patient care were used.This is specifically explained for (Dutch) research involving the CONCOR registry and DNA-bank [16,17]. ",
"section_name": "Clinical Data",
"section_num": "2.1."
},
{
"section_content": "Copy number profiling was performed on DNA isolated from peripheral blood using 180 K (Amadid #023363) Human Genome CGH Microarray slides from Agilent Technologies (Version 5.1, Santa Clara, CA, USA) according to the manufacturer's protocols.The results were classified with Cartagenia BENCH software 5.1.(Cartagenia, Leuven, Belgium). ",
"section_name": "Array-CGH",
"section_num": "2.2."
},
{
"section_content": "In addition, a next-generation sequencing (NGS) targeted structural heart disease gene panel was performed, which consisted of 46 genes, including Noonan-syndrome related genes (BRAF, KRAS, LZTR1, MAP2K1, NRAS, PTPN11, RAF1, RASA2, RIT1, SOS1, and SOS2). ",
"section_name": "Next-Generation Sequencing Targeted Panel",
"section_num": "2.3."
},
{
"section_content": "For ddPCR, the QX200 Droplet Digital PCR system (Bio-Rad, Hercules, CA, USA) was used.The ddPCR reaction was performed in a 21 µL volume consisting of 10 µL 2× ddPCR Supermix for Probes (No UDP) (Bio-Rad), 1 µL 20× target primers/probe mix (FAM), 1 µL 20× reference primers/probe mix, 1 µL (5U) Hind111 restriction enzyme (New England Biolabs, Ipswich, MA, USA), and 1 µL DNA sample at 20 ng/µL.The mixture was loaded into a DG8 cartridge (Bio-Rad) together with 70 µL of droplet generation oil (Bio-Rad) and covered with a DG8 gasket.After processing the droplets in the droplet generator, the samples were transferred to a 96-well PCR plate.PCR amplification was carried out in a T11 Touch thermal cycler (Bio-Rad).The cycling protocol was as follows: 95 • C for 10 min, 40 cycles of 94 • C for 30 s and 60 • C for 1 min followed by an infinite 15-degree hold.After PCR, the plate was loaded on the QX200 droplet reader (Bio-Rad).Data were analyzed using QuantaSoft Analysis Pro software Version 1.2 (Bio-Rad). ",
"section_name": "CNV Detection Using Droplet Digital PCR (ddPCR)",
"section_num": "2.4."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The female index case was born to healthy Caribbean parents after an uneventful pregnancy of 37.5 weeks.She had a birth weight of 3200 g (50th centile), length of 51 cm (85th centile), and head circumference of 32.5 cm (10th centile).On the second day of life, she was admitted to the neonatology ward due to cyanosis with a heart rate of 125/min, respiratory rate of 40/min, and oxygen saturation of 84%.Echocardiography revealed an Ebstein anomaly with an atrial septal defect, hypoplastic right ventricle, and persistent left superior vena cava.Treatment with prostaglandin E2 and nasal continuous positive airway pressure was started.She was then referred to a tertiary pediatric heart center in Colombia for surgery. The patient underwent a Starnes procedure, atrio-septostomy, closure of the pulmonary valve, and placement of a 3.5 mm Blalock-Taussig shunt.She experienced postoperative complications, including renal failure and paralysis of the diaphragm.At the age of one year, she had a follow-up surgery consisting of a bidirectional Glenn shunt.She suffered from bronchiolitis, pneumonia, and asthma and required a gastrostomy tube for feeding.At the age of 9 months, she was examined by a clinical geneticist.At that time, her height was 65 cm (2nd centile), weight was 6820 g (15th centile), and head circumference was 42 cm (15th centile).Dysmorphological examination showed hypertelorism, mid face hypoplasia, long philtrum, uplifted earlobes, and an anteriorly placed anus (Figure 1).An abdominal ultrasound was performed at the age of 10 months to exclude additional organ abnormalities. ",
"section_name": "The Clinical Description of the Affected Patient Is Presented in This Section",
"section_num": "3.1."
},
{
"section_content": "An array-CGH was performed.The Agilent Bench Lab CNV Version 5.1 180 K array-CGH showed a de novo deletion on chromosome 6p25.3 of ~403 kb (Figure 2) and a maternally inherited duplication in Xq27.2 of ~316 kb (arr [GRCh37] 6p25.3(1727928_2131157)x1 dn, Xq27.2(140693307_141009050) x3 mat, in accordance with the ISCN 2020 nomenclature [18].No (likely) pathogenic variants were identified in the NGS gene panel.At the age of 4 years, she developed severe cyanosis caused by an aorta-pulmonary collateral, which was closed by intervention.Because of severe pulmonary hypertension, completion of Fontan circulation was not feasible.She had divergent eyes, a difference in size of the globe, and a right nasolacrimal duct stenosis.She also had delayed motor and speech development and developed convulsions at the age of one year.EEG showed occipital epileptic activity for which anti-epileptic therapy was started. Because of left-sided hypotonia at the age of 2 years, a CT of the brain was performed, which showed no abnormalities.This has resolved spontaneously. ",
"section_name": "Molecular and Cytogenetic Studies",
"section_num": "3.2."
},
{
"section_content": "An array-CGH was performed.The Agilent Bench Lab CNV Version 5.1 180 K array-CGH showed a de novo deletion on chromosome 6p25.3 of ~403 kb (Figure 2) and a maternally inherited duplication in Xq27.2 of ~316 kb (arr [GRCh37] 6p25.3(1727928_2131157)x1 dn, Xq27.2(140693307_141009050) x3 mat, in accordance with the ISCN 2020 nomenclature [18].No (likely) pathogenic variants were identified in the NGS gene panel. ",
"section_name": "Molecular and Cytogenetic Studies",
"section_num": "3.2."
},
{
"section_content": "Genetic investigations in 129 Ebstein cases from the CONCOR data revealed no additional deletions or duplications. ",
"section_name": "Genetic Investigations in Large Ebstein Anomaly Cohort",
"section_num": "3.3."
},
{
"section_content": "In the five Caribbean cases with Ebstein anomaly, SNP arrays were performed, but none of them showed a 6p25.3deletion.One patient had a 16q23.1 deletion, but unfortunately, her parents refused testing, and the other patients all had normal SNP array results. ",
"section_name": "Genetic Investigations in Caribbean Ebstein Cohort",
"section_num": "3.4."
},
{
"section_content": "We report a patient with Ebstein anomaly, a rare, heterogeneous congenital heart defect with largely unknown etiology.Although Ebstein anomaly has been associated with pathogenic variants in a handful of genes and various copy number variants, these account for only a small proportion of cases [4,[11][12][13].We demonstrate here that the index patient has a large, intragenic, and de novo deletion in GMDS that is likely associated with the patient's phenotype.GMDS encodes a short-chain mannose dehydrogenase protein (GDP-mannose 4,6-dehydratase) that catalyzes the first step in a molecular process called fucosylation, which has a function in many processes important in development [20].GMDS is a key player in fucosylation as it is the rate-limiting enzyme [20].Although no pathogenic human single nucleotide variations have been reported for GMDS, a case with Ebstein anomaly and a comparable large intragenic GMDS deletion has been recently reported [6].The inheritance of that deletion was not reported.Both these large intragenic deletions are predicted to lead to loss-of-function of GMDS as they remove a large part of the GMDS coding region.In addition, two patients with Tetralogy of Fallot (TOF), a rare congenital heart disease, have been described to harbor a smaller deletion (of unknown inheritance) in the same region as the two Ebstein patients [19] (see Figure 2).These TOF-associated deletions, however, do not delete any GMDS coding parts, making it difficult to predict their effect.Nonetheless, based on our results coupled with the cases described above, we hypothesize that GMDS, or the GMDS locus, is important for normal (cardiac) development.Animal experiments corroborate this as GMDS knockout mice have an abnormal cranium and snout morphology [21], while Song et al. demonstrated that a GMDS missense variant in zebrafish results in disturbed neural development and reduced Notch signaling (see below) [22].Together, this indicates that GMDS or its products have a role in normal development.One possible mechanism by which GMDS might be involved in development comes from its role as a major player in protein glycosylation (specifically fucosylating).It is known that protein glycosylation regulates various functions and physicochemical properties and that it plays a critical role in maintaining homeostasis.However, alterations of glycosylation are associated with the development and aggravation of many diseases [23].This raises the possibility that altered glycosylation could lead to congenital heart disease as observed in our patient and the other three reported intragenic GMDS deletion patients.A possible link between congenital heart disease and altered glycosylation might be that a group of key proteins in (cardiac) development, the Notch proteins, are actually fucosylated [24].Indeed, reduced fucose levels result in decreased Notch1 activation [23,24], a key player in cardiac development [25,26]. The importance of Notch1 in cardiac development is underscored by the fact that it is one of the most frequently mutated genes in patients with congenital heart disease, and that Notch1 KO mice demonstrate a plethora of congenital heart disease phenotypes [26].One hypothesis could therefore be that GMDS haploinsufficiency leads to decreased GDPfucose production, which in turn affects fucosylation of Notch1, thereby reducing its activity, resulting in cardiac anomalies.Alternatively, it is also possible that the intragenic deletions not only impact GMDS but also its nearest neighbor FOXC1, a forkhead transcription factor [27] located just 10 kb from the last GMDS exon.FOXC1 is an essential component of mesodermal [27] and neural crest development [27], both important for heart development [28,29].While FOXC1 is not affected by any of the deletions directly, it is possible that disruption of conserved enhancer sequences within the GMDS locus also affects the regulation of (spatial-temporal) expression of FOXC1.FOXC1-knockout mice present with a variety of phenotypes, including congenital heart disease, and demonstrate abnormal valve development, which is part of the Ebstein anomaly, suggesting that altered expression of FOXC1 may cause developmental problems of the heart [29].An additional possibility is that GMDS locus deletions have an impact on multiple neighboring genes and thereby cause the phenotype.Regardless, further investigation into the mechanism of how intragenic GMDS deletions can lead to disease is needed. Given the link between GMDS and congenital heart disease, we collected a cohort of 134 Ebstein and Ebstein like cases, in which we investigated the GMDS locus for additional aberrations.No deletions or duplication were identified in this cohort, suggesting that GMDS intragenic aberrations are a relatively rare cause of Ebstein anomaly. In addition to Ebstein anomaly, our index case also presented with seizures and dysmorphic facial features.This is in contrast to the other large GMDS intragenic deletion case, where the Ebstein anomaly was reported as an isolated condition [11].However, as no additional clinical details were reported, it is possible that minor birth defects, such as dysmorphic features, and/or developmental delay were present in that patient.Alternatively, the difference in phenotype could also be caused by differences in genetic background and/or the difference in size and location of the GMDS intragenic deletions.With regard to the two cases with smaller GMDS deletions associated with tetralogy of Fallot [19,22], the patients in that study were grouped into syndromic vs. non-syndromic.However, it is unclear in which group these two cases with GMDS deletions fall.Taken together, there is a degree of phenotypic diversity with regard to the heart phenotype of GMDS intragenic deletion carriers, while the extent of extra-cardiac phenotypes is still unclear. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In summary, we report a patient with Ebstein anomaly with a de novo intragenic GMDS deletion.This locus has previously been sporadically associated with Ebstein anomaly and tetralogy of Fallot.We hypothesize that (large) deletions of this locus lead to loss of function of GMDS, which affects fucosylation, and in turn interferes with downstream developmental signaling molecules (e.g., Notch1) that are dependent on fucosylation for proper function.Given all the evidence, we conclude that GMDS intragenic deletions can be associated with rare cases of congenital heart disease. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "The data presented in this study are available on request from the corresponding author.The data are not publicly available due to privacy reasons. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: Under Dutch law, assessment of the study protocol by our ethics committee was not indicated since only genetic and clinical data collected during regular patient care were used.For Dutch research involving CONCOR-data, this is explained on their website https://concor.net/en/aboutconcor/methodology.html(accessed on 1 June 2022). The parents provided written consent for research use of their data.For Dutch research involving CONCOR-data, this is explained in https://pubmed.ncbi.nlm.nih.gov/16121765/ and on their website https://concor.net/en/aboutconcor/methodology.html(accessed on 1 June 2022). The authors declare no conflict of interest. ",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: Under Dutch law, assessment of the study protocol by our ethics committee was not indicated since only genetic and clinical data collected during regular patient care were used.For Dutch research involving CONCOR-data, this is explained on their website https://concor.net/en/aboutconcor/methodology.html(accessed on 1 June 2022). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The parents provided written consent for research use of their data.For Dutch research involving CONCOR-data, this is explained in https://pubmed.ncbi.nlm.nih.gov/16121765/ and on their website https://concor.net/en/aboutconcor/methodology.html(accessed on 1 June 2022). ",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.7554/elife.98747.3
|
The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation
|
<jats:p>The sebaceous gland (SG) is a vital appendage of the epidermis, and its normal homeostasis and function is crucial for effective maintenance of the skin barrier. Notch signaling is a well-known regulator of epidermal differentiation, and has also been shown to be involved in postnatal maintenance of SGs. However, the precise role of Notch signaling in regulating SG differentiation in the adult homeostatic skin remains unclear. While there is evidence to suggest that Notch1 is the primary Notch receptor involved in regulating the differentiation process, the ligand remains unknown. Using monoclonal therapeutic antibodies designed to specifically inhibit of each of the Notch ligands or receptors, we have identified the Jag2/Notch1 signaling axis as the primary regulator of sebocyte differentiation in homeostatic skin. Mature sebocytes are lost upon specific inhibition of the Jag2 ligand or Notch1 receptor, resulting in the accumulation of proliferative stem/progenitor cells in the SG. Strikingly, this phenotype is reversible, as these stem/progenitor cells re-enter differentiation when the inhibition of Notch activity is lifted. Thus, Notch activity promotes correct sebocyte differentiation, and is required to restrict progenitor proliferation.</jats:p>
|
[
{
"section_content": "The skin is a vital organ that acts as a protective barrier against the external environment, and safeguards against fluid loss.An important component of this barrier function is the presence of a complex mixture of oils, known as sebum, which is produced by the SGs.SGs are part of the epidermis and are typically associated with the hair follicle.These acinar structures have two cell types: basal stem or progenitor cells which encase the differentiated sebocytes (Figure 1a).Sebocyte differentiation begins at the proximal tip of the of the SG, with maturing sebocytes moving upwards, enlarging, accumulating lipids, and ultimately undergoing a highly regulated and specialized form of cell death in which they release their lipid contents into the sebaceous duct (Figure 1b; Kretzschmar et al., 2014;Schneider and Paus, 2010).This process requires the constant turnover of sebocytes, which occurs over a period of 7-14 d in mice (Jung et al., 2015).Both over-and underproduction of sebum have been linked to various skin disorders including acne or dry skin (Al-Zaid et al., 2011;Binczek et al., 2007;Karnik et al., 2009;Lovászi et al., 2017;Rittié et al., 2016;Seiffert et al., 2007;Shi et al., 2015;Smith and Thiboutot, 2008;Stenn et al., 1999), and rare sebaceous carcinomas constitute aggressive tumors leading to high mortality (Buitrago and Joseph, 2008;Nelson et al., 1995), thus SG number and function have to be tightly regulated for proper skin function. The Notch signaling pathway is one of the most studied regulators of cell fate decisions, and is known to be widely involved in epidermal differentiation.The pathway consists of multiple ligands and receptors that typically form a signaling axis in pairs.Notch signaling can regulate cell fate by either inducing or inhibiting differentiation, or by making binary cell fate decisions (Wilson and Radtke, 2006).Classically, these functions of Notch signaling have been studied during development, but increasing evidence suggests that the Notch pathway is also involved in regulating cell fate and cell states in adult homeostatic tissues (Ables et al., 2011;Lafkas et al., 2015;Mosteiro et al., 2023;Sato et al., 2012;Siebel and Lendahl, 2017). While it is known that Notch signaling is not required for embryonic development of the epidermis, it is essential for the postnatal maintenance of the hair follicles and the SGs (Watt et al., 2008).However, the precise role of Notch signaling in adult sebocyte differentiation has not been comprehensively investigated, with most studies examining irreversible deletions of the Notch pathway components in the embryonic ectodermal lineages.While these studies report SG defects, it remains unclear whether these defects are due to a direct effect on the SGs, or whether they are a consequence of general skin defects also observed in these models.For example, SGs are absent in mice with embryonic pan-Notch deletions such as Rbpj (Blanpain et al., 2006), gamma-secretase, Notch1Notch2, and Notch1Notch2Notch3, and are severely reduced in Notch1 and Notch1Notch3 embryonically-deleted skin (Pan et al., 2004), while deletion of Notch2 alone does not affect the SG (Pan et al., 2004).Consistent with the constitutive deletions, loss of Rbpj in adult SGs also results in missing sebocytes, while loss of Notch1 in the adult SGs results in miniaturized lobes that still contain some differentiated sebocytes (Veniaminova et al., 2019).Interestingly, activation of Notch1 in the adult skin results in enlarged SGs (Estrach et al., 2006).Conversely, for the Notch pathway ligands, embryonic and adult deletion of Jag1 (Estrach et al., 2006), and embryonic deletion of Dll1 (Estrach et al., 2008) results in normal SG morphology.Collectively, these data suggest that Notch1 is the dominant Notch receptor involved in regulating sebocyte differentiation, however, it remains unclear which ligand is required. In our previous work, we observed that systemic inhibition of Jag2 using monoclonal therapeutic antibodies resulted in SG defects in adult mice (Lafkas et al., 2015).Given that our therapeutic antibodies selectively, potently, and transiently inhibit each of the distinct Notch receptors and ligands (Tran et al., 2013;Wu et al., 2010;Yu et al., 2020), they constitute ideal tools to dissect the contribution of each of these pathway members to sebocyte differentiation in adult homeostatic skin.Leveraging the use of these antibodies, we demonstrate that specific inhibition of the Jag2 ligand or Notch1 receptor both result in the loss of mature sebocytes in the SG, establishing the Jag2/Notch1 signaling axis as a crucial regulator of sebocyte differentiation in adult homeostatic skin.The loss of mature sebocytes in the SG is concomitant with an accumulation of cells with a basal phenotype, forming epithelial remnants in the SG.Cells in these epithelial remnants are actively proliferating and express stem/progenitor markers indicating that sebocyte differentiation is halted, while stem/ progenitor numbers are increased.Importantly, this phenotype is reversible, as these epithelial cells re-enter differentiation with the return of Notch activity.Thus, Notch activity is required in sebocyte stem/progenitor cells for their proper differentiation, and its inhibition locks these cells in a reversible progenitor state. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To investigate the role of Notch signaling in the homeostatic skin, we treated 8-wk-old mice with a single dose of the various antagonizing antibodies (anti-Notch1, anti-Notch2, anti-Jag1, anti-Jag2, ",
"section_name": "Jag2 is the dominant Notch signaling ligand involved in regulating sebocyte differentiation",
"section_num": null
},
{
"section_content": "Basal stem cell Lrig1 ",
"section_name": "Progenitor cells AR,Lrig1",
"section_num": null
},
{
"section_content": "Proximal tip and the isotype control antibody anti-Ragweed) alone or in combination.We then examined the dorsal, resting phase (telogen) skin at 3, 7, and 14 d post-treatment.We first confirmed that our antibodies could reproduce the requirement of Notch1 in regulating sebocyte differentiation.To this end, we treated mice with Notch1 (aN1) and Notch2 (aN2) blocking antibodies and examined the SGs at 7 d post-treatment.Loss of mature sebocytes was observed specifically after aN1 treatment, while the aN2-treated SGs showed normal morphology (Figure 1c andd).However, the combined treatment of aN1N2 had a more pronounced effect on SG morphology as compared to aN1 alone (Figure 1c andd), indicating that Notch2 also contributes to regulating sebocyte differentiation, possibly as a compensatory mechanism after inhibition of the dominant Notch1 receptor.Next, we investigated which ligand formed the signaling pair with Notch1.Loss of mature sebocytes was observed specifically after treatment with the Jag2 blocking antibody (aJ2), but not after treatment with the Jag1 blocking antibody (aJ1) at 7 d post-treatment (Figure 1e andf).For aJ2 treatment, the loss of sebocytes began at 3 d post treatment, but affected only a small proportion of the SGs at this time point (Figure 1g).However, by 7 ds post-treatment, most SGs had either completely lost all sebocytes, or had only some sebocytes remaining (Figure 1g).The loss-of-sebocyte phenotype was most pronounced with a combined treatment of Jag1/Jag2 blocking antibodies (aJ1J2) (Figure 1g), suggesting that while Jag2 is the primary Notch ligand involved in regulating sebocyte differentiation, Jag1 also plays a minor role in this process, but is unable to produce a phenotype on its own.In conclusion, these data indicate that the Jag2-Notch1 signaling axis is the dominant Notch ligand-receptor pair required for sebocyte differentiation in the adult skin. ",
"section_name": "N1ICD,Lrig1",
"section_num": null
},
{
"section_content": "We also examined the percentage of SGs consisting of bursting sebocytes releasing sebum as a proxy of a functional sebaceous duct.There were no significant differences between treatments (Figure 1-figure supplement 1a and b), hinting at a functionally intact sebaceous duct. ",
"section_name": "Hair follicle",
"section_num": null
},
{
"section_content": "We next characterized the expression of the relevant receptor and ligand in the SG to develop a spatial map of Notch activity in this tissue.Both the Notch receptors and their ligands are transmembrane proteins that interact with each other in neighboring cells to activate the pathway.To examine this interaction, we performed a triple stain for the cleaved (active) form of the Notch1 intracellular domain (ICD), and Notch1 and Jag2 in situ hybridization (ISH) probes at 3 d post antibody treatment.In the control treated mice, Notch1 ICD was observed in the basal stem cell compartment of the SG, but not in the differentiating sebocytes (Figure 2a-c, cell types based on morphology).We saw a similar pattern of N1ICD-positive cells for aJ1 treatment (Figure 2d-f).Notch1 signaling was not active in all basal cells, as only ~50% of them were positive for N1ICD after control and aJ1 treatment (Figure 2g).Consistent with previous studies, the majority of these N1ICD + cells were present near the proximal tip of the SG (Figure 2-figure supplement 1a-c; Veniaminova et al., 2019), where the initial sebocyte differentiation has been proposed to occur (Kretzschmar et al., 2014).However, after aJ2 treatment, Notch1 activity (ICD staining) was absent, or observed only at very low levels in the basal stem cells, while they still expressed Notch1 and Jag2 mRNA (Figure 2h-j).Strikingly, we noticed the expression of both Notch1 ISH and Jag2 ISH in the same cell on most N1ICD + cells (Figure 2k).Interestingly, a majority of all basal cells (including N1ICD-cells), expressed both N1 and Jag2 mRNA (Figure 2-figure supplement 1d).Our data shows that the vast majority of SG basal stem cells express both the ligand and receptor, but only some basal stem cells experience active Notch signaling at any one point in time, as evidenced by the presence of N1ICD.This could be a technical limitation as the triple staining only captures a temporal snapshot of the basal stem cells, with the N1 + J2 mRNA positive cells going on to express N1ICD later, or it could hint towards a more complex regulatory mechanism involved in activating Notch signaling. ",
"section_name": "Notch is active in the sebaceous gland stem cells",
"section_num": null
},
{
"section_content": "Notch activity, as well as Notch1 and Jag2 mRNA, were also observed in the interfollicular epidermis (IFE) and other cells of the hair follicle (Figure 2-figure supplement 1e and f).While there is a reduction in Notch-active cells in these other regions, it does not appear to significantly impact the rest of the skin.There were no significant differences in the width of the IFE or the adipocyte layer between treatments (Figure 2-figure supplement 1g and h).This together with the histological appearance (Figure 2-figure supplement 1i and j) of these regions suggests that proliferation and differentiation in these compartments remain unaffected. ",
"section_name": "Figure 1 continued",
"section_num": null
},
{
"section_content": "To further detail the effect of Notch inhibition on sebocyte differentiation, we examined the expression of mature sebocyte markers.Adipophilin (Adipo) is expressed in all mature sebocytes (Frances and Niemann, 2012;Ostler et al., 2010) and Fatty acid synthase (FASN) is expressed in mid-and late-differentiating sebocytes, with FASN levels decreasing in the most mature sebocytes (Cottle et al., 2013;Figure 1b).Since N1ICD staining disappears at 3 d post antibody treatment, we examined the mature sebocyte markers at this time point.While N1ICD staining was specifically lost in the SG basal stem cells of aJ2-treated skin, mature sebocytes expressing FASN were still observed at this timepoint (Figure 2j, Figure 3a andb andFigure 3-figure supplement 1a).All cells that express FASN also express adipophilin, but since FASN levels decrease with sebocyte maturity, we also examined and focused on adipophilin to mark all sebocytes (Figure 3c andd and Figure 3-figure supplement 1b-e).The number of cells that expressed adipophilin was not significantly different between treatments at 3 d post antibody treatment (Figure 3h).At 7 d post antibody treatment, control and aJ1 treated skin showed normal SG morphology and sebocyte marker expression (Figure 3e and Figure 3-figure supplement 1f, h and i), but both aJ2 and aJ1J2 SGs had lost sebocyte marker expression, and the SG was filled with cells with a basal phenotype (basal-like cells) (Figure 3f and Figure 3-figure supplement 1g, j and k).Consistently, the number of cells expressing adipophilin were significantly lower for aJ2 and aJ1J2 treatment (Figure 3i).Interestingly, we noticed that some of these affected SGs still contained a few mature sebocytes (Figure 3f and Figure 3-figure supplement 1g), which were found at the distal end near the sebaceous duct.The location of these remaining sebocytes suggests that existing mature sebocytes are not affected by the Notch blockade, and go through their normal differentiation process, eventually bursting and releasing the sebum (Figure 3g).Thus, we propose that Notch blockade inhibits differentiation at the basal stem cell level or in a sebocyte progenitor. ",
"section_name": "Loss of Notch activity in the SG stem cells inhibits sebocyte differentiation",
"section_num": null
},
{
"section_content": "To determine whether the epithelial cells that filled the affected SGs at 7 d post-treatment were stem/ progenitor cells, we examined them for stem and early differentiation markers.Lrig1-positive cells form a distinct stem cell compartment that maintains the SG and the upper part of the hair follicle (Frances and Niemann, 2012;Niemann and Horsley, 2012;Page et al., 2013), allowing us to use Lrig1 as a marker for the basal stem cells of the SG, while Androgen Receptor (AR) can be used as an early marker of sebocyte differentiation (Cottle et al., 2013;Figure 4a andb andFigure 4-figure supplement 1a and b).We noticed that the AR-expressing cell population could be divided into two groups: a basal stem cell population that co-expressed Lrig1, but did not express FASN (arrowheads in Figure 4a andc), and an early differentiating sebocyte population that expressed FASN, but did not express Lrig1 (arrows in Figure 4a andc).We hypothesize that the Lrig1+/AR + population is a progenitor cell population, in addition to the Lrig1 + stem cells, similar to the recently identified transitional basal cell population in the SG (Veniaminova et al., 2023).There were no significant differences in the number of Lrig1 positive stem cells per SG, or the AR-expressing progenitor population between treatments at 3 d post-treatment (Figure 4d ande).By 7 d post-treatment, however, the basal-like cells that filled the SG were all positive for Lrig1 (Figure 4f andg andFigure 4figure supplement 1e), with the total number of Lrig1 positive cells per SG increasing significantly for aJ2 and aJ1J2 treated SGs (Figure 4h), while SGs after aJ1 treatment showed normal morphology and marker expression (Figure 4-figure supplement 1c andd).The proportion of AR-expressing cells was not significantly different between treatments (Figure 4i).These results indicate that the basal-like cells that fill the SG after blocking Notch signaling by aJ2 and aJ1J2 treatment, are stem/ progenitor cells.Additionally, we examined the SGs for their proliferative capacity to confirm stem/ progenitor function.In a normal SG, proliferation is restricted to the basal stem cells (Figure 4j).As expected, proliferation was also restricted to the basal stem cells in aJ1-treated SGs (Figure 4-figure supplement 1f and g).Remarkably, most of the basal-like cells in the aJ2-treated SGs were proliferative, while the rare mature sebocytes remaining in the SG were non-cycling (Figure 4k).As the SGs are mostly filled with basal-like cells at this time (Figure 4h), the total number of proliferating cells per SG was significantly higher (Figure 4l).Overall, these data suggest that inhibition of Notch activity by aJ2 treatment retains the stem and progenitor (Lrig1 + and Lrig1+/AR+, respectively) cells in their immature proliferative state and prevents differentiation. ",
"section_name": "Notch activity in the SG stem cells is required to prevent unregulated progenitor proliferation",
"section_num": null
},
{
"section_content": "The therapeutic antibodies employed do not inhibit Notch signaling permanently, as the antibodies eventually become cleared from the animal's system.To determine whether the loss of sebocyte phenotype was reversible, we examined the SGs 14 d post single-dose treatment.Intriguingly, mature sebocytes begin to recover at this time point (Figure 5a and b, compared with Figure 1f andg).We hypothesized that the sebocyte recovery must be due to the return of Notch activity after antibody washout.To test this, we examined the SGs for Notch activity at 7 d post-treatment, since the return of Notch activity must precede the recovery of mature sebocytes.Fittingly, we observed N1ICD expression return in some of the basal-like cells at this time (Figure 5c andd and Figure 5-figure supplement 1a-d).The percent of N1ICD positive cells per SG strongly increased from 1% at 3 d post-treatment to 29% at 7 d post-treatment (Figure 5e), even though the percentage of N1ICD positive cells per SG remained significantly lower than control for aJ2 and aJ1J2 treated SGs at this time (Figure 5-figure supplement 1d).Next, we examined the SGs at 14 d post-treatment for mature sebocyte markers to confirm the sebocyte recovery.Indeed, we saw the return of cells expressing adipophilin in the aJ2 and aJ1J2 treated SGs (Figure 5f and g and Figure 5-figure supplement 1e-h).There was an overall increase in the number of these cells from day 7 to day 14 (Figure 5h), even though the number of these cells remained significantly lower in these SGs compared to controls (Figure 5-figure supplement 1h).Interestingly, the majority of the adipophilin-expressing cells were found in the proximal third of the SG (51% for aJ2 treatment and 66% for aJ1J2 treatment), consistent with the initiation of sebocyte differentiation at the proximal tip.However, there was a significant proportion of these cells found in the middle third (22% and 25%, respectively), and distal third (27% and 9%, respectively) of the SG.This could be due to the newly differentiated cells moving through the SG in a proximal to distal direction, as is the case during normal homeostasis.Alternatively, the stem cells could also be differentiating at sites other than just the proximal tip, as previously demonstrated by multi-color lineage tracing (Andersen et al., 2019).We further examined the SGs at 14 d post-treatment for their AR expression, and confirmed that it had also been restored to its normal pattern (Figure 5i and j and Figure 5-figure supplement 1i-l).We also found that the average number of AR-expressing cells decreased from day 7 to day 14 post aJ2 and aJ1J2 treatment (10-2.95, and 8.56-5.55,respectively) (Figure 5k), returning to a more homeostatic state.Together, these data indicate that Notch inhibition does not result in a permanent cell fate switch, but maintains the stem/progenitor state, allowing the recovery of the differentiation process with the restoration of Notch activity. ",
"section_name": "The block in sebocyte differentiation is lifted upon recovery of Notch activity",
"section_num": null
},
{
"section_content": "Based on our findings, we propose that the Jag2/Notch1 signaling axis is essential for correct sebocyte differentiation in homeostatic dorsal skin, and that inhibition of this signaling retains the basal stem and progenitor cells in a proliferative state, and blocks further differentiation.Thus, Notch signaling is required to prevent unregulated stem/progenitor proliferation, and induction of the sebocyte differentiation program.Moreover, Notch inhibition resulting in complete loss of mature sebocyte differentiation is a reversible phenotype, indicating that there remains a functional progenitor pool present during the studied timeframe. Here, we have leveraged the use of monoclonal therapeutic antibodies designed to inhibit each of the distinct Notch receptors or ligands to study the role of Notch signaling in adult homeostatic tissue.We have shown that Jag2 is the hitherto unknown ligand involved in regulating sebocyte differentiation.Inhibition of Notch signaling using Jag2 blocking antibodies results in the loss of mature sebocytes, with the resulting SG being filled with basal-like cells forming 'finger-like' epithelial remnants. A similar phenotype has been described by a previous study (Veniaminova et al., 2019) that used Lrig1-CreERT2 to irreversibly knock out RBPJ in adult homeostatic skin.This strategy enabled inhibition of Notch signaling specifically in the Lrig1 + stem cell population that normally maintains the SGs, but preserved it in the rest of the hair follicle.Interestingly, the authors saw two contradictory phenotypes as a result: overall loss of SG lobes, replaced by the finger-like epithelial remnants, as well as the presence of persistent SGs that were enlarged.The authors showed that over time, patches of Rbpj mutant cells extended out of their niche into the IFE, where Lrig1 is not expressed, and that loss of RBPJ in the IFE led to enlarged SGs.They argue that while Notch signaling promotes sebocyte differentiation in the SG stem cells, it indirectly suppresses these glands from the IFE.Our results did not show the enlarged SG phenotype, and we only observed the finger-like epithelial remnants.While injections of the Notch blocking antibodies are systemic, we only observed a reduction in the number of Notch-active cells in the IFE, but not a complete loss.This could explain why we didn't observe the enlarged SG phenotype.Additionally, we observed the phenotype as early as 3 d post antibody treatment, while Veniaminova et al., observed the SGs 10 wk after tamoxifen injection.These differences are likely due to the different methodologies used in the two studies. Several studies indicate that Notch signaling is essential for the postnatal maintenance of SGs (Blanpain et al., 2006;Estrach et al., 2008;Estrach et al., 2006;Pan et al., 2004;Watt et al., 2008), but so far only Veniaminova et al., have specifically examined SGs in the adult homeostatic skin.They observed that the stem/progenitor and differentiation markers were intermingled in the finger-like remnants forming an unnatural hybrid state, neither staying in a true progenitor state nor differentiating.In contrast, we observed a complete block in differentiation, with the stem/progenitor cells being locked in an immature proliferative state.These observations suggest that antagonistic antibodies may be able to achieve a more complete inhibition of Notch signaling in the SG stem/ progenitor compartment. Importantly, we were able to show that the loss-of-sebocyte phenotype in the SG is reversible.Having used therapeutic antibodies to block Notch signaling, the inhibition of the signaling pathway was not permanent.As Notch activity returned to the SG after antibody washout, sebocyte differentiation also recovered.These data indicate that the loss of Notch signaling does not impact the stem/ progenitor potential of the SG, but prevents further differentiation.Indeed, a functional progenitor pool accumulates in the SG, primed for differentiation, as soon as Notch signaling becomes active.A recent study by Veniaminova et al., has shown that Lrig1-CreERT2 used to irreversibly knock out Pparg in adult homeostatic skin ablates 99% of the SGs (Veniaminova et al., 2023).Interestingly, they showed that non-recombined cells from other parts of the hair follicle migrate to the SG zone and regenerate the genetically ablated SGs.This regeneration process is dependent on the hair growth cycle, with the SGs primarily regenerating during the anagen (active growth) phase and not in the telogen phase.While our studies cannot rule out the contribution of non-SG cells to the SG recovery seen upon the return of Notch activity, this recovery is not dependent on the hair growth cycle.Sebocytes are able to differentiate while the hair follicle is still in the telogen phase, indicating a lift of the An important open question is how Notch signaling regulates sebocyte differentiation.FASN, which can be used as a readout for sebocyte differentiation, is a downstream target of AR (Schirra et al., 2005).We observed a Lrig1+/AR+/FASN-population in the normal SGs, similar to the basallike cells that fill the finger-like remnants seen after Notch inhibition.This expression pattern indicates that AR requires a co-activator to activate downstream gene expression in the SG.Previous studies have reported that Notch effectors such as HEY1, HEY2, and HEYL can act as co-repressors of AR in prostate cells (Belandia et al., 2005;Kamińska et al., 2020;Lavery et al., 2011).It is possible that certain Notch effectors can act as either co-activators or co-repressors of AR in a context-dependent manner.Further investigation will be needed to understand the exact molecular role Notch signaling plays in regulating sebocyte differentiation. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "aJ2=2.90E-87, aJ1J2=1.73E-92.Chi-square test used for statistical analysis.All treatments were compared against aRW.Total n of SGs quantified per treatment: aRW = 485, aJ1=430, aJ2=394, aJ1J2=364.Scale bars are 100 μm.(c,d) Representative co-stain images for N1ICD and fatty acid synthase (FASN) in SGs from mice (n=5 each) treated with aRW (c) and aJ2 (d), 7 d post-treatment.(e) Quantification of the percentage of N1ICD + cells in SGs from mice (n=5 each) treated with aRW and aJ2, 3 and 7 d post-treatment.Percentage was calculated by dividing the number of N1ICD+ cells by the total number of basal-like cells in each SG.p-values: for comparison between day 3 and day 7 for aRW treatment = 0.297, for comparison between day 3 and day 7 for aJ2 treatment = 1.00E-14.Total n of SGs quantified per treatment: aRW at 3 d = 13, aRW at 7 d = 13, aJ2 at 3 d = 15, aJ2 at 7 d=22.(f,g) Representative adipophilin staining in SGs from mice (n=5 each) treated with aRW (f) and aJ2 (g), 14 d post treatment.(h) Quantification of the number of cells expressing adipophiln in each SG, 7 and 14 d after treatment with aRW, and aJ2.p-values: for comparison between day 7 and day 14 for aRW treatment = 0.292, for comparison between day 7 and day 14 for aJ2 treatment = 1.38E-4.Total n of SGs quantified per treatment: aRW at 7 d = 18, aRW at 14 d = 19, aJ2 at 7 d = 22, aJ2 at 14 d=29.(i,j) Representative co-stain images for androgen receptor (AR) and FASN in SGs from mice (n=5 each) treated with aRW (i) and aJ2 (j), 14 d post-treatment.(k) Quantification of the number of cells expressing AR in each SG, 7 and 14 d after treatment with aRW and aJ2.p-values: for comparison between day 7 and day 14 for aRW treatment = 0.273, for comparison between day 7 and day 14 for aJ2 treatment=9.53E-09.Total n of SGs quantified per treatment: aRW at 7 d=12, aRW at 14 d=10, aJ2 at 7 d=18, aJ2 at 14 d=23.Student's t-test used for statistical analysis.C57BL/6 mice were obtained from Charles River-Hollister, and were used for all experiments.Mice were housed under specific-pathogen-free conditions, and were 8 wk old upon treatment.This time point was chosen to correspond to the resting (telogen) phase of the hair growth cycle, as SG size can vary by hair cycle stage.All mice were injected intraperitoneally with a single dose of blocking antibodies diluted in sterile saline at the following concentrations: anti-Jag1 at 20 mg/kg, anti-Jag2 at 20 mg/kg, anti-Jag1 + anti-Jag2 at 20 mg/kg + 20 mg/kg=40 mg/kg, anti-Notch1 at 5 mg/kg, anti-Notch2 at 10 mg/kg and anti-Notch1 + anti-Notch2 at 5 mg/kg + 10 mg/kg=15 mg/kg.Anti-Ragweed isotype control antibody was injected at concentrations to match the maximum dose of treatment antibodies. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Telogen mouse dorsal skin was collected at 3, 7, and 14 d post antibody injection.For frozen sections, skin samples were fixed in 4% paraformaldehyde in PBS, for 40 min at 4 °C, washed, and then immersed in 30% sucrose in PBS overnight at 4 °C.The tissue was then embedded in OCT (TissueTek), frozen immediately on dry ice, and stored at -80 °C.Additional skin tissue from the same animals was also fixed with 10% neutral buffered formalin and then used to create paraffin-embedded sections.Hematoxylin and eosin (H&E) staining was performed by the Pathology core at Genentech.The triple immunofluorescence stain for Notch1 ICD (IHC), and Notch1 mRNA (ISH), and Jag2 mRNA (ISH) was performed by the histopathology development group at Genentech.ACD LS 2.5 probes were ordered from Advanced Cell Diagnostics.RNAScope LS 2.5 Murine-Jag2_C1 (417518) nucleotides spanning from nt 552-1480 of reference sequence NM_010588.2,and Murine-Notch1_ C2 (404648-C2) nucleotides spanning from nt 1153-1960 of reference sequence NM_008714.3.For positive control, RNAScope LS 2.0 Murine-PPIB probe (313917) nucleotides spanning from nt 98~856 of reference sequence NM_011149.2were used.For negative control RNAScope LS 2.0 DapB probe (312038) nucleotides spanning from nt 414~862 reference sequence EF191515 were used.For Immunohistochemistry, we used N1ICD (Cell Signaling, 4147), at 20 ug/ml working concentration. The triple immunofluorescence for murine Jag2_Notch1 (dual ISH) with anti_Notch1 ICD (IHC) 3 Plex ISH_ISH_IHC in murine tissues using formalin-fixed, paraffin-embedded sections was performed on the Leica Bond-RX complete automation system using the RNAscope LS Multiplex Reagent Kit (322800).Slides were baked and dewaxed on Leica Bond-RX and pretreated with Bond Epitope Retrieval Solution 2 (ER2) (AR9640) from Leica at 100 °C for 40 min.After pretreatment, the probes were cocktailed, and hybridization was performed at 42 °C for 120 min followed by amplification steps and developed with Opal 570 fluor at 1:1000 and Opal 690 fluor at 1:1500 from Akoya using the Opal Polaris 7 Auto Detection Kit (NEL811001KT).The immunohistochemistry was performed upon completion of ISH.The primary antibody incubation was 60 min at room temperature, followed by secondary antibody incubation for 30 min at room temperature with HRP conjugated Goat anti-Rabbit (Perkin Elmer, NEF812001EA).The slides were then developed with Opal 780 fluor at 1:25 (Akoya, SKU FP1501001KT) following the manufacturer's instructions.Slides were imaged using Olympus VS200. All other immunohistochemistry was performed using the following antibodies: N1ICD at 1:500 (Cell Signaling, 4147), FASN at 1:100 (BD, 610963), Adipophilin at 1:500 (Fitzgerald, 20R-AP002), Ki67 at 1:100 (Thermo Fisher Scientific, SP6 RM-9106-SO), Lrig1 at 1:200 (R&D Systems, AF3688-SP), and AR at 1:250 (Abcam, ab133273 [EPR1535(2)]).Slides were imaged using the Leica Thunder microscope. Imaging parameters were identical for all images.Images were processed using Fiji and Adobe Illustrator. ",
"section_name": "Histopathological analysis and immunochemistry",
"section_num": null
}
] |
[
{
"section_content": "We are grateful to C Cottonham, S Hankeova, and G Hernandez for their helpful discussions.We thank the Genentech Research Pathology, Necropsy, and Histology laboratories for their experimental contributions.We appreciate the insightful feedback and comments on the paper from L Mosteiro, E Reyes, and B Biehs. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "No external funding was received for this work. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for Figure 1; Figure 2; Figures 3-5 and Figure 1-figure supplement 1, Figure 2-figure supplement 1 and Figure 5-figure supplement 1. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Competing interests Syeda Nayab Fatima Abidi: S.N.F.A. is an employee of Genentech.Sara Chan: S.C. is an employee of Genentech and holds shares in Roche.Kerstin Seidel: K.S. is an employee of Genentech and holds shares in Roche.Daniel Lafkas: D.L. was a Genentech employee, and is currently employed at Roche, and holds shares in Roche. Louis Vermeulen: L.V. is an employee of Genentech and holds shares in Roche.Frank Peale: F.P. is an employee of Genentech and holds shares in Roche.Christian W Siebel: C.W.S. was a Genentech employee, and holds shares in Roche.C.W.S is currently employed at Gilead Sciences. ",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
}
] |
10.3390/jcdd2040248
|
Embryonic Development of the Bicuspid Aortic Valve
|
<jats:p>Bicuspid aortic valve (BAV) is the most common congenital valvular heart defect with an overall frequency of 0.5%–1.2%. BAVs result from abnormal aortic cusp formation during valvulogenesis, whereby adjacent cusps fuse into a single large cusp resulting in two, instead of the normal three, aortic cusps. Individuals with BAV are at increased risk for ascending aortic disease, aortic stenosis and coarctation of the aorta. The frequent occurrence of BAV and its anatomically discrete but frequent co-existing diseases leads us to suspect a common cellular origin. Although autosomal-dominant transmission of BAV has been observed in a few pedigrees, notably involving the gene NOTCH1, no single-gene model clearly explains BAV inheritance, implying a complex genetic model involving interacting genes. Several sequencing studies in patients with BAV have identified rare and uncommon mutations in genes of cardiac embryogenesis. But the extensive cell-cell signaling and multiple cellular origins involved in cardiac embryogenesis preclude simplistic explanations of this disease. In this review, we examine the series of events from cellular and transcriptional embryogenesis of the heart, to development of the aortic valve.</jats:p>
|
[
{
"section_content": "Congenital heart disease (CHD) is the most frequently occurring birth defect among liveborn humans.Bicuspid aortic valve (BAV) in particular is the most common congenital valvular heart defect, having an overall frequency of 0.5%-1.2%and occurring more frequently in males and white individuals.BAVs result from abnormal aortic cusp formation during valvulogenesis, whereby adjacent cusps fuse into a single large cusp resulting in two, instead of the normal three, aortic cusps [1].BAV is a strong risk factor for accelerated aortic valve disease, principally aortic stenosis, and ascending aortic aneurysm [2,3]. BAV occurs in several genetic syndromes, including Turner syndrome, and is associated with coarctation of the aorta, hypoplastic left heart syndrome, Holt-Oram syndrome, ventricular noncompaction and adult-onset aortopathy [1].In addition, BAV occasionally demonstrates complex inheritance in large families without evidence of syndromic features, other congenital heart defects or a family history of CHD.Yet, some 10% of BAV patients have first-degree relatives with the condition or associated non-valvular abnormalities such as aortic coarctation, thoracic aortic aneurysms, mitral valve disease or ventricular septal defects [4]. Over the past few decades, much progress has been made in understanding the embryological development of the heart, including the contributions of various cell populations and the molecular biology of its very complex spatial and temporal anatomical development.Advances in the identification of transcription factors, signaling molecules and structural genes for heart development have assisted the discovery of genes causing CHD.Culprit genes have been identified using genomewide association studies (GWAS) or candidate gene sequencing [5][6][7], which have elucidated the etiology of some congenital heart defects, notably atrial and ventricular septal defects [8][9][10][11] and Tetralogy of Fallot [12][13][14].However, the underlying pathology of most structural outflow tract (OFT) defects remains unknown because of the complexity of transcriptional and signaling mechanisms in the developing embryo and the numerous cell types involved in tract development.Thus far, for any single structural defect, numerous genes have been implicated, each in only a small percentage of patients.Similarly, there has been limited progress in defining the molecular etiology of BAV.Although autosomal-dominant transmission of this condition has been observed in some three-generation pedigrees, notably involving the gene NOTCH1 [15,16], no single-gene model clearly explains BAV inheritance.Likewise, evidence for specific loci for a genetic etiology of sporadic BAV (wherein no first-or second-generation relatives with BAV can be identified) is limited. In this review, we describe normal development of the aortic valve; the key cellular players; the specifics of cell-cell communication and migration; and pathways possibly involved in BAV development. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "The vertebrate heart has remarkable evolutionary conservation and structural similarity that includes divided pulmonary and systemic circulations, a conduction system and valves to create coordinated unidirectional flow at high pressure.These diverse structural and functional features of the vertebrate heart reflect its origination from cardiac neural crest (CNC) cells, which contribute to portions of the OFT and septum; the mesenchyme that comprises the first heart field (FHF) and second heart field (SHF); the endothelium, which provides growth factor signals and precursor cells for formation of the cardiac valves; and the proepicardium, which provides precursors for the coronary vasculature and mural valve leaflets [17,18].Spatial and temporal integration of these various cell types is required for proper cardiac specification and anatomical morphology during embryogenesis. ",
"section_name": "Progenitor Development of the Vertebrate Heart",
"section_num": "2."
},
{
"section_content": "We start at gastrulation as specification of cardiac progenitor cells occurs by this time.After formation of the hollow sphere of cells known as the blastocyst, the three germ layers of the embryo (the ectoderm, mesoderm, and endoderm) are formed by development of the primitive streak (along the anterior-posterior axis) and gastrulation-the process of conversion of the spherical blastocyst to the complex and differentiated gastrula that forms the organs at about days 15-16 of human embryogenesis (Figure 1).Gastrulation requires cell polarity, axis specification and cell-type specification to establish a three-dimensional map of the embryo.These contributions are from the first heart field (red), second heart field (yellow) and cardiac neural crest (blue).Modified from [19]. Embryonic progenitor cells derive from the epiblast on either side of the primitive streak.The process of forming three germ layers from the blastocyst requires epiblast migration and epithelial to mesenchymal transition (EMT) to form the mesoderm and endoderm.Finally, cell lineages of the heart, pharyngeal arches and vasculature begin to be defined.EMT is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells, which are multipotent stromal cells that can differentiate into a variety of cell types.After undergoing EMT, mesodermal cells move cranio-laterally before converging medially while forming the lateral plate mesoderm (LPM) and other mesodermal derivatives.The LPM splits into the dorsal somatic and ventral splanchnic mesoderm.The dorsal somatic mesoderm gives rise to the body wall and other derivatives, while the ventral splanchnic mesoderm gives rise to all heart components mentioned earlier as well as other derivatives. EMT is primarily mediated by transforming growth factor-β (TGF-β) signaling as well as Wnt (wingless-related MMTV integration sites), fibroblast growth factor (FGF) and the bone morphogenic pathway (BMP), amongst others.Expression of cardiac-specific transcription factors, such as the GATA, MEF2, NK2, TBX and HAND gene families, control cardiac cell fate and the morphogenesis of cardiac structures.Adjacent endodermal cells that are not destined to become cardiac precursors express many of these transcription factors and are paracrine controllers of development.Thus, complexity of the vertebrate heart results from spatial and temporal selectivity of a diverse range of regulatory proteins and regulatory elements on specific cell populations.This specificity in gene regulation probably underlies the restriction of many cardiac defects to specific anatomical regions of the heart [5]. ",
"section_name": "Gastrulation in the Early Embryo",
"section_num": "2.1."
},
{
"section_content": "Mesodermal progenitor cells derived from the LPM arise from the cranial third of the primitive streak during early gastrulation.These mesodermal cells migrate in a cranial-lateral direction to become localized on either side of the primitive streak and undergo EMT to mesodermal cells, then proliferate and migrate in a semicircular fashion.Later, they coalesce in the midline to form the cardiac crescent. The cellular fate of these cardiac mesodermal cells is initially uncommitted, but after migration, they become specified to differentiate into the cardiogenic mesoderm of the FHF and SHF at about day 18 of human embryogenesis (Figure 2).The FHF is formed from the cells that are first to differentiate from the cardiac crescent and are marked by the transcription factor Hcn4.The FHF gives rise to the early heart tube and, later, to much of the left ventricle, portions of both atria and some of the right ventricle [20]. The SHF is composed of undifferentiated progenitor cells from the medial splanchnic mesoderm adjacent to the pharyngeal endoderm.Many transcription factors have been implicated in the development of the SHF, including Mef2c, Tbx1, Islet-1 (Isl1), Hand2, Fgf8 and Fgf10 [20].Once the rudimentary heart tube is created, cells from the SHF contribute to the OFT, giving rise to the smooth muscle of the aortic root, and join with more distal smooth muscle of the ascending aorta derived from neural crest cells.The SHF is also the source of the majority of the myocardium of the right ventricle, components of the interventricular septum, a small part of the left ventricle and the atria [21,22].Specifically, the SHF cells reach the heart tube at both the arterial pole (anterior SHF) and venous pole (posterior SHF).At the arterial pole, the anterior SHF-derived mesoderm supplies the myocardium of the right ventricle up to the right side of the ventricular septum.The SHF also contributes to the semilunar valves and the walls of the great arteries.Progenitors of SHF cells are marked by the transcription factor Isl1 [23].Stages of human cardiac development with color-coding of contributing cell populations, seen from a ventral view.These contributions are from the first heart field (red), second heart field (yellow) and cardiac neural crest (blue).By day 15 post-fertilization, the cardiac crescent is specified to form specific segments of the linear heart tube, which is patterned along the anteroposterior axis to form the looped and mature heart.Cardiac neural crest cells populate the aortic arch arteries (III, IV, and VI) and aortic sac (AS) that together contribute to specific segments of the mature aortic arch.Mesenchymal cells form the cardiac valves from the conotruncal (CT) and atrioventricular valve (AVV) segments.Abbreviations: A, atrium; V, ventricle; RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; PA, pulmonary artery; Ao, aorta; DA, ductus arteriosus.Modified from [24]. ",
"section_name": "Cardiac Mesoderm (FHF and SHF)",
"section_num": "2.2."
},
{
"section_content": "Neural crest cells form a wide variety of cells types, including nervous tissue, melanocytes, cartilage, bone, connective tissue and smooth muscle.CNC cells are a population of multipotent neural crest cells that originate from the dorsal neural tube at the interface of the ectoderm and neural ectoderm during embryogenesis.They migrate bilaterally into the ectoderm and endoderm of the pharyngeal arches as each arch develops and proliferate with the pharyngeal arteries to form the smooth muscle layer of the carotid artery (3rd pharyngeal arch), upper limb arteries and aortic arch (4th pharyngeal arch), and pulmonary arteries (6th pharyngeal arch). A subpopulation of CNC cells continues caudal migration to the anterior domain of the SHF to form the OFT [25] at about day 22 in the human, where they contribute to the aorta, main pulmonary artery, and aortic-pulmonary septum via the truncal cushions [26]; valve formation via the endocardial cushions; and perhaps separation of the ventricular myocardium from the aorta and pulmonary artery [26].The CNC cells at the base of the OFT lie in the medial walls of the arterial trunks, continuous proximally with the remnant of the aorto-pulmonary septum [27].The 4th and 6th pharyngeal arches are connected to the proximal OFT by the aortic \"sac\"-a non-muscular intra-pericardial connection that becomes the ascending aorta by septation of these arches; rotation of the OFT enables connection of the septated arches to their respective ventricles [28].Thus, septation of the elongated spiraling OFT occurs with fusion of the single aorto-pulmonary septum and enlarged endocardial cushions after significant CNC cell colonization of the truncal cushions. CNC cells also provide signals that shape the developing heart.For example, they pattern the aortic arch, ventricular and atrial septae, and OFT [29].Similar to requirements for the EMT process, Wnt, FGF and BMP are necessary for CNC cell migration and programming.Disruption of the endothelin, semaphorin, Wnt/β-catenin-Pitx2, Notch, TGF-β, BMP and Hedgehog pathways results in septal and OFT defects. ",
"section_name": "Cardiac Neural Crest Cells",
"section_num": "2.3."
},
{
"section_content": "The fate of cardiogenic mesoderm cells is shaped by their communication with the notochord and the pharyngeal endoderm of the embryo.The endocardium in particular plays a crucial role in several steps of the development of the early heart, including cell specification and guiding cell migration.It is thought that mammalian endocardial and myocardial cells are derived from EMT of a common multipotent progenitor within the cardiac mesoderm [30,31].The endocardium plays two key roles during OFT morphogenesis: population and proliferation of the endocardial cushions, and regulation of OFT myocardial proliferation.In response to signaling from the conal OFT myocardium, the endocardium undergoes EMT to give rise to a population of mesenchymal cells that seed the initially acellular endocardial cushions, which then remodel into the arterial valves [32]. Signaling between CNC and SHF cells is required for OFT development [33,34], and removal of SHF progenitors results in OFT defects [35].These observations show the importance of multiple extra-cardiac and cardiac cell lineages needed for OFT development.Migration of extra-cardiac lineages into the heart and OFT requires precise temporal and spatial regulation and signaling [36]. ",
"section_name": "Myocardial and Endocardial Cell Contributions",
"section_num": "2.4."
},
{
"section_content": "The OFT extends from ventricular myocardium to the margin of the pericardial cavity and is made up of the infundibulum (conus) nearer the right ventricle, and the proximal aorta and pulmonary artery (truncus) nearer the branchial arches [37,38].The lumen of the OFT is continuous with that of the aortic sac of the pharyngeal arch arteries. The OFT is derived from CNC and FHF cells that give rise to endocardium and myocardium, as well as cells derived from the SHF, and from EMT of endothelial cells (Figure 3) [30,36,38].Additionally, luminal endothelial cells of the OFT vessels undergo EMT to contribute to the endocardial cushions that, together with the migrating CNC cells, form the OFT septum [36].The distal OFT originates from CNC-derived mesenchyme cells [39,40], whereas the proximal OFT originates from endocardial-derived mesenchyme [41].During early embryogenesis, CNC cells migrate ventrally as mesenchymal cells to populate the distal OFT, where they merge with SHF and endocardial cells to form the aorto-pulmonary septum [39].This septum divides the initially single embryonic OFT (also called truncus arteriosus) into aortic and pulmonary arteries, establishing separate systemic and pulmonary circulations. Much of the development of the OFTs is confused by naming conventions of structures present during embryogenesis and by different interpretations of anatomical, histologic and lineage-tracing models in different species [42].It also seems likely that some of the observations in quail-chick models are not applicable to primate embryogenesis and that mouse models offer better insight.Nonetheless, there are some well-established principles to cell origin and specification in the OFT.vascular smooth muscle derived from the second heart field (dotted yellow) and the location of myocardium from derived from the second heart field (striped yellow).The aortic annulus or hinge region is formed where myocardial cells meet the vascular smooth muscle cells of the media of the aorta and pulmonary trunk and endothelial derived mesenchyme is the source of the fibroblastic annular tissue.The media of the aorta and pulmonary trunk is derived from secondary heart field proximally (dotted yellow) and the cardiac neural crest distally (blue).The interface between these populations is at the sinotubular junction.Abbreviations: Ao, aorta; AS, aortic sac; AVC, aorto-ventricular cushions; LV left ventricle; PT, pulmonary trunk; RV, right ventricle.Modified from [36] and [27] ",
"section_name": "Development of the OFT",
"section_num": "3."
},
{
"section_content": "Cells from the branchial mesenchyme, the paired 3rd/4th and 6th pharyngeal arteries, project into the pericardial cavity as an aortic sac.The 3rd and 4th pharyngeal arteries eventually form the aortic arch, while the initially paired 6th pharyngeal arteries fuse to form the pulmonary artery and ductus arteriosus.When the OFT is first formed, it has exclusively muscular walls that extend to the borders of the pericardial cavity, where the tract becomes continuous with the aortic sac.This sac is initially separated from the truncus but elongates to fuse with the distal truncus.Within the lumen, the endocardial jelly concentrates into pairs of facing cushions that continuously extend the length of the OFT, spiraling around one another as they run from the distal end of the right ventricle to the aortic sac.The cushions are proteoglycan-rich structures that eventually become populated by three mesenchymal cell populations: CNC-, SHF-and EMT-derived cells.The core of the protrusion is derived from the SHF, but it is covered by material derived from the neural crest [43]. Septation of the OFT requires formation of the truncal and conal cushions situated in the distal and proximal OFT, respectively.CNC cells form the mesenchyme of the truncal (right-superior and left-inferior) cushions that subsequently fuse to form the aortopulmonary septum, dividing the distal OFT into the aorta and pulmonary trunk [7].Separation of the aortic and pulmonary trunk (aorto-pulmonary septation) occurs when the distal portions of the truncus swellings gradually fuse together in a cranial to caudal direction, to septation.By day 23-25 of human embryogenesis, the heart tube is formed and anterograde circulation begins, with development of the arterial pole over the next 5 days [20].Septation of the aorto-pulmonary trunk results in the formation of the definitive intrapericardial pulmonary and aortic outflow channels, each of which possesses three segments, namely, the intrapericardial arterial trunks distal to the sinotubular junction, the arterial roots with their valves, and the subvalvar ventricular OFT.Separation of the aorta and pulmonary artery is completed by expansion of the aorto-pulmonary septum into the mesenchymal tissue of the proximal truncus swelling, separating the arterial valves (Figure 4).Mouse and chick studies have shown that abnormal EMT resulting in either deficient or excessive OFT cushion formation can give rise to structural OFT defects [36]. ",
"section_name": "Development of the Aorto-Pulmonary Trunk",
"section_num": "3.1."
},
{
"section_content": "The boundary at the dog-leg bend in the OFT identifies the border of the primordial OFT destined to become the sinotubular junction [44].The valves and their supporting sinuses develop from the proximal part of the tract.Aortic and pulmonary valve development begins at approximately days 31-35 in humans, from the endocardial cushions in the OFT and atrioventricular canal of the primitive heart tube [45].In early stages of arterial valve development, endothelial cells overlying the primitive endocardial cushions invade the cushion matrix, resulting in relatively bulky and cellularized endocardial cushions [46]. The semilunar valves arise from the conotruncal and intercalated cushions of the OFT, with the conotruncal cushions give rise to the right and left leaflets of each of the valves.In the aorta, these are the right and left coronary leaflets, while in the pulmonary valve, these are the right and left cusps (Figure 5).From the opposite quadrants of the OFT, the right-posterior and the left-anterior intercalated cushions develop respectively into the posterior aortic (non-coronary cusp) and the anterior pulmonic (anterior cusp) leaflets.These semilunar valve leaflets also derive their mesenchyme primarily from the endocardium [47].The muscular wall of the proximal OFT does not have a cellular contribution to the valve leaflets or their supporting arterial sinuses, but does contribute to their growth in paracrine fashion.The rudimentary arterial valve leaflets and sinuses are formed by creating \"cavities\" in the fused distal parts of the proximal endocardial cushions, along with similar cavities in the new cushions through apoptosis and alterations in the extracellular matrix (Figure 5) [48].Cavitation of the cushions results in a central lumen of each cushion that separates the three valve leaflets, with the peripheral portion arterializing to form the wall of the supporting valve sinuses.Valvulogenesis continues with elongation and thinning of the endocardial cushions by remodeling and compartmentalizing into the collagen-rich fibrosa, proteoglycan-rich spongiosa, and elastin-rich atrialis/ventricularis layers [46].The extracellular matrix composition and organization of the valve leaflets are critical for normal valve function, and dysregulation of extracellular matrix remodeling or structural components can lead to valve malformations [49]. ",
"section_name": "Development of the Aortic and Pulmonary Valves",
"section_num": "3.2."
},
{
"section_content": "The gene regulatory network of valve progenitor cells in the endocardial cushions includes the complex spatial and temporal interplay of transcription factors involved in EMT and mesenchymal progenitor populations [50].Endocardial cushion formation is initiated by signals from the OFT myocardium that cause adjacent endocardial cells to undergo EMT.Many transcription factors and signaling pathways have been implicated in EMT and cushion morphogenesis, including members of the TGF-β superfamily, Notch, BMP and Gata families, Nfatc1, Wnt/β-catenin, Twist-1, Sox9 and others (Figure 6).The complexity of cardiac structure and functioning cells types developed from an apparently few embryonic cell types is surprising.Similarly, the marked localization of single-gene defects to specific cardiac structures appears at odds with the presence of many of these proteins in numerous cell types of the heart and other organs [5].Ascertaining the timing and relative expression of these transcription factors is difficult and has been complicated by misinterpretation of results from older studies using less robust techniques and non-human models.Nevertheless, an expanding list of transcription factors appear to have important roles, or at least are well described.These factors overlap with genes that have been implicated in BAV, but to a limited extent. ",
"section_name": "Transcriptional Regulation of Valvulogenesis",
"section_num": "4."
},
{
"section_content": "Signaling factors, particularly from the Notch and TGF-β families, facilitate EMT and mesenchymal invasion of the OFT cushions [51].The Notch pathway is involved in the majority of cardiac embryogenesis, notably including valve development [52].Notch1, 2 and 4 receptors and their ligands (Jag1/2 and Dll4) are expressed in the OFT and cushions throughout valvulogenesis [51,[53][54][55].When activated, the Notch intracellular domain (NICD) translocates to the nucleus where it creates a complex of NICD, Rbpj, and the inhibitor MAML leading to activation of Notch target genes.In the embryonic valve, HES, HEY, Acta2, Snai2, Smad3, and Runx3 are direct Notch target genes [56].Notch activation also inhibits TGF-β-induced Smad1 and Smad2 signaling, subsequently decreasing expression of their target genes [57].Underscoring the importance of NOTCH, its inactivation or mutation leads to multiple forms of CHD, such as BAV [15], Alagille syndrome [58,59] and hypocellular endocardial cushions [57,60]. Several mouse models have been generated for Notch1/2/4 and their downstream effectors to examine the functions of Notch signaling during valve development.In mice, Notch1/2/4, Rbpj and Maml knockouts are embryonic lethal, with affected embryos having OFT and other cardiac and non-cardiac abnormalities [51,57,61], whereas Notch3 knockouts do not have an abnormal phenotype [62].Demonstrating the complexity of pathways in aortic disease, haploinsufficiency of Notch1 on a mouse NOS3 -/-background causes marked aortic phenotypes, whereas each mutation alone has very limited phenotypic changes [63][64][65].Interestingly, single-gene knockouts of the cardiac ligands of Notch-Jag1, Jag2 and Dll4-also do not show valve abnormalities, perhaps indicating redundant signaling.Importantly, Notch has been implicated in mouse and human calcific aortic valve disease (CAVD), a common form of heart valve disease [66][67][68]. ",
"section_name": "Notch",
"section_num": "4.1."
},
{
"section_content": "BMPs 2-7 and TGF-βs are part of the TGF-β superfamily expressed in the embryonic heart.BMPs bind to cardiac BMP Type I receptors-ALK2 (activin-like kinase) and ALK3-and Type II receptors-BMPRII, ActRII or ActRIIB-that activate an intracellular canonical signaling pathway mediated by Smad4 [69,70].Similarly, three TGF-β ligands (TGF-β 1/2/3) bind to cardiac Type I (ALK5) and Type II (TGFBRII) receptors to activate canonical TGF-β signaling [69] mediated by Smad2 and Smad3.This transcriptional pathway in conjunction with Smad inhibitory proteins regulates transcription of TGF-β superfamily-responsive genes [71].BMP, Notch and TGF-β promote EMT, mesenchymal cell invasion into the cardiac cushions and remodeling of the valves [69,72].Specific to formation of the arterial valves, TGF-β signaling plays an essential role in the initial promotion and cessation of EMT, and in cushion mesenchyme proliferation and in differentiation during heart valve development. BMP, TGF-β and Notch are not only activators of these processes, but also transcriptional repressors via the Snail family of proteins that are required for the initial steps in EMT and cushion development [73].As BMP, TGF-β and Notch all target Snail expression, Snail is likely a critical point of convergence for their signaling in valve development.In the adult heart, abnormal BMP signaling has been found in CAVD [74,75], implicating it in the pathogenesis of this condition.Numerous cardiac tissue-specific knock-down experiments have established the importance of the BMP pathway in the development of the endocardial cushions and valve development [76][77][78].In contrast, cardiac tissue-specific knock-down of TGF-β1 yields little structural defect.Knock-down of TGF-β2 does produce structural OFT, septal and aortic arch defects [79,80], along with hypercellular cardiac cushions and valves [81].Important to the initiation and progression of CAVD, TGF-β signaling is involved in the activation of valvular interstitial cells and their transformation to myofibroblasts in the adult valve [82]. ",
"section_name": "BMPs and TGF-β",
"section_num": "4.2."
},
{
"section_content": "The Gata family of zinc-finger transcription factors binds the \"GATA\" nucleotide motif and plays an essential role in cardiac development [83].With relevance to the aortic valve, Gata4 and Gata5 are expressed in endocardial cells, endocardial cushions and OFT, and Gata6 is expressed in CNC cells [84].Abnormalities in mice with GATA mutations indicate that interaction between Gata4 and Gata5 is necessary for the endocardial cushions to develop, as GATA4 +/-GATA5 +/-double heterozygotes have enlarged semilunar valves compared with single GATA4 +/-and GATA5 +/- heterozygotes [84].Gata4 mutant mice and humans show septal defects, including both atrial and ventricular septal defects [8,[85][86][87][88]. ",
"section_name": "Gata",
"section_num": "4.3."
},
{
"section_content": "In the embryonic heart, Nfatc1 is expressed before EMT of the OFTs and is seen in endocardial cells, but not in transformed mesenchymal cells of the endocardial cushions [41].It is also expressed after rudimentary valve formation, enabling elongation of the valve leaflets through degradation of the extracellular matrix of the endocardial cushions by enzymes such as cathepsin K [89].Nfatc1-deficient (nfatc1 -/-) mice have normal endocardial cushion formation but fail to begin normal remodeling of the endocardial cushion for arterial valve development, and this deficiency is embryonic lethal [90]. In addition to its role in valve development, Nfatc1 also controls NFκB ligand (RANKL), a member of the tumor necrosis factor ligand family that is a key factor for osteoclastogenesis and that signals through its receptor RANK to promote differentiation of bone-resorbing osteoclasts [91].The transcription factor Nfatc1 transduces signals from RANKL for differentiation of osteoclast-mediated bone resorption [91] by controlling expression of β3-integrin [92].Thus, common defects involving Nfatc1 may be at play in BAV, CAVD and ascending aortic aneurysm. ",
"section_name": "Nuclear Factor in Activated T-Cell, Cytoplasmic 1",
"section_num": "4.4."
},
{
"section_content": "The homeodomain factor NKX2-5 is the most commonly mutated single gene in human CHD, accounting for 1%-4% of specific malformations, including cardiac conduction and OFT abnormalities as well as atrial and ventricular septal defects.However, lack of mutational genotype-phenotype correlation points to an early role for this factor in development of the FHF and SHF, and possible phenotypic modification by downstream genes [93].Although Nkx2-5 is widely expressed in the developing heart, its most significant role appears to be in SHF development, specifically, directing SHF specification and morphogenesis of SHF-derived cardiac structures [94,95].Nkx2-5 represses Bmp2/Smad1 signaling and regulates SHF proliferation and OFT morphology [94], and appears to require Mef2c for its action [96].However, for a gene associated with so much human CHD, we know little about its mechanisms of action in cardiac development, especially considering its importance in late natal and post-natal cardiac development [97]. ",
"section_name": "Nkx2-5",
"section_num": "4.5."
},
{
"section_content": "The first non-human BAV was observed in NOS3 deficient mice, where the normal expression of NOS3 expressed in aortic valve endothelium of adult mice was not observed [63].Structural cardiovascular abnormalities of the aorta were not observed in NOS3 -/-mice except when combined with haploinsufficiency of NOTCH1 [65].The role of NOS3 in adult development of aortic aneurysm, coarctation or aortic valve stenosis is not clear as aortic wall NOS expression is lower in bicuspid aortic aneurysm [98] and in the aortic root [99] but not associated with aortic stenosis or aneurysm in NOS3 -/-mice [100]. ",
"section_name": "Nitric Oxide",
"section_num": "4.6."
},
{
"section_content": "The mature aortic valve has cusps composed of three overlapping layers of highly organized extracellular matrix, embedded with valve interstitial cells and covered by a layer of valve endothelial cells [101].The three layers are the fibrosa, spongiosa and ventricularis (Figure 6).The fibrosa, which is located on the arterial aspect of arterial valves, is composed predominantly of fibrillar collagens (Types I and III) that are circumferentially oriented and provide tensile stiffness [46][47][48][49].The ventricularis layer, located on the ventricular side of the arterial valves, is composed primarily of radially oriented elastic fibers that facilitate tissue motion [50,51].The middle layer, the spongiosa, is composed primarily of proteoglycans with interspersed collagen fibers.Proteoglycans, which are also present in the other layers, serve as an interface between the orthogonally arranged fibrosa and ventricularis layers to provide tissue compressibility and integrity. Like other forms of congenital heart disease (CHD), although chromosomal (DiGeorge and Turner syndrome) and Mendelian (NOTCH1) causes of BAV have been identified, these account for a small percentage of BAV cases [102].The genetic mechanisms underlying the majority of \"sporadic\" cases of BAV are not known.Even for these seemingly sporadic cases, epidemiological studies have demonstrated a roughly 10% increased risk of BAV in siblings and offspring, and a similar figure for the occurrence of aortic aneurysm in relatives with, or without, BAV, indicating the potential role of shared genes or even environmental causes [103,104]. The genetic architecture of sporadic BAV likely includes: (i) accumulation of rare and uncommon variants in cardiac developmental genes leading to a mutational load favoring BAV formation; (ii) epigenetic modification of cardiac developmental genes; (iii) common variants in genes that may not be obviously linked to cardiac development but have impact on their function; and perhaps; (iv) environmental causes.Complicating this scenario, many CHD-causing gene variants are likely selected against by a decrease in reproductive fitness.The resulting allelic heterogeneity reduces the power of GWAS for CHD.This may or may not be an issue for BAV, as its effect on reproductive fitness is thought to be limited.Nevertheless, these issues collectively make the discovery of the etiology of BAV difficult (Table 1).BAV is commonly associated with aneurysm of the aortic sinuses, the ascending aorta, and aortic coarctation.Pathological examination of the BAV-associated aortic aneurysm often shows non-inflammatory degeneration of smooth muscle cells often described as cystic medial necrosis [105][106][107].The co-occurrence of BAV with aortic aneurysm and coarctation of the aorta may imply a common genetic mechanism for these diseases.Dysregulation of the canonical TGF-β signaling axis in humans offers one such example.As described above, TGF-β signaling is a key pathway promoting EMT in valvulogenesis and cellular migration, activities critical to normal valvular development.Mutations in the canonical TGF-β signaling pathway may therefore be hypothesized to cause an enhanced rate of BAV.The human condition Loeys-Dietz syndrome (LDS) is caused by mutations in the genes encoding the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), the TGF-β ligands 2 and 3 (TGFB2 and TGFB3), as well as in the downstream transducer of TGF-β signaling, SMAD3.BAV is frequently encountered in patients with LDS, as well as a nearly complete penetrance of thoracic aortic aneurysm [108,109].While overt TGF-β dysregulation may be an uncommon mechanism for typical BAV-associated thoracic aortic aneurysm, minor variation in other pathways that overlap functionality between valvulogenesis and arterial development may be common in patients with BAV-associated thoracic aortic aneurysm.While one commonly encounters arguments that aortic aneurysms are solely the result of abnormal flow patterns around the valve, these arguments cannot properly account for observed individual variation and the lack of correlation between hemodynamic derangement and aneurysm size seen in BAV patients.Therefore it seems likely that genetic variation in addition to hemodynamic factors plays a major role in aneurysm susceptibility in typical BAV disease. As previously mentioned, BAV is the most common of congenital valvular defects and is characterized by a valve having only two commissures (Figure 7).It is commonly associated with aneurysm of the aortic sinuses and the ascending aorta, and aortic coarctation.Pathological examination of the BAV-associated aortic aneurysm often shows non-inflammatory degeneration of neural crest-derived smooth muscle cells often described as cystic medial necrosis [105][106][107].Nevertheless, experimental evidence supporting a common underlying developmental mechanism to explain the association of aortic valve and associated aortopathy is lacking.Type III: fusion between the LCC and NCC to create a fused left-non-coronary cusp (LNC).Both Type II and Type III BAVs result from fusion of the posterior aortic interacted disc with either the inferior (Type II) or superior (Type III) septal cushion.These differences in anatomy imply differences in etiologic mechanisms [110]. Human BAV has been associated with chromosomal variation in DiGeorge syndrome (22q11.2del ) and Turner syndrome (Xp del ).The genetic basis of structural valve abnormalities in DiGeorge syndrome has been attributed to hemizygosity of TBX1 and impaired downstream signaling by Fgf8 [111,112], but the association is imperfect.The cause of BAV, often with coarctation, in 30% of women with Turner syndrome is unknown, although good evidence has implicated a haploinsufficient region lying distal to Xp11.4 [113][114][115].The very high incidence of BAV in Turner women compared with XY men favors existence of autosomal modifier genes for this valvular disease and perhaps a homologous Y chromosomal gene [4].BAV has also been associated with the primary ciliopathies, such as Joubert syndrome [116], and with ventricular non-compaction [117,118].Using linkage, loci within the 5q15-21, 13q33-qter and 18q22.1 chromosomal regions have been identified [4,119], but the responsible genes have not. Smaller abnormalities such as copy number variation, indels and single-nucleotide polymorphisms almost certainly have some responsibility for BAV in the general population, especially in sporadic cases not associated with extensive non-valvular phenotypes.Variants in NKX2-5 (5q34) [120,121], NOTCH1 (9q34.3)[15,16,122,123], FBN1 (15q21.1)[124], MATR3 (5q31.2) [125] and GATA5 (20q13.33)[126][127][128] have been observed in individuals with BAV.However, some of these associations may result from co-existing disease, such as association of FBN1 with ascending aortic disease [129].Unfortunately, none of these findings provide epiphanous insight into the causes of BAV. Study of the cause(s) of BAV is a good example of the limitations of disease mapping using genetic variation to explore human complex disease (Table 1).There may be significant cost and time advantages to discerning the cause of BAV and its co-existing diseases using vertebrate models of the condition.Despite limitations in animal models of human disease, targeted \"knock-in\" candidate gene mutations in mice [130] and zebrafish [131] may provide useful insight by modeling human BAV better than gene knockout mouse models do [130].These individual SNPs or small insertion/deletion mutations are far more frequently observed in human disease than whole-exome or whole-gene deletions.In addition, these smaller variants more accurately replicate human disease and thus have greater potential applicability.The value of large or whole-gene deletion is principally confined to identifying pathways of disease rather than the exact molecular biology of disease.Although these techniques are a necessary albeit limited step, they cannot explain biological functions of the candidate genes identified.Culturing of embryonic and aortic valve interstitial cells will be needed to validate these targets and identify their relationships to subsequent CAVD and aortic disease [3,130]. ",
"section_name": "Etiology of BAV Disease",
"section_num": "5."
},
{
"section_content": "In this review, we have described the series of events from cellular and transcriptional embryogenesis of the heart, to development of the aortic valve.The frequent occurrence of BAV and its anatomically discrete but common co-existing diseases (ascending aortic disease, aortic stenosis and coarctation of the aorta) leads us to suspect a shared cellular origin.However, the extensive cell-cell signaling and interactions of cardiac embryogenesis preclude simplistic explanations of this disease. ",
"section_name": "Summary",
"section_num": "6."
}
] |
[
{
"section_content": "This work was supported by a National Institutes of Health grants 1R01HL114823 (SCB), 1P30GM103342 (RAN), 8P20GM103444-07 (RAN), R01HL127692 (RAN, DM), American Heart Association 15GRNT25080052 (RAN). ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest",
"section_num": null
}
] |
10.3390/cells9061484
|
Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL
|
<jats:p>NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is considered an antigen-driven B-cell neoplasm, characterized by clonal expansion of mature CD5+ B-lymphocytes [1][2][3][4][5][6].Despite its clinical heterogeneity, a consistent feature of CLL cells is the overexpression of NOTCH1 and NOTCH2 [7][8][9][10].NOTCH1 is affected by gain-of-function mutations in a subset of CLL cases (10 to 15%), where it is considered to be an independent prognostic marker associated with disease progression [11][12][13][14][15][16][17].The high nuclear NOTCH2 activity is not only a hallmark of all CLL cases-where it is associated with the expression of the B-cell activation/differentiation marker CD23-but is also functionally linked with CLL cell viability [7,8,18]. The conserved NOTCH gene family (NOTCH1-4) encodes transmembrane receptors that regulate a wide variety of differentiation processes by modulating binary cell fate decisions in response to external signals [19][20][21][22].Canonical NOTCH signaling is induced by ligand binding, the intracellular domain of NOTCH (N IC ) is released by a series of proteolytic events involving γ-secretase followed by translocation to the nucleus, where it acts as context and cell type specific transcription factor on CSL (for CBF1, Suppressor of Hairless, and LAG-1)-responsive genes like FCER2 (CD23) in CLL cells [7,18,[20][21][22].However, non-canonical NOTCH signaling also exists and involves the activation of NFκB [23].In the murine system, Notch2 is implicated in the development of marginal zone (MZ) B2 B-cells and of Cd5+ (B-1a) B-lymphocytes [24], and is indispensable for CLL initiation in Cd5+ (B-1a) B-cells [25]. Deregulation of NOTCH signaling is observed in an increasing number of human neoplasms, where the individual NOTCH receptors act either as oncogenes or as tumor suppressors, depending on the cellular context and microenvironment [20,26,27].Therefore, targeting oncogenic NOTCH, for example with γ-secretase inhibitors (GSI), represents a promising therapeutic strategy in the treatment of NOTCH-associated tumors/leukemias [27][28][29][30][31].In a first attempt to address this issue, we found that the majority of CLL cases express GSI-resistant NOTCH2/CSL transcription factor complexes and did not respond to the selective GSI DAPT [18].In contrast, targeting nuclear NOTCH2 with the Aspergillum-derived NOTCH2/CSL transactivation inhibitor, gliotoxin efficiently induced apoptosis in CLL cells by a mechanism involving the induction of the NOTCH3 and the NR4A1 gene on the mRNA level [32].However, the global effect of gliotoxin on the complex and interconnected signal transduction pathways and the role of NOTCH3 in CLL cells remains to be determined. In the current study, we extended our prior work and compared the anti-neoplastic effects of gliotoxin and the GSI RO4929097 [29,31,33] in a reasonable cohort of well-characterized CLL cases.Here we show that the inhibition of NOTCH2 signaling by gliotoxin is associated with the recovery of a potentially non-canonical tumor suppressing NOTCH3 activity in CLL cells.Furthermore, assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) revealed that gliotoxin treatment is associated with prominent changes in the epigenetic landscape in CLL cells. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Heparinized peripheral blood was obtained from 33 CLL patients after signed informed consent (MUW-IRB approval numbers 495/2003, 11/2005, and 36/2007).Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) centrifugation.CLL cases were screened for characteristic CLL chromosomal aberrations by FISH analysis.The IGHV and NOTCH1 mutational status was determined by Sanger sequencing (LGC Genomics, Berlin, DE).The GSI sensitivity of nuclear NOTCH2 was determined by quantification of DNA-bound NOTCH2/CSL transcription factor complexes in CLL cells ±0.5 µM RO4929097 after one day of incubation using electrophoretic mobility shift assays (EMSA), essentially as described [18].The NOTCH2 (C651.6DbHN)antibody used for the supershift/interference assays was obtained from the Developmental Studies (San Diego, CA, USA).Flow cytometry was performed on a FACSCalibur TM using CellQuest Pro software (Becton Dickinson, San Jose, CA, USA).AnnexinV and propidium iodide staining was performed to estimate the percentages of cells undergoing apoptosis.Apoptosis was calculated as the sum of early apoptotic (Ax+/PI-) and late apoptotic/necrotic (Ax+/PI+) cells using a kit from eBioscience (San Diego, CA, USA).Cell viability/metabolic activity was evaluated by a nonisotopic MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay (Ez4U) (Biomedica, AT). ",
"section_name": "Patients' Characteristics and Sample Collection",
"section_num": "2.1."
},
{
"section_content": "Total RNA was extracted using the TRI Reagent ® isolation system (Sigma-Aldrich, St Louis, MO, USA).M-MLV reverse transcriptase and GoTaq PCR kits (Promega, WI, USA) were used for semi quantitative RT-PCR.The MYC primer sequences used in this study read as follows: forward 5'-GAAAACAATGAAAAGGCCCC-3' and reverse 5'-TTCCTTACGCACAAGAGTTC-3'.Primer sets for NOTCH1, NOTCH2, NOTCH3, FCER2, NR4A1, and ACTB were published elsewhere [32].PCR bands were stained with GelRedTM (Biotium, Fremont, CA, USA) and visualized using the ChemiDocTM gel imaging system from Bio-Rad (Hercules, CA, USA). ",
"section_name": "Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis",
"section_num": "2.4."
},
{
"section_content": "The siRNA duplexes (siRNAs) for NOTCH3 (ON-TARGETplusTM) and the controls (RISC-free Co-siRNA, and siGLO red transfection indicator) were obtained from Dharmacon (Lafayette, CO, USA).Transfection of siRNAs into the CLL cells was performed by using the lipid reagent siLentFect TM from Bio-Rad Laboratories (Hercules, CA, USA).The transfection efficiency was determined by FACS and varied from 70 to 90%. ",
"section_name": "Gene Silencing by RNA-Interference",
"section_num": "2.5."
},
{
"section_content": "Accessible-chromatin mapping on the CLL cells was performed using the ATAC-seq method with minor modifications together with the ATAC-seq processing pipeline, as described previously [34,35].Principle component analysis (PCA) was performed on the quantile-normalized log-transformed values of chromatin accessibility across all the accessible sites discovered in all samples, and DESeq2 was used to detect the differential regions between treatment timepoints across patients [36].Regions with an FDR-adjusted p-value smaller than 0.05 were selected and clustered using the Euclidean distance and complete linkage, from which two clusters representing the earliest branching point were extracted.HOMER [37] was used for de novo motif finding on the region clusters and LOLA [38] for enrichment in previously existing location-based datasets.Genes assigned to the two clusters of regions were enriched using the Enrichr tool [39]. ",
"section_name": "ATAC-Seq",
"section_num": "2.6."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Eighteen CLL cases (Table 1) were subjected to MTT assays and the dose-dependent effect of gliotoxin, RO4929097, and DAPT (0.01 to 10µM) on cell viability was determined after 3 and 7 days of incubation. After 3 days, gliotoxin efficiently decreased CLL cell viability (IC50 between 0.1 µM and 1 µM), whereas RO2949097 and DAPT were only partly effective in a subset of patient samples, including those cases characterized by the NOTCH1∆CT mutation (Figure 1A-C).The mean inhibition of CLL cell viability (±SD) in the NOTCH1 wild type versus NOTCH1∆CT mutated CLL cases at an inhibitor concentration of 0.5 µM after 3 days was as follows: 84% (±23%) versus 87% (±16%) for gliotoxin, 14% (±13%) versus 36% (±14%) for RO4929097, and 10% (±14%) versus 33% (±16%) for DAPT, respectively.After 7 days, the response to RO4929097 was more prominent and 11 out of the 18 treated CLL cases (61%) reached IC 50 levels at a drug concentration of 0.5 µM (Figure 1E) and, thus, were phenotypically considered as GSI sensitive.The NOTCH1∆CT mutated CLL cases and/or CLL cases expressing the GSI-sensitive (GSI-S) nuclear NOTCH2 clustered in this GSI-sensitive group.In contrast, CLL cases expressing GSI-resistant (GSI-R) nuclear NOTCH2 and with the wild type (wt) NOTCH1 status clustered in the GSI-resistant group.Maximal responses were already achieved at 0.1 µM RO4929097 and increasing amounts did not significantly enhance its effect on the CLL cells.The drop in CLL cell viability at higher drug concentrations (10 µM) after this plateau phase might be attributed to off-target effects (Figure 1B,E) [40].The mean inhibition of CLL cell viability (±SD) in the NOTCH1wT versus NOTCH1∆CT mutated CLL cases at an inhibitor concentration of 0.5 µM after 7 days was as follows: 95% (±6%) versus 92% (±5%) for gliotoxin, 40% (±20%) versus 54% (±7%) for RO4929097, and 22% (±17%) versus 38% (±12%) for DAPT, respectively. ",
"section_name": "Dose and Time-Dependent Effects of Gliotoxin and GSI on CLL Cell Viability",
"section_num": "3.1."
},
{
"section_content": "The observed delayed effect of GSI on CLL cell viability in the MTT assays suggests that GSI primarily affect the metabolic activity rather than directly inducing apoptosis.Therefore, we measured the percentage of apoptotic CLL cells (n = 16) after exposure to equal doses (0.5 µM) of gliotoxin, RO4929097, and DAPT by AnnexinV/PI staining. After 3 days, gliotoxin significantly induced apoptosis in all CLL samples (p < 0.001), whereas the effects of GSI were moderate (Figure 2A).After 7 days, GSI had a variable effect on CLL cell apoptosis (Figure 2B).RO4929097 increased apoptosis in CLL cases expressing GSI-S nuclear NOTCH2 (p = 0.003; n = 7) and, surprisingly, decreased apoptosis in CLL cases expressing GSI-R nuclear NOTCH2 (p = 0.001; n = 9) (Figure 2C), irrespective of the NOTCH1 mutational status (Figure 2D).Interestingly, the anti-apoptotic effect of GSI was restricted to CLL samples derived from Rai/Binet I/A and II/B patients (CLL2, CLL7, CLL8, CLL9, CLL11, CLL12, CLL13, CLL15, and CLL17), whereas a pro-apoptotic effect of GSI was mainly observed in CLL samples derived from Rai/Binet IV/C patients (5 out of 7; CLL6, CLL10, CLL14, CLL16, and CLL20). ",
"section_name": "GSI Inhibited Spontaneous Apoptosis in Early Clinical Stage-Derived CLL Samples Expressing GSI Resistant Nuclear NOTCH2",
"section_num": "3.2."
},
{
"section_content": "We next confirmed in a time kinetic RT-PCR experiment in two additional representative CLL ",
"section_name": "The Inhibition of Spontaneous Apoptosis by RO4929097 is Associated with Inhibition of Recovered NOTCH3 mRNA Expression in CLL Cells",
"section_num": "3.3."
},
{
"section_content": "We next confirmed in a time kinetic RT-PCR experiment in two additional representative CLL samples (CLL21, 22) our previous observation that gliotoxin inhibited NOTCH2, to a lesser extent NOTCH1, and induced NOTCH3 mRNA expression within 4 h of incubation (Figure 3A) [32].Interestingly, RO4929097 counteracted gliotoxin-induced NOTCH3 transcription after one day (Figure 3A).This suggests that RO4929097 interrupted a positive feedback loop of NOTCH3 mRNA expression.NOTCH receptors regulate context and cell type specific their own expression and each other in positive and negative feedback loops [19].Moreover, RO4929097 decreased (58 versus 64%) or increased (86 versus 79%) the effect of gliotoxin on the percentage of apoptotic cells, depending on the GSI sensitivity of nuclear NOTCH2 (Figure 3A).Therefore, we asked whether NOTCH3 might also account for the GSI-mediated inhibition of spontaneous apoptosis in early clinical stage-derived CLL long-term suspension cultures. NOTCH3 mRNA was not detectable in frozen samples from freshly isolated CLL cells of our initial drug screening cohort (Figure 3C).After 7 days, however, the anti-apoptotic effect of RO4929097 in two representative Rai/Binet I/A patient samples expressing GSI-R nuclear NOTCH2 (CLL8 and CLL9, Figure 3B) was clearly associated with inhibition of spontaneously recovered NOTCH3 mRNA expression, together with unchanged or even enhanced NOTCH2 mRNA and NOTCH2/CSL DNA-complex expression (Figure 3C, left panel).with a decrease in NOTCH2 mRNA and NOTCH2/CSL DNA-complex expression without any detectable NOTCH3 gene activity after 7 days in culture (Figure 3C, right panel).In accordance with published data [9,16], the NOTCH1 mRNA was GSI sensitive in all cases and was more expressed in the Rai/Binet IV/C-derived CLL cells (Figure 3C).Together, this combined approach suggests that recovery of NOTCH3 mRNA expression is involved in the inhibition of spontaneous apoptosis by GSI in CLL long-term suspension cultures and may be associated with GSI resistance of NOTCH2 and early stage derived CLL samples. ",
"section_name": "The Inhibition of Spontaneous Apoptosis by RO4929097 is Associated with Inhibition of Recovered NOTCH3 mRNA Expression in CLL Cells",
"section_num": "3.3."
},
{
"section_content": "We next analyzed NOTCH3 and FCER2 (CD23) expression in CLL cells (n = 4) in relation to spontaneous as well as gliotoxin induced apoptosis on the mRNA and protein level by RT-PCR and FACS (Figure 4; see Supplemental Figure 1 for a detailed FACS analysis of CLL24 cells).NOTCH3 was almost undetectable on the mRNA (Figure 4A) and on the cell surface protein level (Figure 4B) in freshly isolated CLL cells.After 4 days in culture, we found an increase of surface NOTCH3 expression together with a decrease in surface CD23 on CLL23 and CLL24 cells (Figure 4B).On the mRNA level, NOTCH3 expression was below the detection limit in the 4 days control.The loss of CD23 expression seemed to be a prerequisite for NOTCH3 expression since NOTCH3 was primarily detected on CD23-negative CLL cells.In contrast, the induction of apoptosis by RO4929097 in two representative Rai/Binet IV/C patient samples expressing GSI-S nuclear NOTCH2 (CLL10 and CLL16, Figure 3B) was associated with a decrease in NOTCH2 mRNA and NOTCH2/CSL DNA-complex expression without any detectable Cells 2020, 9, 1484 8 of 18 NOTCH3 gene activity after 7 days in culture (Figure 3C, right panel).In accordance with published data [9,16], the NOTCH1 mRNA was GSI sensitive in all cases and was more expressed in the Rai/Binet IV/C-derived CLL cells (Figure 3C). Together, this combined approach suggests that recovery of NOTCH3 mRNA expression is involved in the inhibition of spontaneous apoptosis by GSI in CLL long-term suspension cultures and may be associated with GSI resistance of NOTCH2 and early stage derived CLL samples. ",
"section_name": "Induction of Surface NOTCH3 Expression by Gliotoxin is Associated with Downregulation of CD23 and Increased Apoptosis of CLL Cells",
"section_num": "3.4."
},
{
"section_content": "We next analyzed NOTCH3 and FCER2 (CD23) expression in CLL cells (n = 4) in relation to spontaneous as well as gliotoxin induced apoptosis on the mRNA and protein level by RT-PCR and FACS (Figure 4; see Supplemental Figure S1 for a detailed FACS analysis of CLL24 cells).NOTCH3 was almost undetectable on the mRNA (Figure 4A) and on the cell surface protein level (Figure 4B) in freshly isolated CLL cells.After 4 days in culture, we found an increase of surface NOTCH3 expression together with a decrease in surface CD23 on CLL23 and CLL24 cells (Figure 4B).On the mRNA level, NOTCH3 expression was below the detection limit in the 4 days control.The loss of CD23 expression seemed to be a prerequisite for NOTCH3 expression since NOTCH3 was primarily detected on CD23-negative CLL cells. Cells 2020, 9, x FOR PEER REVIEW 9 of 19 which might be attributed to the loss of the inhibitory effect of these drugs on CD23 over time (Figure 4A,B).Gliotoxin induced the NOTCH3 gene (Figure 4A), enhanced NOTCH3 surface expression, and upregulated apoptosis in a dose-dependent manner in all cases (Figure 4B).The remaining living CLL cells were enriched for CD23-positive and NOTCH3-negative cells (Figures 4B,C andS1), confirming that CD23 expression is associated with CLL cell viability whereas NOTCH3 expression is associated with CLL cell apoptosis. In summary, we found a direct correlation between the percentage of surface NOTCH3-positive and apoptotic CLL cells and an indirect correlation of these two parameters with the percentage of CD23-positive and living CLL lymphocytes (Figure 4D).In contrast, we detected a low expression of CD23 on freshly isolated cells from Duvelisib (CLL25) or Ibrutinib (CLL26) treated CLL patients (Figure 4B).However, FCER2 (CD23) expression spontaneously recovered on the mRNA and protein level in these 2 samples after 4 days in culture which might be attributed to the loss of the inhibitory effect of these drugs on CD23 over time (Figure 4A,B). ",
"section_name": "Induction of Surface NOTCH3 Expression by Gliotoxin is Associated with Downregulation of CD23 and Increased Apoptosis of CLL Cells",
"section_num": "3.4."
},
{
"section_content": "Gliotoxin induced the NOTCH3 gene (Figure 4A), enhanced NOTCH3 surface expression, and upregulated apoptosis in a dose-dependent manner in all cases (Figure 4B).The remaining living CLL cells were enriched for CD23-positive and NOTCH3-negative cells (Figure 4B,C and Figure S1), confirming that CD23 expression is associated with CLL cell viability whereas NOTCH3 expression is associated with CLL cell apoptosis. In summary, we found a direct correlation between the percentage of surface NOTCH3-positive and apoptotic CLL cells and an indirect correlation of these two parameters with the percentage of CD23-positive and living CLL lymphocytes (Figure 4D). ",
"section_name": "Targeting NOTCH3 Signaling Decreased NR4A1 mRNA Expression and Counteracted Gliotoxin Induced Apoptosis in CLL Cells",
"section_num": "3.5."
},
{
"section_content": "We hypothesized that NOTCH2 and NOTCH3 have opposite roles in the binary cell fate decision between positive and negative selection of the activated CLL cells.Therefore, we investigated the effects of targeting NOTCH3 by RO4929097, or more specifically, by siRNA in PMA-stimulated CLL cells [32].In this model, CLL cells form tight clusters and express the CLL proliferation center marker MYC (Figure 5B), resembling the situation found in lymphoid tissues [41,42].To avoid background effects on NOTCH2 signaling, we selected five CLL cases expressing GSI-R NOTCH2 (CLL7, 8, 9, 13, 21). A time kinetic confirmed that CD23 was expressed on almost all representative CLL9 cells after one day of PMA stimulation (Figure 5A) [18].Interestingly, prolonged stimulation with PMA for 3 days led to the downregulation of CD23, upregulation of NOTCH3, and an increase in the percentage of apoptotic CLL9 cells.This effect was clearly enhanced by gliotoxin treatment (Figure 5A). As expected, gliotoxin induced the NOTCH3/NR4A1 axis, downregulated the NOTCH2/FCER2 (CD23) axis, and inhibited the NOTCH2/CSL transcription factor complex within 4 h of incubation (Figure 5B, left panel) [32].In accordance with the FACS data, the NOTCH3/NR4A1 axis was also upregulated in PMA-stimulated CLL9 cells after 3 days in culture without gliotoxin treatment (Figure 5B, right panel).Neither gliotoxin nor PMA induced DNA-bound NOTCH3/CSL complexes in EMSA.RO4929097 inhibited the NOTCH3/NR4A1 axis, upregulated the NOTCH2/FCER2 (CD23) axis, and enhanced the NOTCH2/CSL transcription factor complex (Figure 5B, right panel), which stands in sharp contrast to the effect of gliotoxin.The NOTCH1 target gene MYC was downregulated by RO4929097 (Figure 5B, right panel) [9,[43][44][45]. The opposite effects of gliotoxin and RO4929097 on the NOTCH2/FCER2 (CD23) axis and the NOTCH3/NR4A1 axis were reflected by the opposite effects of these compounds on CLL cell viability.As shown in Figure 5C, gliotoxin remarkably induced apoptosis (n = 5, mean% ± SD: 85 ± 5% versus 23 ± 6%), while RO4929097 significantly decreased spontaneous (10 ± 2% versus 23 ± 6%; p = 0.009) as well as gliotoxin-induced apoptosis (53 ± 9% versus 85 ± 5%; p = 0.001) in CLL cells. NOTCH3 gene silencing by siRNA decreased the apoptotic effect of the gliotoxin, leading to a 3.5-fold increase in (Ax-/PI-) living CLL9 cells with increased surface CD23 expression after 3 days in culture (Figure 5D).Corresponding RT-PCR analysis confirmed that NOTCH3 gene silencing downregulated the NOTCH3/NR4A1 axis and upregulated the NOTCH2/FCER2 (CD23) axis (Figure 5E), resembling the effect of GSI treatment (Figure 5B, right panel).Interestingly, gliotoxin-mediated upregulation of the NR4A1 gene was completely blocked by an NFκB activation inhibitor (Figure 5F), suggesting that NOTCH3 regulates NR4A1 transcription via non-canonical NOTCH3/NFκB signaling [23,46,47].This would explain the lack of DNA-bound NOTCH3/CSL complexes in EMSA.All other control experiments show similar trends.Collectively, these data strongly suggest that inhibition of the anti-apoptotic canonical NOTCH2/CSL signaling (NOTCH2/FCER2 axis) by gliotoxin recovers a GSI sensitive pro-apoptotic non-canonical NOTCH3 function which may involve NFκB dependent NR4A1 expression (NOTCH3/NR4A1 axis) in CLL cells.A hypothetical model summarizing the proposed counteracting roles of NOTCH2 and NOTCH3 in CLL cells is given in Figure 5G. ",
"section_name": "Targeting NOTCH3 Signaling Decreased NR4A1 mRNA Expression and Counteracted Gliotoxin Induced Apoptosis in CLL Cells",
"section_num": "3.5."
},
{
"section_content": "We have recently shown that genome-wide mapping of gene-regulatory elements using the transposase-accessible chromatin (ATAC-seq) assay is a useful tool to investigate gene regulation in CLL cells [35].Therefore, we analyzed gliotoxin-induced chromatin changes in CLL cells after 3 days of incubation (n = 7, Figure 6A), where we identified 62,760 unique chromatin accessible regions (Figure S2A).These sites represent mainly enhancers and promoters (Figure S2B, a representative locus spanning the CXCR4 gene region is shown in Figure 6B).Unsupervised principal component analysis (PCA) confirmed that IGHV mutation status is the major source of heterogeneity in chromatin accessibility in CLL cells, as described previously (Figure 6C, left panel) [35].However, ",
"section_name": "Gliotoxin Modulates Chromatin Accessibility at Gene Regulatory Elements Containing Potential NOTCH/CSL and NR4A1 Binding Sites",
"section_num": "3.6."
},
{
"section_content": "We have recently shown that genome-wide mapping of gene-regulatory elements using the transposase-accessible chromatin (ATAC-seq) assay is a useful tool to investigate gene regulation in CLL cells [35].Therefore, we analyzed gliotoxin-induced chromatin changes in CLL cells after 3 days of incubation (n = 7, Figure 6A), where we identified 62,760 unique chromatin accessible regions (Figure S2A).These sites represent mainly enhancers and promoters (Figure S2B, a representative locus spanning the CXCR4 gene region is shown in Figure 6B).Unsupervised principal component analysis (PCA) confirmed that IGHV mutation status is the major source of heterogeneity in chromatin accessibility in CLL cells, as described previously (Figure 6C, left panel) [35].However, Principle Components 1 and 4 clearly revealed dose-dependent changes in chromatin accessibility in response to gliotoxin treatment (Figure 6C, right panel). Clustering of significantly changing regions (Figure 7A) segregated the sites into those that lose accessibility (Region Cluster 1, marked in blue) and those that gain accessibility (Region Cluster 2, marked in orange) in response to gliotoxin treatment (representative genomic loci of each cluster are shown in Figure 7B).By overlapping the differential ATAC-seq sites with publicly available transcription factor binding experiments [38], we observed that Region Cluster 1 largely overlaps with the functional NOTCH/CSL binding sites in T-cell acute leukemia cells (Figure S3A).On the other hand, Region Cluster 2 largely overlaps with the functional NFκB binding sites in lymphoblastoid cell lines (Figure S3B). To get unbiased insights into the potential regulators of differentially accessible regions we carried out de novo motif analysis to discover the enriched DNA binding elements.Differential accessible gene-regulatory elements in Region Cluster 1 were found to be enriched for the promoter-associated motif GFY (general factor Y) and the recognition site for ZNF143, both of which contain the NOTCH/CSL consensus binding site TGGGAA [48].In Region Cluster 2, we found an enrichment of consensus sites for the transcription factors ATF3, PU.1, NFκB, FLI1, NR4A1 (nur77), and RUNX (Figure 7C).Clustering of significantly changing regions (Figure 7A) segregated the sites into those that lose accessibility (Region Cluster 1, marked in blue) and those that gain accessibility (Region Cluster 2, marked in orange) in response to gliotoxin treatment (representative genomic loci of each cluster are shown in Figure 7B).By overlapping the differential ATAC-seq sites with publicly available transcription factor binding experiments [38], we observed that Region Cluster 1 largely overlaps with the functional NOTCH/CSL binding sites in T-cell acute leukemia cells (Figure S3A).On the other hand, Region Cluster 2 largely overlaps with the functional NFκB binding sites in lymphoblastoid cell lines (Figure S3B). To get unbiased insights into the potential regulators of differentially accessible regions we carried out de novo motif analysis to discover the enriched DNA binding elements.Differential accessible gene-regulatory elements in Region Cluster 1 were found to be enriched for the promoterassociated motif GFY (general factor Y) and the recognition site for ZNF143, both of which contain the NOTCH/CSL consensus binding site TGGGAA [48].In Region Cluster 2, we found an enrichment of consensus sites for the transcription factors ATF3, PU.1, NFκB, FLI1, NR4A1 (nur77), and RUNX (Figure 7C). By linking the differentially accessible regions to their nearest gene, we found that Region Cluster 1 maps toward the genes involved in the regulation of the actin cytoskeleton (ITGB1, ACTN4, MSN, SSH1, and others), HIF-1/2-alpha signaling (VEGFA, HMOX1, TRFC, PLCG2, and others) and RAP1 signaling (RAPGEF1, RAP1A, VAV2, and others) (Figure 7D, see Table S1).Region Cluster 2 was enriched for genes involved in B-cell activation/differentiation (LYN, IRF4, BCL6, ETV6, ARID3A, POU2F2, THEMIS2, IL21R, and others), NFκB signaling (TRAF1-3, RELB, CARD11, and others), TGFß signaling (TGFB1, SMAD3, PML, and others), and apoptosis (FASL, TP73, BBC3, BID, MIR34A, TNFRSF8, GZMB, and others) (Figure 7D, see Table S2).By linking the differentially accessible regions to their nearest gene, we found that Region Cluster 1 maps toward the genes involved in the regulation of the actin cytoskeleton (ITGB1, ACTN4, MSN, SSH1, and others), HIF-1/2-alpha signaling (VEGFA, HMOX1, TRFC, PLCG2, and others) and RAP1 signaling (RAPGEF1, RAP1A, VAV2, and others) (Figure 7D, see Table S1).Region Cluster 2 was enriched for genes involved in B-cell activation/differentiation (LYN, IRF4, BCL6, ETV6, ARID3A, POU2F2, THEMIS2, IL21R, and others), NFκB signaling (TRAF1-3, RELB, CARD11, and others), TGFß signaling (TGFB1, SMAD3, PML, and others), and apoptosis (FASL, TP73, BBC3, BID, MIR34A, TNFRSF8, GZMB, and others) (Figure 7D, see Table S2). ",
"section_name": "Gliotoxin Modulates Chromatin Accessibility at Gene Regulatory Elements Containing Potential NOTCH/CSL and NR4A1 Binding Sites",
"section_num": "3.6."
},
{
"section_content": "In the last decade, much effort has been spent into the development of GSIs as tools for therapy for NOTCH-associated human neoplasias [29][30][31].However, four NOTCH family members are present in mammalian cells, where the individual receptors may have opposite functions concerning their role as oncogenes or tumor suppressors in a context-and microenvironment-dependent manner [26,30,31].Moreover, transformed cells may express truncated NOTCH forms that do not depend on γ-secretase for processing and function [49,50].A search for an alternative to GSI revealed that the Aspergillum-derived secondary metabolite gliotoxin is a potent inhibitor of DNA-bound NOTCH2/CSL transcription factor complexes, and efficiently induced apoptosis in CLL lymphocytes and in NOTCH2-associated solid tumor cell lines-independent of their sensitivity to GSIs [32,51] In this study, we show that gliotoxin as a nuclear NOTCH2 inhibitor may have an additional therapeutic advantage over GSI as pan-NOTCH inhibitors in CLL.We were able to demonstrate that the GSI RO4929097 targets an unexpected non-canonical tumor-suppressing NOTCH3 activity, which is involved in spontaneous as well as gliotoxin-induced apoptosis in CLL cells. On the CLL cell surface, upregulation of NOTCH3 was associated with downregulation of CD23, suggesting that downregulation of the NOTCH2/FCER2 (CD23) axis, either spontaneously due to the lack of appropriate activation stimuli in vitro [18], or by gliotoxin treatment [32], is a prerequisite for NOTCH3 expression in CLL cells.The strong association between surface NOTCH3 expression and apoptosis suggests that NOTCH3 signaling is involved in the execution phase of CLL cell apoptosis by prolonging the expression of the newly identified NOTCH3 target gene NR4A1, a multi-functional tumor-suppressor gene implicated in the regulation of B-cell tolerance to self-antigens [52][53][54]. ATAC-seq confirmed that gliotoxin targets canonical NOTCH signaling as indicated by reduced accessibility at potential NOTCH/CSL consensus sites (TGGGAA) [48].This includes the promoter-associated motif GFY, and the consensus site for ZNF143, a transcription factor known to modulate NOTCH target gene expression in competition with CSL [55].In contrast, gliotoxin increased the chromatin accessibility at the potential ATF3, PU.1, NFκB, FLI1, RUNX, and NR4A1 binding sites in the vicinity of genes involved in B-cell activation, differentiation, and apoptosis.This might reflect, at least in part, the loss of a NOTCH2-mediated differentiation arrest and the induction of non-canonical NOTCH3/NFκB signaling [23].It has been shown that NOTCH3 activates NFκB, a positive regulator of the NR4A1 gene [46], through an IKKα-dependent alternative pathway [47].This would not only explain the gain in chromatin accessibility at NFκB and NR4A1 consensus sites and the lack of DNA-bound NOTCH3/CSL complexes in response to gliotoxin treatment, but also the NOTCH3-dependent NR4A1 gene activity in CLL cells.In line with this assumption, we found that inhibition of NFκB counteracted gliotoxin-mediated upregulation of NR4A1 mRNA in CLL lymphocytes.However, this important issue needs further mechanistic exploration. NOTCH2 and NOTCH3 signaling antagonize each other in different cell systems [56][57][58][59], suggesting that these NOTCH receptors also have opposite functions in the antigen-dependent regulation of CD5+ (B-1a) B-cell homeostasis. Under physiological conditions, NOTCH2 signaling might be induced by ligand-expressing surrounding cells in order to protect the proliferative/regenerative reservoir of CD5+ B-cells from NR4A1-mediated activation-induced cell death (AICD) [32,52,60].This scenario might take place in the marginal zone (MZ) of the spleen [61], where DLL1-expressing bystander cells have been identified [62].In contrast, apoptotic NOTCH3 signaling might counteract the uncontrolled expansion of CD5+ B-cells in the periphery.In line with this hypothesis, we found that NOTCH3 inhibition by RO4929097, or more specifically, by siRNA, downregulated the NOTCH3/NR4A1 axis, enhanced the NOTCH2/FCER2 (CD23) axis, and counteracted apoptosis in CLL cells.The NOTCH3 gene is frequently epigenetically silenced in B-cell acute lymphocytic leukemia (B-ALL) cells, pointing to a broader tumor-suppressor role of NOTCH3 in B-cells [63].Moreover, the CLL downregulated/deleted MicroRNA-16 [64] has been shown to exert its pro-apoptotic function by NOTCH2 inhibition in pre-eclampsia, where an inverse correlation between NOTCH2 and NOTCH3 expression also has been found [59]. In terms of CLL biology, constitutive active NOTCH2 might dominantly suppress apoptotic NOTCH3 signaling, thereby enabling the (self-) antigen-driven progredient expansion of the malignant clone.The affinity and avidity of the corresponding (self-) antigens might be the basis for the NOTCH2 dominance seen in CLL cells.A short time exposure to B-cell activation mimetic PMA favors the NOTCH2/FCER2 (CD23) axis, whereas long-term stimulation with PMA shifts the NOTCH2/FCER2 (CD23) axis to the NOTCH3/NR4A1 axis in CLL cells.One likely mediator of this bi-phasic PMA effect might be the activation and subsequent downregulation of the B-cell receptor-associated protein kinase C-delta (PKC-δ) [65][66][67], a positive regulator of nuclear NOTCH2 activity [10,18,27,68].Therefore, compounds that interfere with B-cell activation might affect the expression of both NOTCH receptors in CLL cells.This would explain why CLL cells pre-treated with Duvelisib (a dual PI3K-δ/γ inhibitor) [69] or Ibrutinib (a Bruton's tyrosine kinase inhibitor) [2,44] express lower amounts of CD23 and NOTCH3 and are less sensitive to apoptosis. The additional effect of NOTCH1 signaling in this scenario is less clear.NOTCH1 is not detectable in nuclear NOTCH/CSL transcription factor complexes in CLL cells [7,8,18,32].However, NOTCH1 is frequently mutated and/or overexpressed in advanced stage CLL cells, where it has a CLL-driving role by regulating MYC expression [8, 9,16,17,[43][44][45].In this context, NOTCH1 may indirectly account for the relative GSI sensitivity of NOTCH2, keeping in mind that active NOTCH1 is a positive regulator of the NOTCH2 gene in CLL cells (Figure 5G) [12]. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In summary, we show that the nuclear NOTCH2 inhibitor gliotoxin has global effects on the NOTCH signaling network in CLL cells, including the recovery of a newly identified non-canonical tumor suppressing NOTCH3 activity.This proof-of-concept may be the basis for the design of innovative therapies aimed at specifically targeting oncogenic NOTCH signaling in CLL cells. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following are available online at http://www.mdpi.com/2073-4409/9/6/1484/s1, Figure S1: Detailed FACS data of CLL24 cells, Figure S2: Genomic distribution and characteristics of open chromatin sites in CLL cells in response to gliotoxin treatment, Figure S3: Overlap of differential ATAC-seq sites with public transcription factor binding datasets in gliotoxin treated CLL cells, Table S1: ATAC-seq gliotoxin response region cluster 1, Table S2: ATAC-seq gliotoxin response region cluster 2. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": ". ID Age Gender Rai/Binet Stage IGHV Status Cytogenetic Alterations NOTCH1 Mutations NOTCH2 GSI-R/S* Treatment CLL1 76 male II/B U, VH2-5 normal N1∆CT GSI-S no CLL2 69 male II/B U, VH1-69 normal N1∆CT GSI-R no CLL3 70 male IV/C M, VH4-34 Tri12 wt ND no CLL4 64 male II/B M, VH3-23 13q-, 17p-wt ND no CLL5 51 female IV/C U, VH1-69 normal wt ND no CLL6 56 male IV/C M, VH3-21 13q-, 11q-wt GSI-S no CLL7 68 male II/B M, VH3-48 13q-, 11q-wt GSI-R no CLL8 84 male I/A NA normal wt GSI-R no CLL9 81 female I/A M, VH3-15 13q-wt GSI-R no CLL10 73 female IV/C M, VH3-23 normal wt GSI-S no CLL11 66 female I/A M, VH3-48 13q-wt GSI-R no CLL12 70 male I/A M, VH1-8 13q-wt GSI-R no CLL13 66 female II/B M, VH3-23 13q-wt GSI-R no CLL14 75 female IV/C NA 13q-wt GSI-S no CLL15 65 male I/A U, VH1-69 14q32-wt GSI-R no CLL16 52 male IV/C U, VH1-69 normal wt GSI-S no CLL17 55 male II/B U, VH3-11 13q-, 11q-wt GSI-R Ibrutinib CLL18 40 female I/A U, VH3-20 normal wt GSI-S no CLL19 68 female III/B U, VH1-2 13q-N1∆CT GSI-S no CLL20 60 male IV/C U, VH1-46 13q-, 11q-N1∆CT GSI-S no CLL21 52 male II/B NA 13q-ND GSI-R no CLL22 70 female II/B M, VH3-13 13q-ND GSI-S no CLL23 54 male I/A U, VH3-53 normal ND ND no CLL24 77 female IV/C NA 17p-ND ND no CLL25 54 male I/A U, VH4-39 11q-ND ND Duvelisib CLL26 69 male II/B U, VH3-21 13q-ND ND Ibrutinib CLL27 70 male II/B M, VH3-13 13q-ND ND no CLL28 61 female I/A M, VH3-7 normal ND ND no CLL29 77 female II/B M, VH3-74 normal ND ND no CLL30 87 female IV/C M, VH3-11 13q-ND ND Idealisib CLL31 68 female II/B M, VH3-48 13q-ND ND no CLL32 83 female II/B M, VH4-59 13q-ND ND Ibrutinib CLL33 60 male II/B U, VH1-69 13q-/11q-ND ND Idealisib ",
"section_name": "Pat",
"section_num": null
},
{
"section_content": "Funding: This research was funded by the Austrian National Bank, \"Jubilaeumsfonds\"-Grant No. 13012; by the \"Initiative Krebsforschung\", UE71104017, UE1504001, and UE711043037; by a Clinical Research Grant of the Austrian Society of Hematology & Oncology, AP00359OFF and by the WKO price 2017. The authors kindly thank Christoph Bock for his technical support concerning ATAC-seq. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by the Austrian National Bank, \"Jubilaeumsfonds\"-Grant No. 13012; by the \"Initiative Krebsforschung\", UE71104017, UE1504001, and UE711043037; by a Clinical Research Grant of the Austrian Society of Hematology & Oncology, AP00359OFF and by the WKO price 2017. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors kindly thank Christoph Bock for his technical support concerning ATAC-seq. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.31083/j.jin2302034
|
Astragalus Polysaccharide Promotes Neuronal Injury Repair via the Notch1/NFκB Signaling Axis in the Ventroposterior Thalamic Nucleus in Rats with Focal Cerebral Ischemia
|
<jats:p>Background: Ischemic stroke is the most common form of stroke and the second most common cause of death and incapacity worldwide. Its pathogenesis and treatment have been the focus of considerable research. In traditional Chinese medicine, the root of Mongolian astragalus has been important in the treatment of stroke since ancient times. Astragalus polysaccharide (APS) is a key active ingredient of astragalus and offers therapeutic potential for conditions affecting the neurological system, the heart, cancer, and other disorders. However, it is not yet known how APS works to protect against ischemic stroke. Methods: Rats were subjected to middle cerebral artery occlusion (MCAO) to imitate localized cerebral ischemia. Each of four experimental groups (normal, sham, MCAO, and MCAO+APS) contained 12 adult male Sprague-Dawley (SD) rats selected randomly from a total of 48 rats. Following successful establishment of the model, rats in the MCAO+APS group received intraperitoneal injection of APS (50 mg/kg) once daily for 14 days, whereas all other groups received no APS. The Bederson nerve function score and the forelimb placement test were used to detect motor and sensory function defects, while Nissl staining was used to investigate pathological defects in the ventroposterior thalamic nucleus (VPN). Immunohistochemical staining and Western blot were used to evaluate the expression of Neurogenic locus notch homolog protein 1 (Notch1), hairy and enhancer of split 1 (Hes1), phospho-nuclear factor-κB p65 (p-NFκB p65), and nuclear factor-κB p65 (NFκB p65) proteins in the VPN on the ischemic side of MCAO rats. Results: APS promoted the recovery of sensory and motor function, enhanced neuronal morphology, increased the number of neurons, and inhibited the expression of Notch1/NFκB signaling pathway proteins in the VPN of rats with cerebral ischemia. Conclusion: After cerebral ischemia, APS can alleviate symptoms of secondary damage to the VPN, which may be attributed to the suppression of the Notch1/NFκB pathway.</jats:p>
|
[
{
"section_content": "Ischemic stroke refers to cerebrovascular diseases that are caused by obstruction of cerebral artery blood flow leading to local brain tissue hypoxic-ischemic necrosis and to neurological defects.It is the second-leading cause of death and disability worldwide, with an increasing incidence [1,2].Neuropathological studies have shown that the ipsilateral thalamus suffers secondary injury following middle cerebral artery occlusion (MCAO), which may result in thalamic shrinkage and reduced sensorimotor function after ischemia [3].In a prior study, we also discovered that after cerebral ischemia, ventroposterior thalamic nucleus (VPN) neurons were harmed and diminished, which may be a significant contributing cause to the inadequate recovery of neurological function in clinical stroke patients. Astragalus is the root of the leguminous plant astragalus mongholicus, and has long been used in Traditional Chinese medicine (TCM).It is the principal ingredient in the Buyang Huanwu decoction, a traditional TCM remedy for treating strokes.Previous pharmacological studies have shown that astragalus polysaccharide (APS), a key active ingredient of astragalus, can reduce the inflammatory response and immune dysfunction caused by cerebral ischemic injury, while also enhancing the index of immune organs and inhibiting the release of pro-inflammatory cytokines [4,5].The Notch signaling axis is one of the most crucial pathways contributing to the long-term survival of neurons.Hairy and enhancer of split 1 (Hes1) and nuclear factor-κB (NFκB) are downstream target genes in this pathway that are induced by activation of Notch signaling [6].The expression level of Hes1 correlates directly with the expression of Neurogenic locus notch homolog protein 1 (Notch1) [7].NFκB is a key transcription factor that induces cell proliferation and differentiation, inflammatory immunity, and apoptosis, as well as playing a significant part in the pathological process of ischemic stroke [8,9].The current study used a rat model of MCAO.APS was administered and its neuroprotective effect on secondary injury to the VPN after cerebral ischemia was investigated in this model, together with its effect on the Notch1/NFκB signaling pathway.We hope that the findings provide experimental support for the clinical use of APS. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Specific pathogen free (SPF) healthy male Sprague-Dawley (SD) rats (n = 48) with a body weight of 200 ± 20 g were purchased from the Qinglongshan Animal Experimental Center [SCXK, (Hangzhou, Zhejiang, China), 2019-0002].Animals were placed in an experimental animal room of the Wannan Medical College and maintained at 24 ± 2 °C with a light/dark cycle of 12 h/12 h.Free feeding and drinking were allowed, and adaptive feeding was undertaken for one week.Following that, 12 rats were randomly assigned to each of four groups: normal, sham, MCAO, and MCAO+APS.The Laboratory Animal Welfare and Ethics Committee of Wannan Medical College evaluated and approved these animal studies (WNMC-AME-2023134). ",
"section_name": "Animals",
"section_num": "2.1"
},
{
"section_content": "The MCAO model was established using the sutureoccluded method [10].Intraperitoneal injection with 1.5% pentobarbital sodium (2 mL/kg, AM00469, Beijing Chemical Reagent Company, Beijing, China) was first used to anesthetize rats.They were then fixed in a supine position on the operating table under a shadowless lamp and the neck skin was disinfected with iodophor.The layers of tissue were separated to expose the right common carotid artery (CCA) and a glass minute hand was used to carefully separate the vagus nerve and CCA.The separation was continued to the bifurcation of the internal carotid artery (ICA) and external carotid artery (ECA), the CCA and distal ECA were ligated, and a spare wire was set up at the proximal end.Ophthalmic scissors were used to make a tiny incision in the ECA so that a threaded plug could be inserted (A2-2432, Beijing Cinonech Co. Ltd, Beijing, China).The ECA was then cut off and the thread was looped into the ICA through the stump of the ECA.The insertion depth was 18.5 ± 0.5 mm until slight resistance was noted.The tip of the cord was passed through the beginning of the middle cerebral artery and the cord ligated.The incision was then sutured to avoid infection during and after the operation.In the sham group, the blood vessels were separated without being ligated.The room temperature was kept at 22 °C during the operation to allow animals to recover from anesthesia under temperature-controlled conditions.After they awoke from surgery (usually about 2 hours), neurological impairment was evaluated according to the Bederson scoring criteria [11].Rats with scores of 1-3 were included in the study.Following successful establishment of the model, the MCAO+APS group was given an intraperitoneal injection of 50 mg/kg APS (purity >90%, A7970, Solarbio, Beijing, China) once a day for 14 consecutive days, as suggested in the literature [12].The study designs are displayed in Fig. 1A. ",
"section_name": "Establishment of the MCAO Model",
"section_num": "2.2"
},
{
"section_content": "Neurological impairment in rats was assessed 2 hours after surgery and on days 1, 3, 7, and 14.Neurological function was classified into four levels: 0, no neurological im-pairment; 1, the forepaw on the opposite side of the tail suspension test could not be fully extended; 2, the resistance of the opposite front paw to thrust decreased; 3, turned to the opposite side after placing. The forelimb placement test was used to assess the comprehensive ability of visual and proprioceptive perception of rats on days 3, 7, and 14.A square grid (3 cm × 3 cm) was placed horizontally at a height of 1 meter above ground.On the frame, rats were gently pushed from behind to encourage them to crawl from one side of the grid surface to the other within 2 minutes.During the crawling process, one point was awarded if the forelimbs fell into the grid.The number of times the hemiplegic forelimbs fell into the net during the 2-minute crawl was recorded. ",
"section_name": "Neurobehavioral Evaluation",
"section_num": "2.3"
},
{
"section_content": "Following treatment, 6 rats in each group were injected intraperitoneally with 1.5% sodium pentobarbital (3 mL/kg) and 4% paraformaldehyde was then infused before brain extraction.Brain tissue was fixed in 4% paraformaldehyde.After dehydration with a sucrose gradient, the tissue was embedded using optimal cutting temperature compound (OTC) in a -20 °C frozen microtome (HM525NX, Thermo Fisher Science, Waltham, MA, USA).It was then cut into 20 µm slices and washed three times with phosphate buffered saline (PBS) for 5 min each before use.Filtered Nissl staining solution was then added dropwise to the tissue slices containing the VPN, and these were placed in a 37 °C incubator for 30 minutes.Excess staining agent was washed off with double distilled water and the sections were dehydrated, made transparent and sealed.Images were observed and recorded at 400× magnification.The morphology and quantity of Nissl-positive neurons in each visual field was examined using ImageJ software (1.41r,National Institutes of Health, Bethesda, MD, USA). ",
"section_name": "Nissl Staining",
"section_num": "2.4"
},
{
"section_content": "Frozen sections of brain tissue containing VPN were baked in an incubator in a 60 °C incubator for 2 hours.H 2 O 2 (3%) was added to remove endogenous peroxidase and the slices were incubated for 10 minutes at 37 °C.To repair the antigen, the slices were boiled in 10 mM sodium citrate buffer (pH 6.0) for 2 minutes at high temperature and for 14 minutes at low temperature.Non-specific antibody binding was first blocked by incubating with 5% goat serum (AR0004, Boster Biotechnology, Wuhan, Hubei, China) for 30 min.Incubation with rabbit anti-Notch1 Affinity Biosciences, Cincinnati, OH, USA) was conducted at 4 °C.Horseradish peroxidase (HRP)-conjugated antirabbit IgG and 3,3 ′ -diaminobenzene (PA 110, TIANGEN Biotech, Beijing, China) were then used for staining and color development.The slices were permeated with xylene and sealed with neutral gum.ImageJ software was used to examine the expression of Notch1, Hes1, and phosphonuclear factor-κB p65 (p-NFκB p65) proteins and to obtain a positive cell count. ",
"section_name": "Immunohistochemical Staining",
"section_num": "2.5"
},
{
"section_content": "The remaining 6 rats from each group were anaesthetized by intraperitoneal injection with 1.5% pentobarbital sodium.After decapitation, the brain was removed and placed on an ice box.According to a Stereotaxic map of rat brain described by Paxinos et al. [13], a surgical blade was used on an ice box to cut 2.16 and 3.84 mm behind the anterior fontanel of the brain tissue to collect contains VPN area of the brain tissue, and then the VPN was extracted with microscopic tweezers as described in Fig. 1B.Proteins from the VPN were extracted using radio immunoprecipitation assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors.The sample protein (40 ug) was run for two hours on an sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis according to the molecular weight of the target protein. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane using a continuous flow of 300 mA for 1.5 hours.The membranes were blocked for two hours before incubation overnight at 4 °C with primary antibodies against Notch1, Hes1, p-NFκB p65, NFκB p65 (1:1000, AF5006, Affinity Biosciences, Cincinnati, OH, USA), and GAPDH (1:1000, AF7021, Affinity Biosciences, Cincinnati, OH, USA).Following this, the membranes were incubated with goat anti-rabbit IgG (H+L) secondary antibodies (1:1000, A0208, Beyotime Biotechnology, Shanghai, China) for 1 hour at room temperature, washed, then analyzed with an ECL kit (P0018FS, Beyotime Biotechnology, Shanghai, China).GAPDH was used as the internal reference for Notch1 and Hes1, and NFκB p65 was used as the reference for p-NFκB p65.The strip gray values were quantified by ImageJ software. ",
"section_name": "Western Blot",
"section_num": "2.6"
},
{
"section_content": "SPSS 26.0 software (IBM Corp., Chicago, IL, USA) was used for statistical analysis of the data, and GraphPad-Prism 9.0 (Dotmatics, Boston, MA, USA) for the creation of images.The measurement data were evaluated by a normality test and by a homogeneity test of variance.Data with a normal distribution were reported as the mean ± standard deviation.The K-W test was used to compare non-normally distributed data, with p < 0.05 used as the threshold for statistical significance. ",
"section_name": "Statistical Analysis",
"section_num": "2.7"
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The Bederson nerve function score revealed that rats in the normal group and in the sham operation group had no neurological function defects (Fig. 1C).In contrast, the MCAO group had severe neurological damage and considerably greater neurological impairment scores than those of the sham group at 2 hours after cerebral ischemia and at days 1, 3, 7, and 14 (p < 0.001).The neurological function scores of the MCAO+APS group were lower than those of the MCAO group on days 7 and 14 (p < 0.05). The forelimb placement test showed that rats in the normal and sham operation groups would grasp the grid line tightly when crawling, and almost never fall into the grid (Fig. 1D).Rats in the MCAO group showed a significantly higher number of hemiplegic forelimb falls into the grid on days 3, 7 and 14 compared to the sham group (p < 0.001).Rats in the MCAO+APS group showed significantly fewer hemiplegic forelimb falls into the grid on days 7 and 14 than those in the MCAO group (p < 0.05). ",
"section_name": "APS Induced the Restoration of Sensory and Motor Function in MCAO Rats",
"section_num": "3.1"
},
{
"section_content": "Nissl staining revealed that neurons in the VPN of rats in the normal and sham groups showed a complete morphology, structure, and quantity, with clear outline, centered nuclei, and complete Nissl bodies (Fig. 2A,B).However, in the MCAO group, the volume of neurons in the affected side of the thalamus was smaller, the edge of the cell membrane was blurred, and both necrolysis and nuclear pyknosis were observed.Furthermore, the number of Nissl-positive neurons was significantly lower in the MCAO group than in the sham group (p < 0.001).The morphology of neurons in the MCAO+APS group was mostly normal, and the number of Nissl-positive neurons was higher than in the MCAO group (p < 0.01). ",
"section_name": "APS Effectively Improved Neuronal Injury in the VPN",
"section_num": "3.2"
},
{
"section_content": "Immunohistochemistry was used to investigate the expression of Notch1, Hes1, and p-NFκB p65 in the right VPN of rats in each group (Fig. 3).Scattered positive cells showed a brown-yellow color.There were no discernible differences in expression between the normal and sham groups (p > 0.05).The MCAO group showed a higher number of positive cells for Notch1, Hes1, and p-NFκB p65 than the sham group (p < 0.001).However, the MCAO+APS group showed decreased expression of Notch1, Hes1, and p-NFκB p65 compared to the MCAO group (p < 0.05, p < 0.001, and p < 0.01, respectively). The results obtained with Western blot analysis mirrored those from immunohistochemistry (Fig. 4).The relative expression levels of Notch1 and Hes1 in the VPN were significantly higher in the MCAO group than in the sham group (p < 0.01), as was the expression of p-NFκB p65/NFκB p65 (p < 0.05).However, Notch1 and Hes1 expression in the VPN from the MCAO+APS group was significantly lower than that of the MCAO group (p < 0.01), while the expression of p-NFκB p65/NFκB p65 was also lower (p < 0.05). ",
"section_name": "APS Inhibited the Expression of Notch1, Hes1 and p-NFκB p65 in the VPN of MCAO Rats",
"section_num": "3.3"
},
{
"section_content": "The incidence of stroke continues to increase [14].However, therapeutic options for cerebral ischemia are very limited, and the recovery of neurological function in clinical stroke patients is often not ideal [15].The search for effective treatment methods and drugs for ischemic stroke has therefore been a popular area of research.The MCAO model used in this study was demonstrated to be successful by the increased nerve defect score observed in these rats.Following APS treatment, the neurological deficit function score in the MCAO+APS group of rats decreased.Moreover, they experienced less falls of hemiplegic forelimbs into the grid, suggesting that APS can promote early recovery of sensorimotor function in rats.A previous study using a rat model of MCAO showed that APS could inhibit neuroimmune disorders and inflammation induced by cerebral ischemic injury, reduce the extent of edema in brain cells, and play a protective role in brain tissue [12].This is consistent with the findings of the present study. According to the literature and to previous laboratory studies, it is important to treat secondary damage in the thalamus following cerebral infarction.The thalamus is the sensory center under the cortex.In rat models of MCAO, secondary damage to the thalamus leads to cognitive dysfunction and sensory defects after focal cerebral infarction, with the damage occurring in the VPN [3,[16][17][18].Nissl staining in the present study showed damage to the morphological structure of neurons in the VPN region of the MCAO group, as well as a reduced number of Nissl-positive neurons.However, the morphological structure of cells in the ischemic region of the MCAO+APS group showed significant improvement compared to the MCAO group, together with an increased number of Nissl-positive neurons.These results suggest that APS could help to attenuate the damage to neurons caused by cerebral ischemia. Inflammation occurs during the entire process of ischemic stroke, and the pathogenic mechanism of ischemic stroke injury is extremely complex [19].The generation of free radicals and the resulting oxidative stress that occurs during ischemic stroke can cause brain damage and activation of an inflammatory response.Both the Notch signaling pathway and NFκB are associated with the inflammatory response after ischemic stroke [20,21].The Notch intracellular domain is released into the cell and moves to the nucleus once the Notch signaling pathway is engaged, thereby inducing transcriptional activation and expression of Hes1 and NFκB [6].Hes1 is commonly used as a biomarker for Notch1 pathway activation [22].NFκB is a key transcription factor involved in the pathological development of ischemic stroke, cell proliferation and differentiation, as well as inflammatory immunity and apoptosis [14,23].During cerebral ischemia, gamma-secretase is activated immediately and the Notch1 level increases, causing an increased level of p65 subunit in the p50/p65 dimer of NFκB.This induces leukocyte infiltration and increases the secretion of inflammatory factors such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, which then escalate the inflammatory response in the neuron [24].Xu et al. [25] reported that Clematichinenoside could protect neurons in ischemic rats by inhibiting the Notch1/NFκB signaling pathway, thereby reducing the inflammatory response and the hence the extent of motor, learning and memory injury.The anti-inflammatory effect of Clematichinenoside is thought to be related to its suppression of Notch1/NFκB signaling pathway activation.Huang et al. [26] reported that APS causes liver cancer cell death by inhibiting the expression of Notch1.We predicted that the APS nerve protecting effect on cerebral ischemia rats would interfere with the Notch1/NFκB signaling pathway and so lessen inflammatory harm.In the present study, immunohistochemical and Western blot methods showed that Notch1, Hes1, and p-NFκB p65 expression levels in the VPN of MCAO experimental animals were higher than in the sham group.This indicates that thalamic ischemia may trigger activation of the Notch1/NFκB signaling pathway.However, the APStreated group showed lower expression levels for Notch1, Hes1, and p-NFκB p65 than the MCAO group.These findings imply that APS can reduce neuronal damage following cerebral ischemia, suppress Notch1/NFκB protein production in VPN during ischemia, and aid in the restoration of sensorimotor function in rats.Nevertheless, further experimentation is required to determine the optimal dosage of APS and to confirm that it prevents the release of downstream inflammatory factors in the Notch1/NFκB signaling pathway. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "APS can promote the recovery of motor and sensory functions and attenuate secondary thalamic injury in rats with cerebral ischemia.This effect may be linked to the inhibition of Notch1/NFκB protein expression, thereby reducing neuronal injury in the VPN region of cerebral ischemia.These findings provide an experimental foundation for further work on the clinical application of APS for ischemic stroke. ",
"section_name": "Conclusion",
"section_num": "5."
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgment",
"section_num": null
},
{
"section_content": "This work was supported by the Research Fund Project of Wannan Medical College (No.WK2022XS45), the Collegiate Major Natural Science Research Projects of Anhui Province (No.KJ2020A0603) and the Teaching and Research Project of Anhui Provincial Department of Education (2019jxtd072). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The datasets used and 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": "HL, HM, and WL conceived and designed the experiment.WL and ZN conducted the experiments and analyzed the data.WL drew up the first draft.FW and HM analyzed the data and revised the manuscript.HL gave final approval to the upcoming release.All authors contributed to editorial changes in the manuscript.All authors read and approved the final manuscript.All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. The animal studies were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Wannan Medical College (No.WNMC-AME-2023134). The authors declare no conflict of interest. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "HL, HM, and WL conceived and designed the experiment.WL and ZN conducted the experiments and analyzed the data.WL drew up the first draft.FW and HM analyzed the data and revised the manuscript.HL gave final approval to the upcoming release.All authors contributed to editorial changes in the manuscript.All authors read and approved the final manuscript.All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "The animal studies were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Wannan Medical College (No.WNMC-AME-2023134). ",
"section_name": "Ethics Approval and Consent to Participate",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflict of Interest",
"section_num": null
}
] |
10.1186/s12979-024-00415-6
|
B-1 derived anti-Thy-1 B cells in old aged mice develop lymphoma/leukemia with high expression of CD11b and Hamp2 that different from TCL1 transgenic mice
|
<jats:title>Abstract</jats:title><jats:p>Human old aged unmutated chronic lymphocytic leukemia U-CLL are the TCL1<jats:sup>+</jats:sup>ZAP70<jats:sup>+</jats:sup>CD5<jats:sup>+</jats:sup> B cells. Since CD5 makes the BCR signaling tolerance, ZAP70 increased in U-CLL not only TCL1<jats:sup>+</jats:sup> alone. In mice, TCL1 (TCL1A) is the negative from neonate to old aged, as TC<jats:sup>–</jats:sup>. V<jats:sub>H</jats:sub>8-12/V<jats:sub>k</jats:sub>21-5 is the anti-thymocyte/Thy-1 autoreactive ATA B cell. When ATA μκTg generation in mice, ATA B cells are the neonate generated CD5<jats:sup>+</jats:sup> B cells in B-1, and in the middle age, CD5<jats:sup>+</jats:sup> can be down or continuously CD5<jats:sup>+</jats:sup>, then, old aged CLL/lymphoma generation with increased CD11b in TC<jats:sup>–</jats:sup>ZAP70<jats:sup>–</jats:sup>CD5<jats:sup>–</jats:sup> or TC<jats:sup>–</jats:sup>ZAP70<jats:sup>+</jats:sup>CD5<jats:sup>+</jats:sup>. In this old aged TC<jats:sup>–</jats:sup>ATA B microarray analysis showed most similar to human CLL and U-CLL, and TC<jats:sup>–</jats:sup>ZAP70<jats:sup>+</jats:sup>CD5<jats:sup>+</jats:sup> showed certain higher present as U-CLL. Original neonate ATA B cells showed with several genes down or further increase in old aged tumor, and old aged T-bet<jats:sup>+</jats:sup>CD11c<jats:sup>+</jats:sup>, CTNNB1<jats:sup>hi</jats:sup>, HMGB<jats:sup>hi</jats:sup>, CXCR4<jats:sup>hi</jats:sup>, DPP4<jats:sup>hi</jats:sup> and decreased miR181b. These old aged increased genes and down miR181b are similar to human CLL. Also, in old age ATA B cell tumor, high CD38<jats:sup>++</jats:sup>CD44<jats:sup>++</jats:sup>, increased Ki67<jats:sup>+</jats:sup> AID<jats:sup>+</jats:sup>, and decreased CD180<jats:sup>–</jats:sup> miR15O<jats:sup>low</jats:sup> are similar to U-CLL. In this old aged ATA B, increased TLR7,9 and Wnt10b. TC<jats:sup>+</jats:sup>Tg generated with ATAμκTg mice occurred middle age tumor as TC<jats:sup>+</jats:sup>ZAP70<jats:sup>–</jats:sup>CD5<jats:sup>+</jats:sup> or TC<jats:sup>+</jats:sup>ZAP70<jats:sup>+</jats:sup>CD5<jats:sup>+</jats:sup>, with high NF-kB1, TLR4,6 and Wnt5b,6 without increased CD11b. Since neonatal state to age with TC<jats:sup>+</jats:sup>Tg continuously, middle age CLL/lymphoma generation is not similar to old aged generated, however, some increased in TC<jats:sup>+</jats:sup>ZAP70<jats:sup>+</jats:sup> are similar to the old age TC<jats:sup>–</jats:sup> ATA B tumor. Then, TC<jats:sup>–</jats:sup> ATA B old age tumor showed some difference to human CLL. ATA B cells showed CD11b<jats:sup>+</jats:sup>CD22<jats:sup>++</jats:sup>, CD24 down, and hepcidin Hamp2<jats:sup>++</jats:sup> with iron down. This mouse V8-12 similar to human V2-5, and V2-5 showed several cancers with macrophages/neutrophils generated hepcidin<jats:sup>+</jats:sup> iron<jats:sup>low</jats:sup> or some showed hepcidin<jats:sup>–</jats:sup> iron<jats:sup>+</jats:sup> with tumor, and mouse V8-12 with different V<jats:sub>k</jats:sub>19-17 generate MZ B cells strongly increased macrophage<jats:sup>++</jats:sup> in old aged and generated intestine/colon tumor. Conclusion, neonate generated TC<jats:sup>–</jats:sup>ATA B1 cells in old aged tumor generation are CD11b<jats:sup>+</jats:sup> in the leukemia CLL together with lymphoma cancer with hepcidin-related Hamp2<jats:sup>++</jats:sup> in B-1 cell generation to control iron.</jats:p>
|
[
{
"section_content": "In human and mice, the Lin28b + Let7 -axis plays a major role in fetal/neonatal development, whereas Lin28 -Let7 + cells are more important in adults [1].In old age, function of the adaptive immune system is diminished [2], whereas increased function of the innate immune system is more important such as myeloid cell differentiation and fetal/neonatal origin B-1 B cells.In old age in human, chronic lymphocyte leukemia (CLL) is the most common type of leukemia in Western countries.Human CLL is CD5 + B cells and is often found with mutated IGH gene (M-CLL) which has a significantly better overall survival than patients with unmutated (U-CLL) [3,4].U-CLL often found as IGHV1-69, which can originate in the fetal/neonatal stage from cord blood [5].Overall, CLL is lower in Africa than in Western countries, however CLL originating in Africa showed higher incidence of U-CLL with V1-69 than M-CLL [6].Since original fetal/neonate generation levels and adult B and T cell interaction with containing original B-1 cells, early origin background is important to old age in U-CLL. In mice, CLL is rarely observed.However, NZB mice show CLL generation [7] and NZB cells showed high NTA(natural thymocytotoxic autoantibodies) expression associated with CD5 + B cells [8].Most mice generate NTA expression, and SM/J and NZB mice express higher NTA than BALB/c and C57BL/6 [9].We first found NTA expression in SM/J mice at the immature T cell stage drives positive selection of B cells with high expression of V H 8-12/V k 21-5 BCR, which showed anti-thymocyte/ Thy-1 autoreactive (ATA) B cells [10].We then, generated ATAμκTg in C.B17 mice (BALB/c-Igh b ), in which ATA B (Igh a ) cells are the largely product of fetal/neonatal B cell development (B-1) as CD5 + B1a, normally constituting B-1 cell subset in adult, as B1 B cells [11].In adult B cell development (B-2) from BM, the expression of V H 8-12/ V k 21-5 BCR drives negative selection, resulting in developmental arrest [11].Then, these B-1 origin V H 8-12/ V k 21-5 ATA B cells can generate leukemia/lymphoma in old aged with increased expression of CD11b [12,13].This paper now showed with CD11b-CD22 ++ , and also found that very high level of hepcidin-Hamp2 in ATA B cells in old age. Mouse ATA B leukemia/lymphoma are TC -, α-Thy-1 ATA B cell, and Hamp2 ++ .These differences between human versus mice.In human CLL, TCL1 + positive and U-CLL CD5 + B cells with ZAP70 + .TCL1 (TCL1A,B) are expressed in the embryonic stage in mice and human, originally [14,15].Then, in mice, TCL1 (TCL1A) is down regulated in fetal liver and then negative in neonate, becoming TCL1 -through adulthood [16].In human, Pro-B, Pre-B, and immature B cells are TCL1 + , and in spleen, TCL1 is still positive in immature B cells, then decreased in mature B cells to minimal levels [17].Then, vascuration made to increase TCL1 in human cells and original B-1 unmutated CD5 + B cells can increased with TC + [18,19], also adult B cells can generated mutated CD5 + B cells with TC + .CD5 binds to SHR-1 related to the BCR, CD5 makes the BCR signaling tolerance [20,21].Thus, not only TCL1 + which BCR signaling hyperresponsive [22], ZAP70 + made more responses in human U-CLL for further response with autoimmune predisposition, showed TC + ZAP70 + CD5 + [23].In aged mice, B cells are continuously TCL1 -(TC -).Some original TC -CD5 + B1a cells can downregulate CD5 in aged [24,25].Thus, ATAμκTg B lymphoma/leukemia can be generated from TC -ZAP70 -CD5 -, or TC -ZAP70 + CD5 + (or CD5 -) B1 cells.We previously observed that transgenic expression of TCL1 + (TC + )Tg in ATA B cells induced CLL/ leukemia in middle aged mice [24], which originated from TC + ZAP70 -CD5 + or TC + ZAP70 + CD5 + (or CD5 -) B1 cells.Since human U-CLL is TC + ZAP70 + CD5 + , we further compared TC -and TC + Tg ATAμκTg mice by microarray analysis, to determine why TC + Tg mice showed lymphoma/leukemia development in middle age instead of old aged.In this paper, we found that RagKO ThyKO ATAμκTg mice which were TC -CD5 -Thy-1 - and lacked mature T and B cells, showed high ZAP70 + B1 cells with early state of CLL/lymphoma.In addition to being ZAP70 + , we found old aged generated ATA B TC -ZAP70 -CD5 -B1 cells with CD11b ++ , most of which were similar to old aged human TC + CLL/U-CLL cells. Thy-1(CD90) is heavily N-glycosylated, glycophosphatidylinositol anchored cell surface protein.Thy-1 controls inflammatory cell recruitment in human and mice [26,27], and Thy-1 plays an important role in the initial stages of virus infection [26].Fetal hepatocytes are important in Thy-1 + human and mice [28,29].However, human Thy-1 is not generate in T cells begining in thymus, which differs from mouse T cells [30].Thus, the original B-1 cells can generate ATA B cells in the thymus of mice [10] but not in human.However, Thy-1 is continuously important in human and mice with Thy-1 expressed on nerve cells (neuron) in brain, fibroblast, endothelial cells, monocytes, myeloid cells, and kidney [30][31][32], and serum Thy-1 is present.Thy-1 moves to endothelial cell to control the initial state of virus infection, and CD11b (Mac-1) helps to Thy-1 activated to endothelial move [33].In humans, this CD11b is mostly expressed by neutrophils/ monocytes (CD11b + Gr-1 + ) to move Thy-1 to endothelial tissues [33,34].Since Thy-1 is important in the nervous system of mice and human, when lacking Thy-1 in brain, strongly inhibited in the dentate gyrus [35].Mouse thymocyte autoantibodies from ATA B1 cells react with Thy-1 in thymus, then original peritoneal cavity pB1a with increased CD11b but not in spleen B1a, and in old age CD11b + ATA B cells, and then, ATA B cells exhibit multiple specificites [36]. Mouse ATA B cell lymphoma/leukemia express high levels of Hamp2 ++ which is hepcidin.Hepcidin is found as Hamp1 and Hamp2 in mice, and human hepcidin is similar to mouse Hamp1 [37].Hepcidin is an innate antimicrobial agent that is induced by most invasive bacteria and also by virus, then limits bacterial proliferation by reducing iron such as in plasma and extracellular fluids, and kills bacteria [38].Hamp2 is more reactive as an antibacterial and antivirus activity than Hamp1 [39,40].The most important function of hepcidin (also mouse both Hamp1 and Hamp2) is to regulate iron [41].Hepcidin is mostly produced by macrophages/monocytes, and TLR induced hepcidin by T cells and B cells [42].ATA B cell tumors in old aged show high Hamp2 expression and decreased iron.This mouse ATA V8-12 V H is related to human V2-5 which is also cancer related with hepcidin + or iron + (Fig. 5D).V H 8-12/V K 21 ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "CD11b ++ B CLL/lymphoma develops in old age of mice from B-1 origin V H 8-12/V k 21-5 ATA Tg-expressing cells. ",
"section_name": "Results and discussion",
"section_num": null
},
{
"section_content": "As previously shown (Fig. 1A), expression of V H 3609(V H 8-12)/V k 21-5 transgene (ATAμκ Tg) in the PreB/immature stage drives positive V H 8-12/V k 21-5 selection in B-1 cells through a process involving Arid3adriven fetal/neonatal B-1 cell development to induce mature CD5 + B1a cells and constitute B1a cells in adults [43].In adult B cell development from BM (B-2), the expression of V H 8-12/V k 21-5 BCR in immature B cells drives negative selection at the mature B cell stage, resulting in arrest [11].As shown in Figure S1A, at the 2 mo stage of ATA B cell development, the PerC, mLN, and intestinal PP cell populations were mature B1a, but BM and Spl cells were dominantly immature.ATA B cells in PBL are immature at 2 mo, but an increased fraction of mature are at 8 mo, which corresponds with increased serum IgM, IgG1, IgG3 by 12 mo of age, whereas IgA is already high by 2 mo.However, when Pla2g2a was deleted at the neonate stage, the generation of mature ATA B cells was reduced including through 14 mo of age [12] (Fig. S1A).Thus, ATAμκTg B cells originally generate neonatal ATA B cells pB1a increase in PBL as mature in middle age and old age.The original anti-thymocyte/ Thy-1 autoreactive mAbs from these B cells are polyspecific and can also bind multiple tumor antigens [36]. CD5 recruits the SHP-1 protein tyrosine phosphatase to inhibit BCR signaling [20,21].Thus, CD5 + B1a cells have more anti-apoptotic signals, serve as a source of autoantibodies, and are considered innate lymphocytes [44].The Lyn-CD22-SHP-1 pathway promotes BCR tolerance [45].CD11b also regulates BCR by binding to CD22 [46].Macrophages in peritoneal cavity can induce B1a cells into PerC [47], which are mainly CD5 + CD11b + .When these PerC B1a cells move to spleen, complement component C3 in spleen can decrease CD11b expression as CD11b - [48], but CD5 expression, thus autoantibody production are retained.However, LPS or CpG can downreguate CD5 in adult B1 cells to generate CD5 - B1 cells [24] (Fig. S1B).These CD5 -CD11b -B1 cells in spleen exhibit strong BCR signaling.In middle age, both TCL1 -(TC -) and TCL1 + Tg (TC + Tg) ATA B cells can become CD5 + or CD5 -, although TC -ATA B cell tumors are most CD5 -, TC + Tg ATA B cells are mostly continuously CD5 + (Fig. S1B).The TC + Tg ATA B cells generate lymphoma/leukemia at middle age [24], whereas TC - ATA B cell tumors are in old age.In older humans, TC + CLL can develop, in which most tumor cells are CD5 + .However, 7-20% human CLL B cells are CD5 - [49], and these human CD5 -B cells induce splenomegaly [50], which is consistent with increased splenic CD5 -B1 cells in the mice. Mouse TC + Tg expressing ATA B cells generate slightly more lymphoma/leukemia in middle age than TC -cells (Fig. 1A).However, TC -ATA B tumor cells in old age showed the recovery of increased CD11b expression (Fig. 1B) [13] and react with CD22 (shown in Fig. 5C).At 2 mo age, CD11b -splenic B1a cells stimulated with CpG increased expression of CD11b (or LPS, but low), and the combination of CpG with IL-10 further increased CD11b [13] (Fig. S1C).As previously showed (in Figure S1C), splenic aMyIIA V H Q52 B1 cells and aPtC V H 11 B1 cells (V H knock in) also increased CD11b expression in old age [13,25].PerC B1a cells originate as CD5 + CD11b + , then become CD5 + CD11b -upon migration to spleen, then further to CD5 -CD11b -, and middle aged CD11b increased, then, CD5 -CD11b ++ tumor cells develop in spleen from ATA B cells in old aged mice.In human TC + CD5 + CLL, 18% of tumor cells are CD11b + but most are CD11b - [51], which differs from the TC -mouse.Some mouse TC -ATA B cells progress to lymphoma/leukemia at old age together with increased myeloid cells [13] (Figs. 1A and5E), which is consistent with some human CLLs that develop with myeloid cells [52,53]. In ATAμTg (V H 8-12/D/J h 2) mice, CD5 + B1a ATA cells generated lymphoma/leukemia in old age, but at a lower rate than ATAμκTg mice [24] (Fig. 1A).Mice co-expressing V H 8-12 and V k 19-17/J k 1 (as V H 8-12/V k generated strong MZ B cell development, and some V H 8-12 FO B cells were also generated [54].The V H 8-12/V k 19-17 BCR is a natural anti-intestinal goblet cell work (AGcA), V H 8-12/V k 19-17μκTg mice strongly generated MZ B cells [54], and some MZ B AGcA cell-derived tumors in old age (Fig. S3).MZ B cells are the major constituent of the marginal zone, together with myeloid, dendritic, and stromal cells [55].Generally, macrophage/neutrophils CD11b + Gr-1 + cells generate hepcidin to control iron [56], and also hepcidin low iron + .Then, old aged in AGcA Tg mice generated high macrophage (CD11b + Gr-1 + ) (Fig. 1A), and as showed in Fig. 5E and Fig. S3, this macrophages made strong intestine/cold tumor in old aged, not hepcidin related positive, rather iron + increased. The mouse V H V8-12 BCR is homologous to human V2-5 (Fig. 1C).Human V1-69 is expressed in unmutated CLL (U-CLL), but V2-5 is low in CLL [57,58].Although human V2-5 is very low in cord blood [59], in depth analysis showed V4-59, V4-34, and V2-5 in cord blood [60], suggesting that V2-5 reacts in neonates.Importantly, it is also well known that HIV-1 (human immunodeficiency virus) is associated with increased V2-5 (2F5) and V1-69 (4E10) in humans [61] (Fig. 1C).V2-5 is also found in Ankylosing Spondylitis (AS) [62] and Alzheimer's disease (AD) [63,64].AD was also found to be associated with mouse V8-12 as human V2-5 [65].Then, it has long been known that dominant V2 (most V2-5) expression is associated with increased susceptibility of multiple sclerosis (MS) as brain-gut axis with iron + [66].Several cancers in human have used V2-5 as listed in Fig. 5D.V2-5 immunoglobulin are variable, most hepcidin + and some showed hepcidin low iron + .Thus, in this paper, first mouse TC -ATAμκ Tg lymphoma/leukemia compared to human old aged TC + CLL/U-CLL, and TC + ATAμk Tg middle aged may difference from TC -in old aged.Then, since TC -ATAμκ Tg B cells found Hamp2 ++ generated in old age different from human CLL, thus, checked with human V2-5 positive cancers with hepcidin + or some iron + lists (Figs. 4 and5). Rag1KO Thy1KO ATAμκTg mice generate ZAP70 + CD5 -ATA B-CLL/lymphoma at early mature and middle age Previously shown Fig. 2A,B,C.Neonate ATAμκTg mature ATA B cells arise first in the peritoneal cavity (PerC) with the support of macrophages [47] and pleural cavity, and present in spleen, intestine, mLN, LN, liver, and blood of aged mice, which also exhibit strong ATA IgM in serum and plasma cells [11].ATA B cell tumors in old aged mice are Spl ++ , mLN ++ , LN + , and Liver ++/+ [13].In intestinal microbiota, ATA IgM is present in intestine and highest in colon (Fig. 2A).At 2 mo, mature B1 cells are already present in PerC, mLN, and intestinal Peyer's patches (PPs) (Fig. S1).In contrast, as previously shown, in Thy-1KO ATAμκTg mice, the original ATA Tg does not generate CD5 + B1 cells, rather, B-2 non-ATA B cells are increased [55] (Fig. 2B).Rag1KO ATAμκTg mice engineered, mature T and non-ATA B cells were not generated, however, MZ ATA B cells are strongly generated and also ATA B1 cells, both of which are CD5 - [55] (Fig. 2C). ATAμκTg mice with both Rag1KO and ThyKO background also generated B1-CLL/lymphoma at mature and middle age without CD5 expression, and these tumors expressed higher levels of ZAP70 than TC -or TC + ATAμκ Tg mice, which are most CD11b -(75%) (Fig. 2D).These tumors express cyclin D2, cMyc, and CTLA-4 as high as originally generated initial B1 cells.Although CTLA-4 is associated with higher CD80 than CD86 in normal mouse B1a cells [67], ZAP70 hi RagKOThy1KO mice showed higher CD86 than CD80 and also increased CD24 expression.Mature T cells and non-ATA B cells are absent in Rag1KO mice, but NK and ILC cells and myeloid cells are continuously present.TC -CD5 -B1 ATA B cells with most CD11b -are generated with ZAP70 ++ at early age including BCR signaling and found in high numbers in spleen and liver but not in mLN.These early RagKOThy1KO ATA B cell tumors are different from Rag + Thy1 + ATA B cells, which are generated at the neonate stage and adult animals controlling tumor development with the presence of mature B-2 and T cells together with Thy-1 + , but tumors still develop in old age. ",
"section_name": "Mouse V8-12 homologous to human V2-5",
"section_num": null
},
{
"section_content": "Neonate-generated B1a cells are known to express high levels of Nod1 and remain continuously Nod1 + , as compared to the low levels in B-2 generated cells [68] (Fig. 3A).These B1a cells are also slightly higher in TLR7 and TLR9 than FO B at 2 mo (Fig. 3A).When FO B and pB1a cells were stimulated, FO B cells were found to express high levels of ZAP70 mRNA by anti-IgM, but this did not occur in B1a cells (Fig. 3A) since CD5 expression in B1a does not require BCR signaling.However, combining CiE-DAP for Nod1 reaction with anti-IgM increased ZAP70 expression, but not Nod2, in B1a cells also in FO B. Treatment with TNFa increased ZAP70 expression in both B1a and FO B, and further increases ZAP70 when TNFa was combined together with Nod1 expression.Nod1 is important for B1a cell increase, since at 4 mo, Nod1 -/-are low ATA B cells in ATAμκTg mice.In addition to Nod1, IL5 is also required for B1a cell generation and maintenance in contrast to FO B cells [69], and expression of CD1d is essential for NKT cell reactivity are similar between B1a and FO B cells as previously shown [12].ATA B CLL generated at middle age is continuously Nod1 + , IL5R + and CD1d + , also in the presence of TC + Tg.In TC -ATA B cell tumors, expressions of these decreased in old aged mice [12] ( Fig. 3B).Compared, CD11b expression is increased on CLL developing in middle aged spleen, and further increased on tumors at old age [12,13] (Fig. 3C).Original B1a cells at 2 mo express more CXCR5 than CXCR4, whereas TC -ATA B cell tumors at old age showed decreased CXCR5 and increased CXCR4, as compared to TC + Tg mice (Fig. 3C).Similarly, in aged humans, CLL that arises CXCR4 expression higher than CXCR5 [70]. We next performed microarray gene expression analysis of tumor to compare TC -versus TC + Tg ATA B cells and ZAP70 -versus ZAP70 + cells in Fig. 4A,B.We also compared gene expression in CD5 -vs CD5 + ATA B1 cells from 12 mo TC -ZAP70 -mice in Fig. 5A. Increased ZAP70 expression in TC + mice as in TC -mice are similar to human CLL/U-CLL, however, old aged TC -ATA B cell tumors increased than middle age TC + Gene expression in ATA B cell tumors compared between old aged TC -ZAP70 -CD5 -, TC -ZAP70 + CD5 + , vs middle aged TC + ZAP70 -CD5 + mice (Fig. 4A), and between TC -ZAP70 -CD5 -vs TC + ZAP70 + CD5 + mice (Fig. 4B).Nod1 expression was decreased in old aged TC -tumor cells, and T-bet and CD11c were increased.T-bet + CD11c + are consistent with old aged in humans [71,72].Tumor from TC -ZAP70 -CD5 -mice expressed the highest levels of CD11b and cyclin D2, and TC + (both ZAP70 -and ZAP70 + ) ATA B cells lacked CD11b, but TC + ZAP70 + increased cyclin D2 (Fig. 4B2,B1).Normal B1a cells show higher levels of cyclin D2 than FO B cells (Fig. 2D), and cyclin D2 is expressed in human CLL [73].Since CB.70 mice have a p16 INK4a mutation [74], cyclin 2 (G1) expression can be more increased, and promote moving from G1 to G2/M [75].Then, both ZAP70 + with TC -and TC + are higher in cyclin D1 (G1), cyclin E1 (G1/S), cyclin A2 (G2/M) (Fig. 4A1,B1) (also higher cyclin D3 and cyclin B1 (G2/M); data not shown) by p16 INK4a mutation. ZAP70 + tumor under both TC -and TC + genotypes exhibited increased expression of TNFa, CD40, IL4, several Tubb (β-tubulin) genes, cMyc, Fzd2, Ki67, and CD86 (Fig. 4A1,B1).Among the Tubb genes, WT fetal/neonatal B1a are higher in Tubb6 [76], and all B cells are positive for Tubb5 [77].Tubb5 is high in TC + ZAP70 + tumor, but Tubb6 is higher in both TC -tumors, as compared to TC + tumor (Fig. 4A1,B3).cMyc was also originally higher in B1a cells (Fig. 2D) and ZAP70 + increased as human U-CLL than M-CLL.Fzd2 is β-catenin dependent, and human CLL and ZAP70 + U-CLL showed expression of Wnt10a and Wnt6, which generated Fzd2 [78].Ki67 (NKI67) is a proliferation-related antigen.High Ki-67 index is associated with many cancers, and human CLL ZAP70 + /U-CLL are also Ki67 hi [79].We also found that CD86 expression was higher than CD80 in ZAP70 + tumors (Fig. 4A1 andB1), which was also found in ATAμκTg Rag1KO Thy1KO mice that are ZAP70 ++ (Fig. 2D).As in Figs.4B1, human U-CLL are most similar to ZAP70 + TC -and TC + tumors as increased CD40, IL-4, cMyc, Fzd2, Ki67 (see Fig. S4-1 for human CLL/U-CLL references).Since human CLL with normal p16 INK4a , human CLL expression of cyclin D1 is limited, and cyclin A2 is not increased, and CD86 + cells are only small portion of the CLL [80]. TC + ZAP70 + also increased as in TC -cells including TC -ZAP70 -cells (Fig. 4B2).B1a cells showed slightly higher TLR7, TLR9, and TLR4 expression than FO B cells at the 2 mo stage.As intracellular Nod1 is decreased in old aged, TC -promote TLR7 and TLR9 increases (Fig. 4A2), and TC + ZAP70 + cells also showed increased TLR7 (Fig. 4B2).In contrast, middle aged TC + Tg tumor cells are strongly high for TLR4 (Fig. 4A2). Fig. 3 Nod1 is important in early B1a cells and Nod1, IL-5R, CD1d decrease in old aged and increased CD11b and CXCR4, different from middle aged TC + Tg ATA B cells.A Nod1 high in neonate and adult B1a. 2 mo FO B versus pB1a for ZAP70 generation after 20 hour later by anti-IgM without or with CiE-DAP (Nod1) or MDF (Nod2), or TNFa without or with CiE-DAP.ZAP70 hi in a-IgM is only FO B cells not CD5 + pB1a, but Nod1 add increased ZAP70.Down: 4 mo ATAμκ Tg mice with NOD1 +/-or NOD1 -/-.ATAid is originally generated V H 8-12/V K 21-5, then, low ATA B cells in Nod1 -/-.Thus, Nod1 is important.B Nod1, IL-5R, CD1d comparison between TC -versus TC + Tg ATAμκTg, in 2 mo and meddle aged leukemia CLL and old aged TC -CLL/lymphoma.Old aged TC -tumor showed down these lists, not middle aged TC -and TC + Tg.C Conclution of TC -ATAμκTg ATA B in 2 mo verus old age tumor stage in spleen, with increased CD11b in TC -ATA B in flow cytometry analysis.Down: In microarray analysis, TC -old age ATA B cells are CXCR4 increase versus CXCR5 down, different compared with TC + Tg.Down right: Several TC -versus TC + Tg ATAuk Tg lymphoma/leukemia pictures used for Fig. 4: 16 mo and 17 mo TC -ZAP70 -CD5 -(Spl ++ , mLM ++ , LN + ), 22 mo TC -ZAP70 + CD5 + (Spl ++ , colon ++ , LN + ), 10 mo TC + ZAP70 -CD5 + (Spl ++ , mLN + , PerC ++ ), 16 mo TC + ZAP70 + CD5 + (Spl ++, Liver ++ , PerC ++ ), and 12 mo samples Fig. 5 (See figure on next page.) Wnt/β-catenin family members are Wnt1,2,3,8,10 with LRP5/6, and non-β-catenin (Wnt/Ca2 + ) family members are Wnt 4,5,6,7,11,16 with ROR1/2.TC -tumors showed dominant expression of Wnt/b-catenin Wnt10b (Fig. 4A2) and also TC + ZAP70 + tumor showed dominant expression of both Wnt10a and 10b (Fig. 4B2).Previous studies showed that human CLL express Wnt3, 4, 5a, 5b, 6, 7, 10, 11, 14, 16 and up-regulated in CLL are Wnt3,5,6,10,14,16 [81].Wnt5a/5b are the most important in U-CLL [82] and other human cancers also express Wnt5a, whereas Wnt5a was very low in the mouse ATA B tumors.Rather, Wnt5b and Wnt6 were high in both TC -ZAP70 + and TC + cells (Fig. 4A2,B2).Wnt10 induces expression of Fzd1 and Fzd5, and Wnt5b induces Fzd4 and Fzd6.ROR1 is commonly expressed in human CLL as a component of the Wnt5a-ROR1-ERK1/2 pathway [83], however, mouse TC + ATA B tumors showed very low ROR1, but instead expressed ROR2.Higher in TC -ZAP70 + and TC + ZAP70 + increased Wnt5b-ERK2, also in human U-CLL [54].Myd88 generated from TLRs and higher in TC -with TLR7, 6 (Fig. 4A2), then, increased Myd88 in TC + ZAP70 + cells occurred together with TLR7 increase (Fig. 4B2).TC -ZAP70 -CD5 -showed increased STAT3, IL-10, IL-6 more than TC -ZAP70 + CD5 + , and TC + ZAP70 + CD5 + cells showed increased these (Fig. 4B2).Thus, middle age TC + ZAP70 + increased as in some TC -cells (including TC -ZAP70 -CD5 -) than TC + ZAP70 -.TC -positive cells and increased TC + ZAP70 + CD5 + in ATA B tumor are the most similar to human CLL data, and CD44 ++ Ki67 + as ZAP70 + higher (also TC -ZAP70 -positive) are significant in U-CLL (Fig. 4B2, References in Fig. S4-2). In TC + (ZAP70 -and ZAP70 + ) ATA B cells, TLR4, Wnt5b/Wnt6, and Fzd6, Rac2 showed high positive than TC -, and Runx2, NF-kB, p300 were also high (Fig. 4A2).Runx2 and p300 are NF-kB generation [84,85].Then, TC + showed high APRIL, contrast to TC -showed high BAFF.Tuft1 (tuftelin) is multiple cancers as metastasis, and up-regulating the Rac1/β-catenin pathway [86].Tuft1 can induce NF-kB [87], and TC + showed high Tuft1 than TC -cells.Although Wnt5a/b from the origin as non-βcatenin, however, Wnt5a/b can be changed to binding to β-catenin [88,89], thus Wnt/b-catenin-TCF/LEF binding can occur.TC + showed LRP5 + (different to TC -LRP6 + ) and high LEF1 than TC -(both ZAP70 -and ZAP70 + ) (Fig. 4A2,B2). CD38, CD43, CD44 in B1a cells are originally higher than FO B cells (Fig. S2B), and increased these in old aged TC -tumor than meddle aged TC + cells, as also shown in flow cytometry analysis (Fig. 4A3).CD38 is different from human than mice, since human FO B cells are negative, then GC B cells increased CD38 [90], and human U-CLL showed high CD38 ++ and CD44 ++ [91,92].Since high CD44 ++ in human ZAP70 + CLL, CD44 is the highest in TC -ZAP70 + than TC -ZAP70 -, and also TC + ZAP70 + increased (Fig. 4B2).CD43 showed human CLL positive [93], as found in mouse TC -tumor.TC + showed higher CD21, CD23, CD24, CD27, and CD49d than TC -.CD21 and CD23 are original low/negative in PerC B1a cells, and TC -ATA B tumor were similar low/and increased in TC + ZAP70 -cells (but, TC + ZAP70 + down).Human U-CLL can be CD21 low [94], and some CLL also showed low CD23 [95].CD24 are all normal B cells + and increased CD24 have been known for cell growth/cancer [96], and increased CD24 was found in TC -Rag1KOThy1KO mice in mature &middle aged ATA B tumor (Fig. 2D), then, old aged TC -showed decreased (Figs.4A3 and5D).CD27 are low/-for all normal B cells compared to CD27 + T cells in mice, and TC + cells ( ZAP70 -and ZAP70 + ) showed slightly increased CD27.In human CD27 are different from mice, since human CD27 are positive in B cells, and original early human B1 cells are CD27 + [97] and CLL CD27 + [98], however, U-CLL can become CD27 low/-by principal component analysis [99].CD49d are all B and T cells positive, and increased in TC + Tg cells, in comparison, TC -cells showed down regulated CD49d (Fig. 4A3).Most human CLL are CD49d + , however few human CLL can became down regulated CD49d in age [100].These CD case papers in TC -> TC + and TC -< TC + are also listed in Fig. 5A. Although ZAP70 + increased in TC + cells as in TC - cells similar to old aged human CLL and U-CLL, many showed increased old aged TC -cell tumor than middle aged TC + cells (Fig. 4B3), incruding Hamp2 increased in TC -.It is possible that TC + > TC -data could be for TC -decrease in old aged.Thus, next in Fig. 5, 12 mo TC - cells analysis with normal CD5 + ATA B mice versus CD5 (See figure on next page.)Fig. 4 Microarray mRNA analysis by ATA B lymphoma/leukemia.TC -ZAP70 -CD5 -, TC -ZAP70 + CD5 + vs TC + ZAP70 -CD5 + and TC -ZAP70 -CD5 -vs TC + ZAP70 + CD5 + .ATA B in spleen.A A1: Higher of TC -ZAP70 + CD5 + list.A2,3: higher of TC -ZAP70 -than TC + ZAP70 -CD5 + (TC ->TC + ) and higher TC + ZAP70 -CD5 + than TC -(TC + > TC -) list.A3: add flow cytometry analysis of TC -versus TC + ATA B lymphoma/leukemia together with WT FO B cell.B B1: TC + ZAP70 + CD5 + similar to high TC -ZAP + CD5 -cells.B2: TC + ZAP70 + CD5 + also several high TC -ZAP70 -CD5 -cells.TC + >TC -are not listed by genes TC + ZAP70 + CD5 + decreased to TC -.B3: TC + ZAP70 + CD5 + cells and also TC + ZAP70 -CD5 + are not increased to old aged TC -cells (ZAP70 -CD5 -, ZAP70 + CD5 + ) as TC -> TC + decreased CD5 -ATA B mice with spleen increased mice, compare with original Fig. 4 TC -> TC + and TC + > TC - data.Also, further analyses to check with human old aged CLL related, including CXCR.This TC -12 mo analysis checked with original TC -vs TC + ATA B tumor data in Fig. 4. Further, add microarray analysis about β-catenin/CTNNB1, Damp/HMGB1, CXCR (CXCR4, CXCR5) (also CXCR3 in Fig. S2A), DDP4(CD26), AID, CD1d, and CD180 with original Fig. 4A samples, and TC + ZAP70 + tumor comparison are in Fig. S2.Under 12 mo TC -ZAP70 -CD5 -cell, Nod1, IL5R, and CD1d are decreased similar to old aged TC -ATA B tumor, and CTLA4 down as both TC -and TC + Tg cells (Fig. S2A) and human CD38 ++ as U-CLL are CTLA4 decreased (Fig. S4-3).This TC -ZAP70 -CD5 -cell most similar to increased in old aged increased TC -and TC + ZAP70 + > TC + ZAP70 -and TC -> TC + ATA B tumor generation (except low Wnt10a), and similar to low TC - cells than TC + cells. ",
"section_name": "Decreased Nod1, IL5R, CD1d, and increased CD11b in the B1 cells in old aged CLL",
"section_num": null
},
{
"section_content": "β-catenin/CTNNB1 and HMGB1 are TC -cells high, and TC -ZAP70 + are more higher, and TC + ZAP70 + are also increased (Fig. S2A).Damp, as damage-associated molecular patters (tumor promoting, and antitumor effects), present HAGB1 (high mobility group box 1) [101], and Damp/HAGB1 generate TLR pathway (TLR2,4,7,9), also generate CXCL12 with CXCR4 [102].High β-catenin CTNNB1 and HMGB1 in TC -and TC + ZAP70 + cells are in human CLL positive and high HMGB1 in CLL plasma [103]. About detail of CXCR in Fig. S2A.CXCR4 is TC -high and TC + low, as TC + ZAP70 + also low.Then, decreased CXCR5 in TC -, and TC + ZAP70 -with continuously high CXCR5 as original 2 mo B1a cells, however, decreased CXCR5 in TC + ZAP70 + .CXCR3 and CCR7 were also higher in TC -than TC + ZAP70 -, and TC + ZAP70 + with decreased CXCR5 showed slightly increased CXCR3 but CCR7 was not increased (Fig. S2A).Thus, TC -ATA B tumor showed CXCR4 + , CXCR3 + , CCR7 + and low CXCR5.In human CLL, CXCR4 higher than CXCR5 [70] different from mouse TC + Tg, and CLL also showed CXCR3 + [104] and CCR7 + in CLL.DPP4 + (dipeptidyl peptidase-4)(CD26) are majority of all B cells + , and DPP4 also increase CXCR4 [105].DPP4 are higher in old aged TC -with CXCR4 hi , and low in middle aged TC + cells (both ZAP70 -and ZAP70 + ) as CXCR4 low.Human CLL are high DPP4, and with high DPP4 in serum with U-CLL [106,107].AID (activation-induced cytidine deaminase) original in fetal liver [108], and autoreactive B cells in immature B cells (T1/T2 B cells) in B-2 cells are AID + , then down for mature FO B, and B1a are also originally low AID (Fig. S2A), then, TLR9 induces AID [109].In human, U-CLL showed high AID than mutated M-CLL [110], and mouse TC -ATA B tumor in old aged, also showed high AID, not TC + cells (both ZAP70 - and ZAP70 + ) (Fig. 5A, Fig. S2A).CD1d and CD180 are decreased in old aged TC -ATA tumor, compaired to middle aged TC + (both ZAP70 -and ZAP70 + ) (Fig. 5A, Fig. S2A).CD180 is originally increased from immature to mature FO B and MZ B cells, also B1a cells CD180 + .Then, TLR7,9 signaling pathway significantly downregulate CD180 [111] and mouse old aged TC -ATA B tumor showed the lost CD180.In human old aged CLL, survival mature M-CLL are CD180 + , contrast to U-CLL are CD180 - [112], and aged memory B cells became CD180 -.Conclusion, TC -> TC + and also down TC -in old age were most similar to 12 mo spleen infection in TC -CD5 - cells, and these are most similar to old aged human CLL/U-CLL (TC -> TC + samples for human CLL/U-CLL references in Fig. S4-2). TC + > TC -tumor lists found to be mostly down in 12 mo TC -CD5 -cells than normal TC -CD5 + B1a.Thus, most TC + tumor appeared to be not increased by tumor than TC -.However, 12 mo TC -CD5 -increased high NF-kB1 and also increased in Runx2 and p300 as similar to TC + > TC -.Thus, clearly, TC + cells are more increased NF-kB1 and NF-kB related Runx2 and p300 [84,85] than TC -.In Fig. 5B showed that NF-kB is also Fig. 5 12 mo TC -CD5 down ATA B spleen ++ cells similar to old aged TC -ATA B lymphoma/leukemia.TC + Tg ATA B lymphoma/leukemia are NF-kB1 high.Old aged TC -ATA B are CD11b ++ CD22 ++ , CD24 low , and Hamp2 ++ generation, and V8-12 with V k 19 AGcA MZ B are 12 mo Hamp2 + and old age generated macrophage ++ with intestinal tumor.A and B. 12 mo TC -CD5 -ATA B with spleen ++ generate similar to old aged TC -CD5 -ATA B tumor.TC + Tg ATA B CLL/lymphoma are higher NF-kB and TCL1 induced miR21 and miR155 and down miR29.C TC -lymphoma/leukemia showed CD11b ++ CD22 ++ and generate Hamp2 ++ (TC -ZAP70 -are more IL10 and IL-6, and TC -ZAP70 + are high IL-22R) with iron down, as USF2 + , TFR + , FPN -.D TC -ATA B lymphoma/leukemia generated high Hamp2 and CD44 ++ CD24 low .Human V2-5 positive papers, as kidney, Breast cancer, Pancreatic cancer, SARS-CoV-2, and SLE.Breast cancer are CD44 ++ CD24 lo .E Some ATA B tumor showed together with high myeloid cells (CD11b + Gr-1 + ) [13], and V H 8-12/V k 19 AGcA generate MZ B with Hamp2 + in 12 mo, then old aged generate macrophages ++ in spleen with intestine/color tumor ++ (See figure on next page.)higher in TC + ZAP70 + than TC -.TCL1 leads AKT [113] and mostly AKT2-NF-kB [114].AKT2 are similar in TC - to TC + ZAP70 -, however, TC + ZAP70 + are increased.Human TCL1 showed higher miR21 and miR155, and lower miR29, and in ATA B cells, these miR showed TC + > TC -(Fig.5B) similar to human TCL1 + CLL [115,116].However, down miR15a and miR16 to mediate Bcl2 increase as found in human CLL [117], and similar by down in TC -than TC + (Fig. 5B).Low CD21, CD23, CD24, CD27 and CD49d in TC -ATA B tumor mice are also low in 12 mo TC -CD5 -spl ++ cells, and these low data can be found in some in U-CLL (CD21, CD27) [94,99] or low generated (CD23, CD49d) can occur in few CLL [95,100].Since normal CD5 + ATA B cell and TC + Tg tumor with CD5 + samples are most human TC + CD5 + CLL positive, these in human CLL references also showed in Fig. S4-3.Hamp2 ++ , CD11b ++ and down CD24 in old aged TC -ATA B tumor are similar to 12 mo TC -CD5 -cells, but these are not in human CLL. Increased CD11b ++ CD22 ++ and Hamp2 ++ Figure 5C.Originally, B1a cells are CD5 + which bind to SHP-1 as Lyn-CD22-SHP-1.CD22 is from BCR [45], then binding CD5-SHP1 or CD11b-CD22 inhibits BCR signaling [46].When loss as CD5 -and CD11b -in spleen B1 [48], middle aged become CD11b + such as by CpG bacteria or CpG + IL-10 + increase, and in old aged, TC -ATA B showed increased CD11b ++ , then increased CD22, and TC -ZAP70 -CD5 -are more higher CD11b ++ CD22 ++ (Fig. 5C).CD22 ++ found in HCL (hairy cell leukemia) [118], however, human CD5 + CD11b -CLL is CD22 lo not require CD22 ++ , thus different [118].TC -CD11b ++ CD22 ++ is allowing the migration and maintaining autoreactive B cell tolerance, and SLE (Systemic lupus erythematosus) found CD11b ++ CD22 hi (Fig. 5D) also autoreactive [46].α-Thy-1 ATA B cells with CD11b ++ can Thy-1 moving to endothelial to control virus infection as by CD11b + Gr-1 + endothelial tissues (33.34). Hamp2 also increased in TC -ATA B cells in old aged.In hepcidin, mouse ATA B cell tumor did not show Hamp1 but Hamp2 ++ .Hepcidin is important for systemic iron homeostasis, and hepcidin generations are the most CD11b + Gr-1 + macrophage and monocytes, and TLR induced hepcidin in T cells and B cells [42].Hepcidin control iron is most in liver, however, mouse Hamp2 is in both liver and pancreas with antibacterial and antivirus active types, and B cells including B1 cells can move from spleen to pancreas with high Hamp2 [119], and Hamp2 is not change erythropoiesis different from human hepcidin [120].In hepcidin, IL-10, IL-6, and IL-22 generate Stat3, then regulate for the induction of hepcidin [121,122].In mice, fetal and neonate showed IL-10 and IL-6 [123,124].IL-10 is original B1a cell generation, however, IL-6 did not require for B1a, rather, IL-6 -showed increased B1a cells [125].Different from Arid3a which is important for neonate mature B1a generation, Arid5a is more adult immature B cell stage and increased IL-6 for B cells [12,126].IL-6 reacts with Th2 and Th17, and mostly increased in Th17 [127].Since in old age, T-bet and CD11c increased [2], T cells changed Th1 and Th17 increase, and Th2 decreased with B cell responsive as IL-4R down (Fig. 5C).High IL-6 in old aged ATA B cell tumor in TC -ZAP70 -CD5 -cells are the Arid5a generation with IL-6 + [126] (Fig. 5C) and also IL-10 ++ (Fig. 4A2).Iron regulation require USF2-hepcidin to generate iron [128,129].Strongly USF2 high in TC - cells in ATA B cells (Fig. 5C).In TC -ZAP70 -were high IL-10 and IL-6 than TC -ZAP70 + cells, and higher IL-22 in TC -ZAP70 + in USF2 and IL-22R (Fig. 5C).In Th17 cell, IL-17 and IL-22 generation [130] and TC -ATA B tumor showed highest IL-22 to receptor IL-22R in the highest TC -ZAP70 + cells as Hamp2 highest generation (Fig. 4A2).IL-22 also generated by innate lymphoid cells ILC3 [131].Cleary, Hamp2 ++ TC -ATA B tumor cells decreased iron stage, by TFR1 + high (as origin iron) and negative ferroportion FPN (SLC40A1) as iron reduction with TFR1 + FPN - [132].Thus, old aged TC -ATA B tumor increased Hamp2 ++ and decreased iron (Fig. 5C).[138] In the SLE group, common usage frequencies of the V gene and J gene, and the proportion of IGHJ6 are higher in SLE group Mouse V8-12 ATA B cells (anti-thymocyte/Thy-1 and polyspecific) are not only CLL, but old aged ATA B cells related to cancer Spl ++ , mLN ++ , LN + , Liver ++/+ . ",
"section_name": "TC",
"section_num": null
},
{
"section_content": "In human, although T cells are completely absent Thy-1, Thy-1 are present.Thy-1 can move to endothelial stage for tumor suppressor, such as in virus infection, and this Thy-1 move to endothelial cells with dominantly from CD11b in CD11b + Gr-1 + presenting monocytes /macrophase [33,34] in human.In both human and mice, Thy-1 is in brain neuron together with CD11b + macrophage [139,140], thus Thy-1 is important.In human, Thy-1 + is also present in kidney in normal in epithelial cells (ECs) [141], also, urine as a liquid by-product of metabolism generates hepcidin + [142] (Fig. 5D).As show in Fig. 5D, several cancers also showed Thy-1 + and hepcidin + .In breast cancer, Thy-1 + are present in epithelium ECs together with some MSC (mesencymal stem cells) and in endothelium cells (ECs), and hepcidin + and iron deficiency [143].In SARS-CoV-2, epithelium ECs and MSC with Thy-1 + , and hepcidin + with iron decreased [144].In SLE, most MSC are CD90 (Thy-1), CD105, CD73 positive, and hepcidin + and iron decreased [145].Human pancreas is different from mouse in Hamp2 + in pacreas [119], and in human pancreatic cancer, fibroblasts and endothelium ECs present Thy-1, however, hepcidin low and iron overload [146], with high macrophage and neutrophil interaction in pancreatic tumor microevironment [147], not normal increases hepcidin.Thus, pancreatic cancer is the different paper to generate high macrophage with low hepcidin and iron overload. CD44 is homing receptor and original pB1a are higher than FO B cells (Fig. S2B), and more increased in old aged ATA B tumor.CD24 (30F1) is a glycophosphatidylinositol (GP1)-anchored protein, with all normal B cells express.CD24 overexpressed in tumor and human CLL [96], and mature & middle ATA B tumor under Rag1KOThy1KO mice showed also higher CD24, then, old aged ATA B tumor showed down CD24 (Fig. 5D).CD44 hi CD24 -/low are well known for human breast cancer [148], originally generated by EMT (epithelial-mesenchymal-transiton) [149].EMT plays a key role in the regulation of cell motility and invation.In pancreatic cancer, CD44 hi CD24 hi , but when non-B cell EMT presented CD44 hi CD24 - [150].DAMP/HMGB1 can present EMT [151], and AID also increased EMT [152].Since old aged ATA B tumor showed DAMP/HMGB1 + and AID + , it is possible that EMP increased with changed to CD24 low with CD44 ++ .Ki67 is increased from HMGB1 [153], and TC -ZAP70 + showed both highest Ki67 and HMGB1, then lowest CD24 in TC -ZAP70 + than TC -ZAP70 - (Fig. 4A3).However, also, p16 INK4A reduction increase EMT [154], and C.B17 mice are p16 INK4A mutation and human breast cancer are often p16 INK4A mutation (49%) [155], thus, ATA B CD24 low with EMT may be the more generated incruding by p16 INK4A reduction in old age. Similar to old aged mouse ATA B tumor, human breast cancer showed CXNNB1 + , HMGB1 + , Ki67 + , CXCR4 + and serum DPP4 + .Pancreatic cancer showed CTNNB1 + mutation, HMGB1 + , Ki67 + , CXCR4 + , DPP4 + .SARS-CoV-2 showed, CTNNB1 hi , and serum HMGB1 hi and dominant T cells showed Ki67 + , and CXCR4 + , DPP4 ++ .In SLE, CTNNB1 low but urokinase-type plasminogen activator made CTNNB1 + , and serum HMGB1 + , and Ki67 + , CXCR4 + , DPP4 + .Then, SLE originally known by presented CD5 + B cells, then, CD5 decreased B cells can occurred, also CD1d downregulated.As found in mouse old aged ATA B tumor, SLE are known the autoimmune disease and generated CD5 -, CD1d -, CD22 hi , CD11b ++ , AID + , CD180 -, CD21 -, CD27 -(Fig.5D), also T-bet hi CD11c hi and TCR7 + TCR9 + .All reference in Fig. 5D listed in Fig. S5: Brest cancer, Pancreatic cancer, SARS-CoV-2, and SLE. Figure 5E.B cells can cross talk fibroblast and became to epithelial cells [156], and B-1 B1 B cells and B-2 MZ B cells interacts macrophage and dendritic cells [157,158], and the spleen MZ B cells clearly contains macrophage receptor.As human CLL showed some increased myeloid cells, old aged mouse ATA B tumor showed increased CD11b + Gr-1 + myeloid cells/macrophage with 12% (Fig. 5E) [13].V8-12 μTg cells showed increased in MZ B with V k 19-17 as AGcA, and generated V8-12/V k 19-17μκTg mice strongly AGcA MZ B cell generation at first 3 wk as early B-2 stage [54] (Fig. S3), and this AGcA MZ B cells showed increased Hamp2 at 12 mo, as ATA B1 cells Hamp2 + , however V11-2 aPtC B1a cells are not increased Hamp2 (Fig. 5E).In old aged, originally AGcA MZ B cells can become tumor 7%, but macrophage with CD11b + Gr-1 + are most strongly increased in spleen by MZ B cells as spleen ++ (56%), and high intestine/colon tumor generated (Fig. 5E, and pictures in Fig. S3), also resulting in a upper respiratory swollen neck (Fig. S3).Human HIV-2 virus are V2-5 positive [61] and accumulated macrophage and hepcidin increases with iron decrease, thus maintenance of viral set-point [159,160].Ankylosing spondylitis (AS) with most V H 2 (dominant V2-5) [62] are the neutrophils progression, and Alzheimer's disease in brain (AD) found in V2-5 are with macrophage polarization subsets, most in bacteria [161].Both AS and AD showed hepcidin + plays with an important role [162,163].Multiple sclerosis (MS), also V H 2 (dominant V2-5) positive [66], as a brain-gut axis and blood-brain, with important macrophage [164], however, with low hepcidin and crucial role of iron in serum [165].Clearly, increased iron in brain in MS in age [163], and MS is intestinal barrier occur with iron [166,167].Aging spleen macrophage can increase tumor-derived factor with iron + in mice [168] and macrophage-iron + in old/damaged generate red blood cells, generate brain functions (brain-spleen axis) and generated colon (spleen-gut axis), then gut-brain-spleen axis [166].These iron + in tumor generation in human is TFR1 hi and FPN + , not FPN lo [169].Human V2-5 + cancer with macrophage/ neutrophils generate mostly high hepcidin with iron deficiency, however, pancreatic cancer and multiple sclerosis (MS) are macrophage generate with high iron with low hepcidin, similar to mouse V8-12 + AGcA MZ B increased macrophage to generate intestinal/colon tumor.Clearly, human V2-5 homologous to mouse V8-12.Mouse old age ATA B cells showed CD11b ++ with control Thy-1 and directly Hamp2 ++ and some increased macrophage, and MZ B showed strongly macrophage increased in spleen with tumor-derived factor. IgVH in TC + Tg lymphoma/leukemia TC + Tg ATA B cells are different from TC -old aged tumor.We previously published TC + Tg generated tumor and found most VHs are as B-1 cell CLL tumor [24] (Fig. 6).In this TC + Tg C.B17 mice, generation of ATA B cells were low (than SM/J and NZB mice), rather, showed several increased IgV H . Dominantly high in J558 (V1-55, V1-52), V H 12 anti-PtC (V12-3), Q52 anti-MyIIA (V2-9).In J558 V1 cells, TC + Tg generation (V1-55, V1-52, V1-82, V1-9, V1-19, V1-78), and in normal C57BL/6 B1a cells V1-55 and V1-53 are high beginning at 2-3wk [170], and V1-14 as anti-Sm for B1a cell.All these V1 showed slightly increased level in human V1-46.Human CLL is low V1-46, however, ratavirus-specific B cells showed high V1-46 [171], and, when repertoire of rotavirus generation, young children showed highest V1-46 [172]. V H 12 (V12-3), Q52 (V2-9, V2-2), 36-60 (V3-2, V3-8, V3-6) showed slightly reacted to human V4-59 (lower V6 than V4-59).In both cord blood and adult blood in human, V1-69 and V4-59 are the higher list, and in cord blood with depth analysis, V4-59 > V4-43 > V2-5 [173].Also, in older subjects, V4-59 is also positive.In human CLL, V4-59 is the medial level with mutated or some non-mutated [174].V4-59 also occurred in HCL (hepatitis C virus) [175], and MCL (mantle cell lymphoma) showed V3-21(mutant) and V4-59 (not/low mutate) [176].S107 (V7-3, V7-1) are positive with human V3-49, V H 11 (V11-2) is positive with V3-74, J606 (V6-4) and X24 (V4-1) are positive with V3-7, and 7183 (V5-17, V5-9, V5-12, V5-6) showed different V3 positive.In human CLL, V3-7 and V3-23 are positive (V3-7 are mostly mutant, and V3-23 are low mutant or mutant), and V3-49, V3-21, V3-74 are low in CLL [174].Mouse aPtC V11-2 are well known for mouse B1a cells, and human V3-74 showed high in celiac disease (CeD)-specific antibodies in gut (gastrointestinal tract), and human aPC showed V3-74 [177].Since mouse V1-55 and V1-52 (similar to human V1-46), and V12-3 and V2-9 (similar to human V4-59) were higher in TC + Tg cells, we next checked the region for higher these V H cells in TC + Tg B-1 tumor cells. High increased B-1 VH cells generated in TC + Tg are not only related to fetal/neonatal generation As shown in Fig. 7A, neonate liver HSC (hepatic stellate cells) generate myeloid progenitor (CMP) and lymphoid progenitor (CLP).Innate immune system in CMP generate myeloblast (GMP) and erythrocyte and megakaryocytes-platelets generation (MEP).In CLP, B-1 cells generated under Lin28b + Let7 -and Arid3a + allowed generation of CD5 + B1a cells.Several B1a binds to erythrocyte under aPtC (phosphatidyleholine): V H 11 (V11-2), V H 12 (V12-3), J558 (V1-53), and aMyIIA (non-muscle myosin IIA) Q52 (V2-9) also can bind to erythrocytes and platelets.These B1a binding to erythrold cells are high in neonate than adult stage [43] (Fig. 7A).In TC - aged, aPtC (V11-2, V12-3, V1-53) and aMyIIA (V2-9) showed lymphoma [25,178], and in old aged V11-2 μTg mice (V H 11t) generated MBC (monoclonal B cell lymphocytosis) in PBL [13], and aMyIIAμκTg (as ON25) generated aMyIIA B CLL/lymphoma [25] (and Fig. S1C).Since human V1-67 and other IgH in U-CLL can bind to MyIIA [179], mouse neonate TC -aMyIIA B cell became similar to human TC + U-CLL.J558 (V1-52) reacted for E-coli from early B1a cells, also generation of lymphoma.J558 (V1-55) is not found in earliest neonate stage, then, at 2-3 wk high increased in C57BL/6 mice [170], and when LPS + generation, strongly CLL/lymphoma generation occur by V1-55 in age in C.B20 mice [180].In TC + Tg in C.B17 mice, highest V12-3 and V1-55, and higher V2-9 and V1-52 in CLL [24]. Erythrold cells are lower in 2 mo than neonate stage [43] (Fig. 7A).Originally Lin28b + in neonate generated B1a V H 11 and V H 12 are continuously incrused in 2 mo Lin28b -Spl and PerC.In contrast, when Lin28b + Tg generate, V1-55 became highest in 2 mo adult, contrast downregulate V H 12 and V H 11 [43] (Fig. 7A).In adult BM, pro-B to pre-B cell generation requires TdT (Terminal deoxynucleatidyl transferanse) for N-necleotides to the V.D. and Jexon of the BCR, in contrast, N-region can not require in fetal and neonate [181].TdT decrease as TdT KO mice together with Lin 28b + Tg mice showed negative in V1-55, contrast, increased in several earliest generated B1 related cells such as V H 11 and Q52 (V2-9), although not increased in V12-3.When B-1 pro-to pre-B cell stage by binding to surrogate light chain (SLC) associated in B-2 stage, clearly that V11-2, V2-9, V1-52 are negative, and middle by V8-12, then positive by V1-55 and V12-3, as previously identified [24].Thus, B-1 V12-3 and V1-55 can continue to generate to B-2 immature B cells not only early B-1 stage, then, TC + Tg aloud increased.Anti-PtC V H 11 and V H 12 are different.Both react with BrMRBC (bromelain-treated mouse erythrocyte), however, V H 11 as a stage of TMA(trimethylammonum) present, and anti-TMA response reflects the V H 11 usage in original mice [182], in contrast, V H 12 showed high reaction with SRBC(sheep red blood cells) as sheep erythorocytes with continuously increase [182].Since TC + Tg mice continuous generate TC + , more generated in V12-3 and V1-55 in age, but, early strongly neonate stage V2-9 (aMyIIA) and V1-52 can also increased in age (Fig. 7A). In neonate ATA B cells, TC + Tg ATAμκTg ATA B mice showed continuously slightly higher Let-7 as adult Lin28 -Let7 + than TC -, thus, TC + Tg changed.TC -ATA B cells showed lower miR150 and miR181b than TC + Tg (Fig. 7B).miR150 and miR181b are originally low in neonate B1 cells since Lin28 + downregulate miR150 and miR181 [183,184], and well known that original miR150 - makes in human B1a cells [185], and human U-CLL are miR150 low [186].In adult miR181 are positive, then, TC + human old aged, miR181 can be decreased, and showed miR181b low is making progression and cell death resistance with human cancer, and miR181 down regulation showed increase TCL1 in human [187].Thus, old aged mouse TC -ATA B tumor with down miR181b similar to human CLL progression [188], without increased TCL1 in mice.TC -ATAμκTg tumor showed increased CD11b and Hamp2 in middle aged CLL, and further increased in old aged, different from TC + Tg. (Fig. 7C). Old aged TC -ATA B cell lymphoma/leukemia with old aged human CLL/U-CLL, and increased Hamp2 as in human cancer hepcidin + Summarized in Fig. 7D.TCL1 are positive in embryo, originally.Then, in mice, TCL1 (as TCL1A) down regulated at 18 day in fetal liver as TC - [16], thus, TC + Tg generation showed continuously TC + fetal/neonate and aged.Originally generated B1 cells in TC + Tg are sertein IgH cells increased in CLL Lynphoma/Leukemia at middle aged with increased NF-kB in reaction to bacteria or viruses generated.Middle age TC + Tg mice tumors are not all similar to old aged.In TC -B1a cells at neonate-early age stage, mature IgH cell levels are different from mouse background, then, certain original TC -B1a cells VHs can generate in old age lymphoma/leukemia.In old aged, both mouse and human generate more T-bet + CD11c + as different T cells, and increased myeloid cell differentiation [189].Then, increased TCL1 in human CD5 + B cells can occur with sensitive bacteria increase generating CLL.Since CD5 + cells leads to stop of BCR signaling, ZAP70 + further required for immediate strongly in human TC + ZAP70 + CD5 + U-CLL for more aggressive with a significantly short time than M-CLL.In mice, increased ZAP70 can several increase than ZAP70 -, but CD5 down with high CD11b ++ CD22 ++ in TC -ZAP70 -CD5 -can also occurred lymphoma/leukemia generation in old age, and high Hamp2 ++ . In ATAμκTg mice in neonate and early aged, B1a cells increased Nod1 + , IL5R + , CD1d + , CTLA4 + , CXCR5 + (in pB1a hi ), cyclin D2 + , cMyC + , and CD11b + in pB1a (not sB1a), as also normal B1a cells.Since C.B17 mice has p16 INK4a mutation, further high cyclin D2.In old aged ATA B cells, decreased originally Nod1, IL-5R, CD1d, CTLA4, CXCR5, also CD49d in B1a cells.In contrast, cyclin D2, cMyc, also CD38, CD44, CD43, STAT3, BAFF, were more increased in old aged and CD11b increased.And, high T-bet + CD11c + and miR181b low in old aged are similar to human old aged CLL.In old aged TC -ATA B mice, ZAP70 -CD5 -and ZAP70 + CD5 + (also CD5 -), are generated with increased cells or down cells similar to human CLL/U-CLL (Fig. 7D).Different from human CLL, ATA B cells showed often CD5 down by bacteria in aged, then with increased CD11b 7 TC + Tg B1 B cells in neonate to early age are changed than TC -.Old aged TC -ATA B cells similar to human CLL/U-CLL and further some different.A Summary.In B-1 B cell generations, aged ability for lymphoma or CLL/lymphoma [13,25,178,180], and V1-55 in C57BL/6 mice is high at 2-3 wk, than 1 wk [170].Down: 2 mo Lin28 Tg + changed are high V1-55 and decreased V1-55 with negative TdT KO Lin28 Tg + persentage, as TdT requaded for B-2 from Pro-B (TdT + ) move to Pre-B.When V1-55 and V12-3 B-1 Pro-B to Pre-B cell stage with heavy chain with surrogate light chain (SLC) association to B-2 analysis.Thus, increased in TC + Tg.B 12 mo microarray relative miRNA level Let-7 showed slightly higher in TC + Tg ATA B cells than TC -, and TC -ATA B are low miR150 and miR181b.C High increased CD11b and Hamp2 in old age TC -ATAμκTg mice.D Concludion of difference of TC + Tg and TC -mice to generated lymphoma/leukemia, and in C.B17 mice TC -ATAμκTg ATA B from neonate, middle age, and old aged.Old age ATAB lymphoma/leukemia generation with most similar to human TC + CLL/U-CLL and different to CLL with CD11b ++ Hamp2 ++ , CD22 ++ , CD24 ",
"section_name": "V2-5 human cancers with hepcidin",
"section_num": null
},
{
"section_content": "Neonate V H 8-12/V k 21-5 CD5 + ATA B cells generated old aged leukemia/lymphoma without TCL1.ATA B cells can decrease CD5 in middle age and increase CD11b ++ CD22 ++ in ZAP70 -CD5 -, generating old age leukemia/lymphoma with autoimmune disease and control Thy-1, also ZAP70 + CD5 + with CD11b + CD22 + .This mouse TC -ATA B cell tumors in old age most similar to human old age TC + CLL/U-CLL, and middle aged TC + Tg ATA B mouse tumors are not similar to old aged in TC -mice, but increased ZAP70 + made several increased data as in old aged TC -.Certain original generated in mouse B1 cell genes decrease or increase in old age.Then, ATA B cells are hepcidin-related Hamp2 increased with iron low as similar to human cancer hepcidin + iron low .Mouse V8-12 similar to human V2-5, and V2-5 related to several cancers with hepcidin + iron low generated macrophage/neutrophils, and multiple sclerosis are hepcidin -iron + generated from macrophage including brain-gut/colon axis.Mouse V H 8-12/V k 19-17 generate AGcA MZ B cells increased macrophage with intestine/colon axis tumor with old age.Conclusion, mouse with V8-12 in old aged, B-1 cells generated CLL/ cancer and B-1 cells directly with Hamp2 ++ Iron low , contrast, B-2 MZ B cells increased macrophage in spleen with intestinal/colon tumor.Since V H 8-12/V k 2-5 B1cells can also interacts macrophages, mouse V8-12 similar to human V2-5 generated macrophage/neutrophil with hepcidin + iron lo or hepsicin -iron + . ",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "C.B17 mice is the BALB/c background with changed as IgM b .Originally generated anti-thymocyte/Thy-1 (ATA) B cells (IgM a ) in SM/J mice [10], and generating ATAμκTg and ATAμTg under C.B17 mice with ATA B lymphoma/leukemia generation in old age [10,11,24].Thy-1KO or Rag1KO under ATAμκTg were generated [55], and ATAμTg generated MZ B cells as AGcA B cells and AGcAμκTg generated dominant MZ B cells were published [54], and continuously analyzed for AGcAμkTg mice in old age, with high macrophage in spleen and generated in intestinal tumor.Eμ-TCL1 transgenic mice (TC + Tg) in C.B17 mice, also with ATAμκTg or ATAμTg, and showed generated lymphoma/leukemia [24].In this paper, microarray gene-expression analysis of ATA B lymphoma/leukemia by old age TC -versus middle age TC + Tg in ATAμκTg tumor and 12 mo TC -CD5 down ATAμκTg comparison. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "mRNA expression analysis using Agilent Technologies whole genome arrays.Total RNA, isolated as described previously [190], was used for production of fluorescentlabeled probe and then hybridized to the array.RNA purity and integrity were evaluated using the 2100 Bioanalyzer (Agilent Technologies) and NanoDrop 1000 (Thermo Fisher Scienti c) before probe generation.Experimental samples were labeled with either Cy3 or Cy5, and a common reference RNA, a pool of total RNAs from several mouse organs generated in the laboratory, was also fluorescent labeled.Each sample was done twice with dye-swap as technical replicates.Hybridized slides were scanned on an Agilent Technologies scanner, and fluorescent intensities of hybridization signals were extracted using Agilent Technologies Feature Extraction software.Statistical analysis was performed in the Fox Chase Biostatistics Facility. miRNA expression analysis.Total RNA was isolated using TRIzol reagent (Invitrogen) and washed with 80% ethanol to better retain small RNAs.The RNA quality and integrity were evaluated using the 2100 Bioanalyzer and NanoDrop 1000. 100 ng total RNA was dephosphorylated with calf intestinal phosphatase and then end-labeled with Cyanine-3-pCp using T4 RNA ligase.Labeled RNA was hybridized to the Agilent Technologies mouse miRNA microarray at 55 °C for 20 h.Slides were washed according to the Agilent Technologies miRNA protocol and scanned.Signals were extracted as described above. ",
"section_name": "Microarray gene-expression analysis",
"section_num": null
},
{
"section_content": "Flow cytometry analysis, sorting and monoclonal antibody reagents, including CD11b (M1/70), CD40 (1C10), CD80 (16-10A1), CD86 (GL-1), CD38 [90], CD43 (S7), CD44 (IM7), CD21 (7G6), CD23 (B3B4), CD24 (30F1), CD27 (LG.3A10),CD49d (R1-2), Gr-1(RB6-8C5).Nod1 -/-mice were originally made [68], and generated in 4 mo ATAμκTg Nod1 +/-and Nod -/-under C.B17 mice.Animal experiments were conducted under a protocol approved by the FCCC Institutional Animal Care and Use Committee (IACUC). ",
"section_name": "Flow cytometry analysis",
"section_num": null
},
{
"section_content": "Gene expression was quantitated by real-time PCR, using TaqMan assays from Applied Biosystems, an ABI 7500 real-time thermal cycler, and ABI software (Life Technologies).Relative gene expression levels were normalized using β-actin values for mRNA as a standard. For spleen FO B versus pB1a in 2 mo, check of increased ZAP70 by anti-IgM, TNFa (aa80-235), DAP (C12-iE-DAP) (from InvivoGen), MDP (from InvivoGene).FO B (1 × 10 5 , 25λ) and pB1a cells (1 × 10 5 , 25λ) and together with anti-mouse IgM (15λ), DAP(15λ), MDP(15λ), TNFa (100 ng/ml, 15λ), then 20 h later, RT-PCR analysis with ZAP70. ",
"section_name": "Quantitative RT-PCR assay",
"section_num": null
},
{
"section_content": "This intestinal microbiota positive analysis was published [25].Crossreactivity of ATA IgM with intestinal microbiota as small intestine and large intestine as colon.For microbiota staining, fecal microbiota were prepared from C.B17.scid mouse intestine and incubated with 2 ug/ml IgM at room temperature for 60 min, followed by FL-anti IgM at 4 °C for 20 min, and then analyzed by flow cytometry.Monoclonal IgM (anti-KLH MM-30, BioLegend) was also used as a control. ",
"section_name": "Intestinal microbiota",
"section_num": null
},
{
"section_content": "This was published for aMyIIA as negative and control IgH (SP6, V627) [25].IgH retrovirus production: Selected IgH VDJ segments were amplified by PCR, cloned into an IgH-μ construct, and inserted into the pMIG retroviral vector (MSCV-IRES-GFP).Each IgH-pMIG plasmid and the pCL-Eco retroviral packaging vector were co-transfected into the Phoenix packing line and 24 h supernatant was collected, filtered, and stored at -80 °C until use.To analyze Ig heavy chain associated with SLC (examine surrogate light-chain)): Pro-B Abelson line N38 was transduced with IgH-pMIG retroviral supernatant.In GFP + (green fluorescent protein) cells, IgH-μ transduction was examined by intracellular IgM staining, and extent of SLC assembly was examined by surface SLC staining, using the conformation-dependent anti-surrogate light-chain antibody SL156.To test the capacity for pre-BCR-mediated proliferation, IgH-pMIG retroviral supernatant was added to pro-B cell sultures of Rag1 -/- BALB/c BM on OP9 stromal cells together with IL-7.IgH (SP6, V627) as a positive control was set to 10. ",
"section_name": "Heavy chain with surrogate light chain (SLC) association analysis",
"section_num": null
}
] |
[
{
"section_content": "We thank several Fox Chase Cancer Center Facilities (Lab Animal, Flow Cytometry, Transgenic, DNA Sequencing) for technical support.Dr. Kerry Campbell in FCCC for comments on the manuscripts. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "No datasets were generated or analysed during the current study. ",
"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/ s12979-024-00415-6. Ethics approval and consent to participate Experiments using animals were conduced under approved by the FCCC Institutional Animal Care and Use Committee (IACUC). The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12979-024-00415-6. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Experiments using animals were conduced under approved by the FCCC Institutional Animal Care and Use Committee (IACUC). ",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1371/journal.pone.0004434
|
Tumorigenic Potential of Olfactory Bulb-Derived Human Adult Neural Stem Cells Associates with Activation of TERT and NOTCH1
|
Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation.Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with maintenance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity.Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.
|
[
{
"section_content": "Due to their ability to self-renew and to differentiate towards the neuronal phenoype, human adult neural stem cells (NSCs) provide an attractive tool for transplantation-based therapy of neurodegenerative diseases that avoids the ethical issues raised by the use of human embryos.However, proliferation and self-renewal properties make NSCs sensitive targets for malignant transformation [1].Some evidence suggests that adult mouse NSCs are quite resistant to transform even in high-passage cultures under mitogen stimulation [2].In contrast, neural precursors from the adult rat subventricular zone (SVZ) have recently been shown to transform into tumorigenic cell lines after expansion in vitro [3].Moreover, several arguments advise caution before grafting NSCs in patients that include, a) evidence that glioblastoma may arise de novo from the oncogenic transformation of NSCs [1,4], b) common molecular determinants regulating neurogenesis and tumorigenesis [5][6][7], and c) generation of glioma-like lesions following growth factor stimulation of the adult SVZ [8]. The forebrain SVZ and the dentate girus of the hippocampus are two areas of persistent neurogenesis in the adult brain.These regions contain dividing cell populations that have been recognized as NSCs and transit amplifying progenitors (TAPs).The former are relatively quiescent cells with the capacity of self-renewal.TAPs proliferate more rapidly and differentiate into migratory neuroblasts and oligodendrocyte precursors.In rodents, TAPs move along the rostral migratory stream to the olfactory bulb (OB).In humans, a lateral ventricular extension of the migratory stream to the OB has recently been demonstrated and NS/progenitor cells (NS/PCs) have successfully been isolated from the OB, which therefore represents an accessible source of neural precursors [9][10][11].Using xenograft models, we found that human adult OB-derived NS/PCs are capable of initiating tumor formation.Although an oncogenic potential has previously been described in rodent NSCs [3,[12][13] and in human adult mesenchymal stem cells [14], the present work provides the first demonstration that human adult NS/PCs arising from normal brain may be tumorigenic in vivo. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Tumorigenic human adult NS/PCs arising from an OB adjacent to meningioma The OB was harvested from five adult patients who had undergone surgery for extracerebral benign lesions (Table S1).Unilateral division of the OB, which is often necessary for surgical exposure, is well tolerated by the patients because the olfactory function is preserved.Immunohistochemistry showed that the human adult OB contains a few hundreds of putative NS/PCs (Fig. 1).Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF.Under these conditions, the OB cells generated primary neurospheres with latencies that ranged from 6 to 8 weeks (Table S2).An exception was Case OB3 where primary neurosphere formation was observed as early as 3 weeks of culturing.Primary neurospheres were dissociated into single cells and plated one cell per mini-well (Fig. 1).Clonal cell cultures were established by dissociation of secondary neurospheres and passaged up to P30 in mitogens.The ability to form spheres after serial passaging, the number and diameter of spheres produced during each passage, and the cloning efficiency were similar among different cultures (Table S2).Upon removal of mitogens and serum exposure, the NS/PC cultures obtained from four of the OB specimens arrested their growth and gave rise to adherent cells that expressed neuronal, astrocytic, and oligodendrocytic markers (Figs. 2A-2B).In contrast, OB3 NS/PC cultures lost both growth factor dependence and potential to differentiate as neurons between P4 and P6.Notably, the OB3 patient harbored a meningioma adjacent to the OB.Losing growth factor dependence and capacity to differentiate by NSCs may indicate transformation.On soft agar assay, an in vitro correlate of transformation, the OB3 NS/PCs developed colonies (Fig. S2).Then, we assessed tumorigenicity in vivo using hetero-and orthotopic xenografts in immunodeficient mice.Two to 3 weeks after grafting, NS/PCs from all OB3 cultures developed subcutaneous tumors with a 88.6 percent take (Fig. 2C and Table 1).Histologically, these tumors showed glioblastoma features, like perinecrotic pseudo-palisading and vascular proliferation.Tumorigenicity of OB3 NS/PCs was demonstrated both at early (P6) and at late passages (P30).Subcutaneous injection of OB1, OB2, OB4, and OB5 NS/PCs resulted in amorphous tissue grafts with embedded scarce cells showing heterogenous morphology and occasional GFAP staining without neoplastic features (not shown).Intracerebral injection of OB3 NS/PCs also produced tumors which developed at 63.1 percent of injection sites by 4 to 6 weeks after grafting (Fig. 2D and Table 1).Histologically, these tumors featured anaplastic astrocytoma with predilection for growing into the ventricles.Intracerebral injection of OB1, OB2, OB4, and OB5-derived NS/PCs did not result in tumor formation (Table 1). In principle, taking NS/PCs from patients with pre-existing tumors nearby the organ where the cells are obtained is inappropriate.For example, human adult non-tumorigenic NSCs surrounding low-grade glioma tissue transform in vitro into highly tumorigenic cancer stem cells [15].In patient OB3, errant meningioma cells infiltrating the OB or adhering to its surface might have overwhelmed the NS/PCs in culture.This hypothesis, however, seems unlikely because of the following, 1) the phenotype of meningioma cells (EMA+/GFAP2/NG22/O42) differed both from that of OB3 NS/PCs (EMA2/GFAP+/NG2+/O4+) and from OB3-derived tumor xenografts (EMA2/GFAP+/ NG22/O42); 2) sphere generation in serum-free cultures occurs in glioblastoma, anaplastic astrocytoma, medulloblastoma, and ependymoma but not in meningioma, and 3) meningioma-derived NS/PCs are expected to develop xenografts with the histological appearance of meningioma or sarcoma not of glioblastoma.In brain pathology, concurrent adjacent meningioma and astrocytic tumors have been described raising the hypothesis that meningioma-released agents may work as growth factors for the glial cells of surrounding brain tissue [16].Thus, the NS/PCs resident in the OB adjacent to meningioma may undergo chronic pressure for growth becoming highly sensitive to mitogens in vitro. ",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "Transplantation technologies of adult human NS/PCs imply strategies where minimal donor material is highly expanded in vitro to the adequate cell number before implantation.In general, NSCs can be expanded either as floating neurospheres in serumfree medium supplemented with mitogens or as adherent monolayers in medium containing both mitogens and serum [17].Neuronal and oligodendroglial differentiation of adherently growing NSCs can be enhanced by growth factor withdrawal and exposure to triiodothyronine (T3) and ascorbic acid [18].Then, we propagated GFP-positive OB1, OB2, OB4, and OB5 NS/PCs between P7 and P10 either under mitogens or under mitogens and 5% serum (Fig. 1).In mitogens and serum, the NS/PCs became adherent, continued to proliferate, and either maintained an undifferentiated phenotype or differentiated, mainly as astrocytes (Fig. 3A).When such adherent serum-stimulated (SS) NS/PCs were returned to serum-free medium with mitogens, they formed floating neurospheres within one week maintaining their clonal efficiency.Upon removal of mitogens and exposure to 1% serum supplemented with T3 and retinoic acid, the SS-NS/PCs slowered down their growth and further differentiated towards the neuronal, astrocytic, and oligodendrocytic lineages (Fig. 3B).Aberrant coexpression of neuronal and glial markers by the SS-NS/PCs was not seen. To examine the behavior of NS/PCs in the CNS environment, we engrafted GFP-positive NS/PCs, which had been expanded either as neurospheres or as adherent monolayers, onto the spinal cord of ciclosporine treated rats or onto the striatum of SCID mice.Surprisingly, 85.7 percent of the rats engrafted onto the spinal cord with the clonal SS-OB2a NS/PCs showed progressive palsy of their hindlimbs by 2 to 4 weeks after grafting.These animals developed highly infiltrating intramedullary tumors that histologically were reminiscent of neuroesthesioblastoma, a malignant neoplasm of the OB that is supposed to arise from an ancestral neuroblast (Fig. 3C and Table 1).The tumor xenografts expressed markers for neuronal, astrocyte, and oligodendrocyte cells.Intracerebral grafting of the SS-OB2a NS/PCs also resulted in tumor formation with 76.9 percent take (Fig. 3D and Table 1).Importantly, the clonally-derived OB2a NS/PCs which had been expanded as neurospheres homed in the host parenchyma showing ramified morphology and neuronal marker expression without generating any tumor (Figs.3C-3D).Similar findings were seen in animals engrafted with OB1, OB4, and OB5 NS/PCs irrespective the technique used for their propagation in vitro.Thus, the oncogenic transformation of human adult NSCs may occur whether a combination of expansion/ selection stimuli, like mitogens and serum, are simultaneously applied to these cells in vitro.Consistently, mouse embryonic NS The OB was obtained from adult patients who underwent neurosurgical operations.On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133.The nestinexpressing cells colocalize glial fibrillary acid protein (GFAP).These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells.In the external plexiform layer, a few proliferating cells (n, 200-300) are detected by Ki67 labeling.Dissociated OB specimens were cultured in serumfree medium supplemented with the mitogens EGF and bFGF.Primary neurospheres were dissociated into single cells and plated one cell per miniwell.Clonal cell cultures were established by dissociation of secondary neurospheres.Clonal cultures from each OB were passaged up to P30 in mitogens.NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo.At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP.The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents.doi:10.1371/journal.pone.0004434.g001 ",
"section_name": "Transformation of human adult NS/PCs following propagation in mitogens and serum",
"section_num": null
},
{
"section_content": "Somatic stem cells are thought to possess proficient mechanisms that allow replicative potential and chromosomal stability.However, chromosomal rearrangements have been detected in long-term expanded adult murine NSCs which apparently do not result in a malignant phenotype [2].We performed kariotype analysis on NS/PCs at regular time points and found chromosomal rearrangements with repeated passages under mitogen stimulation (Figs.4A andS1).Chromosomal changes were found both in tumorigenic and in non-tumorigenic NS/PCs suggesting that these cells need additional requirements to achieve tumorigenicity in vivo.It has been reported that the tumor-like growth properties of the stem cells associate with changes either in oncosuppressors or in oncogenes [2,[5][6]15].Then, we set up a custom real-time RT-PCR array to analyze the expression of 92 mRNAs related with cell proliferation and cancer in tumorigenic relative to non tumorigenic NS/PCs both under proliferating culture conditions and under serum-induced differentiation (Fig. 4B).Relative to the non tumorigenic OB1a and SS-OB1a NS/PCs, tumorigenic OB3a and SS-OB2a NS/PCs showed upregulation of genes related to cell proliferation and inhibiting apoptosis, though solely hTERT and NOTCH1 were overexpressed independently from mitogen stimulation.Tumorigenic OB3a and SS-OB2a NS/PCs did express the hTERT protein, which was undetectable in non tumorigenic NS/PCs, consistent with that reported in normal NS cells (Fig. 4C) [19].Immunofluorescence with anti-NOTCH1 antibody on tumorigenic OB3a and SS-OB2a NS/PCs demonstrated either increased cytoplasmic staining or abnormal nuclear staining (Fig. 4D).Following NOTCH1 blockade with the c-secretase inhibitor X (GSI), OB3a and SS-OB2a NS/PCs lost their ability to form soft-agar colonies suggesting a functional role of NOTCH1 in tumorigenicity of these cells (Figs. 4C andS2).Although the xenografts grown after injection of OB3a and SS-OB2a NS/PCs were histologically reminiscent of different tumors, in both of them molecular analyses pointed to hTERT and NOTCH1 as critical pathways.Telomerase is highly expressed in the majority of human cancers including glioblastoma, where it is believed to contribute to tumor progression because telomerase-dependent telomere maintenance provides cells with an extended proliferative potential [20].Glioblastoma stem cells, which express telomerase under proliferating serum-free conditions, transiently lose telomerase activity in serum-containing media; however, these cells regain telomerase at passages coincident with their exponential growth phase [21].NOTCH is known to promote the proliferation of nonneoplastic NSCs and to inhibit their differentiation; it is also highly activated in embryonal brain tumors, such as medulloblastoma, where it is required both for maintaining the stem cell fraction in vitro and for tumor formation in vivo [22].Up-regulation of hTERT and NOTCH1 in both tumorigenic OB3a and SS-OB2a NS/PCs suggests that a common mechanism may underly the malignant transformation of these cells, and that the histological differences between the OB3a-derived glioblastoma and the SS-OB2a-derived neuroesthesioblastoma may reflect different stages at which the NS/PCs have undergone neoplastic transformation in culture.In the OB3a-derived glioblastoma, the tumorigenic hit may have occurred in an astrocytic-committed precursor cell, whilst in the SS-OB2a-derived neuroesthesioblastoma the cell of origin may be a less differentiated NS/PC that has retained its multipotentiality. Gene therapy trials using human hematopoietic stem cells after retroviral transduction have demonstrated a risk of insertional mutagenesis and oncogenic transformation [23].However, we do not believe that that the tumorigenic transformation of the OBderived NS/PCs may be a consequence of the use of the lentiviral vector that integrated the GFP gene into the genome of these cells.The following arguments do not favour this hypothesis, 1) the OB3 cells, which in vivo gave origin to glioblastoma-like tumors, were not transduced with lentivirus to express GFP; 2) both the OB2 cells and the SS-OB2 cells were transduced with lentivirus, however, only the latter cells developed tumor in vivo, whilst the GFP-positive OB2 cells did not; and 3) the lentivirally transduced OB1, OB4, and OB5 NS/PCs were not tumorigenic in vivo. To conclude, human adult NS/PCs cultured under mitogen stimulation are prone to develop chromosomal rearrangements.In vivo tumorigenicity is heralded by, 1) short latency in primary neurosphere formation, 2) persistent growth after removal of mitogens, 3) loss of serum-induced neuronal differentiation, and 4) up-regulation of hTERT and NOTCH1.The tumorigenic transformation of human adult NS/PCs isolated from an OB adjacent to meningioma raises the possibility that unusual levels of growth factors in the in situ condition, i.e. prior to ex vivo culture, may prime tumorigenicity.This indicates that the tumorigenic potential of the OB3 NS/PCs may be a specific feature of this cell line and not generalizable.In the case of SS-OB2 NS/PCs, however, specific culture conditions seem critical to transformation.Mitogens used simultaneously with factors favouring cell specification may disrupt the regulatory mechanisms that control self-renewal of NSCs and differentiation of TAPs.Therefore, culturing techniques where both proliferation and differentiation of NS/PCs are simultaneously enhanced should be evaluated further in future and discouraged if confirmed as linked to in vivo tumorigenicity. ",
"section_name": "Cytological and molecular characteristics of tumorigenic NS/PCs",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "The OB was harvested from adult patients undergoing craniotomy at the Institute of Neurosurgery, Catholic University, Rome (Table S1).Informed consent was obtained according to protocols approved by the Ethical Commettee of the Catholic University.Immediately after removal, the OBs were dissociated in Papain 0,1% (Sigma-Aldrich, St. Louis, MO) for 30 minutes at PCs (P6) cultured in serum-free medium supplemented with mitogens (left) and in medium containing 1% serum without mitogens (right).Doublepositive cells for nestin and GFAP were counted positive for each antigen and also for both antigens (nestin/GFAP).The OB3a cells do not differentiate towards the neuronal lineage in response to serum stimulation.C, Subcutaneous xenografs of OB3a cells in nude athymic mice.a, Subcutaneous nodules two weeks after grafting (arrows in a).Histological features of glioblastoma (b, H&E).Expression of astrocytic cell marker GFAP (c) and negative staining for the neuronal cell marker neurofilament (d).D, Intracerebral tumor xenografts of OB3a cells in SCID mice.Pattern of brain invasion by OB3a cells one week after grafting into the striatum (a, H&E).Low (b) and high (c and d) magnifications of intraventricular anaplastic astrocytoma-like tumor by two weeks after grafting (b and c, H&E d, anti-HNA immunoreaction).A, Scale bar 250 mm; b, Scale bar 80 mm; c, Scale bar 50 mm; d, Scale bar 30 mm. doi:10.1371/journal.pone.0004434.g00237uC.Dissociated cells were cultured in the presence of human recombinant EGF (20 ng/ml; PeproTech, Rocky Hill, NJ), human recombinant bFGF (10 ng/ml; PeproTech), and LIF (20 ng/ml; Immunological Sciences, Rome, Italy) in DMEM/F12 (1:1) serum-free medium (Invitrogen, Carlsband, CA) containing Lglutamine 2 mM, glucose 0.6%, putrescine 9.6 ug/ml, progesterone 0.025 mg/ml, sodium selenite 5.2 ng/ml, insulin 0.025 mg/ ml, apo-transferrin sodium salt 0.1 mg/ml, sodium bicarbonate 3 mM, Hepes 5 mM, BSA 4 mg/ml, heparin 4 ug/ml [24].Primary neurospheres were dissociated with Accutase (Invitrogen) for 4 minutes at 37uC, serially diluted, and plated one cell per mini-well onto 96-well plates.Mini-wells containg one single cell were marked after microscopic confirmation and assessed for secondary neurosphere generation after one week.Secondary neurospheres were subsequently dissociated, plated at the density of 10 3 cells/cm 2 in serum-free medium containing EGF and bFGF, and passaged up to P30.All experiments were done on at least two clonal cultures from each OB.Between P7 and P10, parallel cultures were established in which cells were grown as adherent monolayers in medium containing EGF and bFGF supplemented with 5% fetal calf serum (Hyclone, Logan, UT).For cell growth experiments, dissociated cells were plated on Matrigel at the density of 10 3 cells/cm 2 either in serum-free medium containing EGF and bFGF or in medium where mitogens were replaced with 1% serum or in medium containing mitogens and 5% serum (Hyclone).Cells were counted with hemacytometer every 48 hours.Cell viability was determined colorimetrically by MTS-assay (Supplementary Methods S1).Differentiation assays were performed by 14 days after plating on Matrigel coated glass coverslips in the absence of EGF and bFGF and in the presence of 1% fetal calf serum (Hyclone) supplemented with 39-59-cyclic adenosine monophosphate (cAMP) 50 mM, all-trans retinoic acid 5 mM (Sigma Aldrich), and triiodothyronine (T3) 30 nM (Sigma Aldrich).Immunostaining of NS/PCs was performed as described [15].We used antibodies against nestin (Chemicon, Temecula, CA), CD133 (CD133/2; Miltenyi, Bergisch, Germany), GFAP (Dako, Glostrup, Denmark), b tubulin III (Chemicon), neurofilament RT-97 (Developmental Studies Hybridoma Bank, Iowa City, IA), MAP2 (Chemicon), NG2 (Chemicon), O4 (Chemicon), hTERT (Novocastra Laboratories), and NOTCH1 (Chemicon). ",
"section_name": "Isolation, Culturing, and Immunophenotyping of NS/PCs",
"section_num": null
},
{
"section_content": "Enhanced green fluorescent protein (GFP) gene transfer in the NS/PCs was performed at P6 using a variant of third generation lentiviral vectors as described [25]. ",
"section_name": "Generation of Fluorescent NS/PCs",
"section_num": null
},
{
"section_content": "Studies involving animals were approved by the Ethical Committee of the Catholic University School of Medicine, Rome.The NS/PCs were grafted either subcutaneously in nude athymic mice, or into the brain of severe combined immunodeficient (SCID) mice, or onto the spinal cord of ciclosporine treated rats (Supplementary Methods S1).For implantation, the NS/PC cultures were splitted 24-48 hours prior to transplant and injected as single cell suspensions.After two to 6-week survival, the animals were sacrificed with an overdose of barbiturate.Either the subcutaneous graft or brain or spinal cord was removed and processed for histology as described [25]. ",
"section_name": "Grafting of NS/PCs in Immunodeficient Rodents",
"section_num": null
},
{
"section_content": "Immunohistochemistry was performed on deparaffinized sections using the avidin-biotin-peroxidase complex methods as ).Gene expression in tumorigenic OB3a and SS-OB2a cells is presented relative to the non tumorigenic OB1a and SS-OB1a cells, respectively, both under proliferating culture conditions and under serum-induced differentiation.Upregulated genes (2 folds, red), downregulated genes (2 folds, green), unchanged genes (gray).C, Expression of hTERT protein in OB-derived NS/PCs.Western blot analysis of HUVEC (lane 1), OB1a (lane 2), OB2a (lane 3), SS-OB2a (lane 4), OB3a (lane 5), and TB10 human glioblastoma (lane 6) cells.Immunohistochemical analysis of hTERT expression in the human adult OB and OB-derived NS/PCs.The hTERT protein is absent in the adult OB (a) as well as in the non-tumorigenic OB1a (b), OB2a (c), and SS-OB1a (e) cells.hTERT is strongly expressed in the nuclei of both tumorigenic OB3a (d) and SS-OB2a (f) NS/PCs.Scale bar 40 mm.D, Immunofluorescence analysis of NOTCH1 expression (left) in OB1a (a), OB3a (b), OB2a (c), and SS-OB2a (d) NS/PCs.NOTCH1 signaling is required for the formation of colonies in soft agar (right).Exposure to c-secretase inhibior X (GSI) after seeding in soft agar significantly reduced clonigenic potential of tumorigenic OB3a and SS-OB2a NS/PCs (P,0.0001,Student t-test).doi:10.1371/journal.pone.0004434.g004 described [25].The following primary antibodies were used, anti-GFAP (Ylem, Avezzano, Italy), anti-neurofilament (Ylem), anti-NG2 (Chemicon), anti-CD133/1 (Miltenyi), anti-nestin (Santa Cruz Biotecnology), anti-human nuclei antigen, (HNA; Chemicon), anti-epithelial membrane antigen (EMA; Ylem), anti-Ki67 (Dakocytomation), anti-hTERT (Novocastra Laboratories).Endogenous biotin was saturated by biotin blocking kit (Vector).For antigen retrieval, paraffin sections were microwave-treated in 0.01 M citric acid buffer at pH 6.0 for 10 min.For hTERT antigen retrieval, paraffin section were microwave-treated in EDTA buffer at pH 8.0 for 10 min. ",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "NS/PC cultures at P3-P8 were incubated in medium containing 10 ng/ml colcemid for 18 hours.The cultures were then lifted and centrifuged.Pellets were osmotically shocked with 0.075 M KCl and fixed with 3:1 methanol:glacial acetic acid.Standard cytogenetic G bands were performed and a mean of 20 methaphases per cell lines were analyzed. ",
"section_name": "Chromosome analysis",
"section_num": null
},
{
"section_content": "We used a 7900HT instrument equipped with SDS2.2 software to perform a custom real-time RT-PCR array (Microfluidic Card, Applied Biosystems, CA).Briefly, cells were plated on Matrigel precoated 100 mm dishes and processed as described above.Preparation of total RNA and cDNA was performed using Ribo Pure kit (Ambion, Austin, TX) and high capacity cDNA Reverse Transcriptase kit (Applied Biosystems), respectively.For data analysis, the mathematical process for deriving relative quantification values was used as described by the manufacturer's guide (Applied Biosystems). ",
"section_name": "Macroarray Analysis",
"section_num": null
},
{
"section_content": "Cell pellets were lysated in a modified RIPA buffer (Tris-HCl 10 mM pH 7.5, NaCl 10 mM, NP-40 0.2%, EGTA 1 mM, EDTA 1 mM, DTT 1 mM and protease inhibitor cocktail; Sigma-Aldrich) on ice for 5-10 min.Nuclear extracts were resuspended in Urea buffer (10 M Urea, Tris-HCl 50 mM pH 7.5, DTT 25 mM) sonicated and normalized using Bradford Assay (Promega Corp).Protein extracts were analyzed by polyacrylamide gel electrophoresis and Western blot.Proteins were probed with rabbit polyclonal anti-TERT (1:1000; Santa Cruz Biotecnology) and monoclonal anti-b-actin (1:5000; Sigma-Aldrich).As control, HUVEC cells at passages 4 to 5 (Bio-Wittaker, Walkersville, MD) and TB10 glioblastoma cells were used. ",
"section_name": "Western Blot",
"section_num": null
},
{
"section_content": "Table S1 Found at: doi:10.1371/journal.pone.0004434.s001(0.02 MB DOC) .The OB2a, SS-OB2a, and OB3a NS/PCs were seeded with a mixture of Top Agar (0,5%)-proliferation medium on top of the base layer.The plates were then incubated at 37u in humidified incubator for 3-4 weeks and colonies were counted.Every week fresh medium mixed with Top-agar was added together with 5 mmol/L csecretase inhibitor X (GSI; L-685.458) or DMSO as control.Three plates for each NSC/PC culture were used.Found at: doi:10.1371/journal.pone.0004434.s005(9.58 MB TIF) ",
"section_name": "Supporting Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Table S2",
"section_num": null
}
] |
[
{
"section_content": "We thank Teresa M. Natale for her contribution in karyotype analysis. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by grants from Fondi d9Ateneo (Progetti D1), from ATENA Onlus, and Nando Peretti Foundations, and from Mrs. Paola Krajnik.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: PC GM LML RP.Performed the experiments: PC MB LRV GP NM ET.Analyzed the data: LM.Contributed reagents/materials/analysis tools: CC.Wrote the paper: RP. ",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.3390/cancers14010207
|
Efficacy of Front-Line Ibrutinib and Rituximab Combination and the Impact of Treatment Discontinuation in Unfit Patients with Chronic Lymphocytic Leukemia: Results of the Gimema LLC1114 Study
|
<jats:p>The GIMEMA group investigated the efficacy, safety, and rates of discontinuations of the ibrutinib and rituximab regimen in previously untreated and unfit patients with chronic lymphocytic leukemia (CLL). Treatment consisted of ibrutinib, 420 mg daily, and until disease progression, and rituximab (375 mg/sqm, given weekly on week 1–4 of month 1 and day 1 of months 2–6). This study included 146 patients with a median age of 73 years, with IGHV unmutated in 56.9% and TP53 disrupted in 22.2%. The OR, CR, and 48-month PFS rates were 87%, 22.6%, and 77%, respectively. Responses with undetectable MRD were observed in 6.2% of all patients and 27% of CR patients. TP53 disruption (HR 2.47; p = 0.03) and B-symptoms (HR 2.91; p = 0.02) showed a significant and independent impact on PFS. The 48-month cumulative rates of treatment discontinuations due to disease progression (DP) or adverse events (AEs) were 5.6% and 29.1%, respectively. AEs leading more frequently to treatment discontinuation were atrial fibrillation in 8% of patients, infections in 8%, and non-skin cancers in 6%. Discontinuation rates due to AEs were higher in male patients (HR: 0.46; p = 0.05), patients aged ≥70 years (HR 5.43, p = 0.0017), and were managed at centers that enrolled <5 patients (HR 5.1, p = 0.04). Patients who discontinued ibrutinib due to an AE showed a 24-month next treatment-free survival rate of 63%. In conclusion, ibrutinib and rituximab combination was an effective front-line treatment with sustained disease control in more than half of unfit patients with CLL. Careful monitoring is recommended to prevent and manage AEs in this patient population.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemia in the adult population.About 21,250 new cases of CLL have been estimated in the United States Cancers 2022, 14, 207 3 of 14 for 2021.CLL mainly affects aged subjects, with an average age at diagnosis of around 70 years [1].During the last years, relevant advances in the understanding of the biologic mechanisms associated with the proliferation and survival of CLL cells have led to the clinical use of ibrutinib, a small molecule that inhibits the Bruton tyrosine kinase (BTK).From the first studies, ibrutinib has been proven to be highly effective, regardless of age, prior treatment, and high-risk biologic features of the leukemic cell [2,3].After that, several randomized trials demonstrated the superiority over chemoimmunotherapy of front-line ibrutinib as a single agent, or combined with an anti-CD20 monoclonal antibody [4][5][6][7].The excellent therapeutic activity of this agent has revolutionized the treatment approach of CLL, and today, ibrutinib is a standard of care for CLL patients of all ages, both in the relapsed/refractory and in front-line settings.However, despite the excellent response rates and prolonged responses, treatment discontinuation, mainly due to adverse events (AE), is a relevant problem limiting the effectiveness of this agent [8][9][10]. Based on the improved outcomes observed with the addition of rituximab to chemotherapy [11][12][13][14] and on the efficacy of ibrutinib and rituximab [15], the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) group, in 2015, started a prospective, multicenter study to investigate the safety and efficacy of a front-line treatment, consisting of six courses of the ibrutinib and rituximab combination followed by ibrutinib single agent, in unfit patients with CLL.Herein, we report the long-term results of this schedule in 146 unfit patients with CLL, the safety profile of treatment, and the reasons and prognostic impact of treatment discontinuation. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Methods",
"section_num": "2."
},
{
"section_content": "Between March 2015 and April 2017, 159 unfit patients with CLL were enrolled in the GIMEMA LLC1114 study, a prospective, phase 2, multicenter, single-arm study.Inclusion criteria included previously untreated CLL requiring treatment according to the International Workshop on CLL (iwCLL) criteria [16].Patients were defined as unfit in the presence of a Cumulative Illness Rating Scale (CIRS) [17] score >6, and or a creatinine clearance <70 mL/min. In addition, the absence of Richter transformation, active infection, or secondary malignancy was also required in patients enrolled in the study.The assessment of the biologic profile included fluorescence-in-situ-hybridization (FISH) and the IGHV and TP53 mutation status as previously described [18,19]. ",
"section_name": "Patients",
"section_num": "2.1."
},
{
"section_content": "Treatment consisted of ibrutinib, 420 mg once daily given continuously, and rituximab, 375 mg/sqm, every week, on day 1 of month 1 and day 1 of months 2-6.Patients received ibrutinib single agent until one of the following events disease progression or severe toxicity, or for a maximum of 6 years. All patients received Pneumocystis carinii prophylaxis with trimethoprim-sulfamethoxazole. ",
"section_name": "Treatment",
"section_num": "2.2."
},
{
"section_content": "The response was assessed according to the iwCLL criteria [16] 2 months after the last administration of rituximab.The response assessment included clinical examination, PB examination, BM aspirate and biopsy, and total body CT scan.In patients who achieved a complete response (CR), a centralized assessment of MRD, in both the PB and BM, was performed by an eight-color flow cytometry assay with a sensitivity of at least 10 -4 according to the internationally standardized European Research Initiative on CLL criteria [20].MRD was further assessed in PB and BM by allele-specific oligonucleotide polymerase chain reaction (PCR) in patients in complete remission (CR) with undetectable MRD (uMRD) by flow cytometry.The response was monitored every 6 months during the follow-up. ",
"section_name": "Response",
"section_num": "2.3."
},
{
"section_content": "The primary endpoint of the study was progression-free survival (PFS).The secondary endpoints included the overall responses rate (ORR), the CR rate, the rate of CRs with undetectable MRD (uMRD) in the PB and BM, overall survival (OS), and survival outcomes according to the clinical and biological features of the patients.The safety profile and reasons for permanent discontinuation of treatment were also analyzed.AEs were graded according to the Common Terminology Criteria for Adverse Events, version 3 [21]. ",
"section_name": "Study Endpoints",
"section_num": "2.4."
},
{
"section_content": "Patients' characteristics were summarized using cross-tabulations for categorical variables or using quantiles for continuous variables.In the univariate analysis, non-parametric tests were performed for comparisons between groups (Chi-Squared and Fisher Exact test in the case of categorical variables or response rate; Mann-Whitney and Kruskal-Wallis tests in case of continuous variables).Survival distributions were estimated using the Kaplan-Meier Product Limit estimator.Differences in survival curves were evaluated using the Log-Rank test.Cox regression models were performed in univariate and multivariate analyses to assess the effect of clinical and biologic factors on PFS and OS.Hazard ratios (HR) and a 95% confidence interval were reported as parameter results of the Cox regression models.The multivariate models were all considered relevant. Curves of the cumulative incidence of treatment discontinuations by specific causes (e.g., adverse events) were estimated using the proper non-parametric method.The Gray test was applied for comparing the curves of cumulative incidence and the Fine and Gray regression model was used in the univariate and multivariate analyses to assess the effects of covariates on the survival outcome in cases of competitive risks. All analyses were analyzed on an Intention-To-Treat basis.All tests were two-sided, accepting p < 0.05 as indicating a statistically significant difference.Confidence intervals were calculated at the 95% level.All of the analyses were performed using the SAS software (release 9.4; SAS Campus Drive, Cary, NC 27513, USA) and R system software (R Foundation for Statistical Computing c/o Institute for Statistics and Mathematics, Wirtschaftsuniversität, 1020 Wien, Austria).Details about data collection are reported in Supplementary Figure S1. ",
"section_name": "Statistical Analysis",
"section_num": "2.5."
},
{
"section_content": "This study has been carried out according to the Helsinki Declaration and was approved by the Ethical Committees of all of the participating institutions.All of the participants gave their written informed consent.This study is registered at ClinicalTrials gov, Identifier: NCT02232386. ",
"section_name": "Ethics",
"section_num": "2.6."
},
{
"section_content": "One hundred and fifty-nine CLL patients were enrolled in this study.The patient disposition is described in Supplementary Figure S1.Thirteen patients were considered not eligible and were excluded from the study before receiving any study drug (not eligible, 8; AE, 2; death, 1; refused treatment, 1; medical decision, 1).One hundred and forty-six patients with a median follow-up of 49.1 months (IQR, 39.4-54) represent the intentionto-treat population assessed for treatment response and safety.The baseline clinical and biological characteristics of patients are summarized in Table 1.Briefly, the median age was 73 years (range 37-88), the median CIRS score 6, the median creatinine clearance 62.7 mL/min, and 37.9% of the patients had an ECOG performance score of 1-2.Unmutated IGHV status was observed in 56.9% of patients and TP53 disruption (del17p and/or TP53 mutation) in 22.2%. ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Patients received a median number of six courses of the ibrutinib and rituximab combination (range 1-6), and 137 (94%) completed the planned six courses of treatment. Here, 127/146 patients (87%) achieved a response at the end of the ibrutinib and rituximab combination.Responses were confirmed by CT scan and included a complete response (CR/CRi) in 33 (22.6%) patients, a partial response (PR) in 76 (52.1%), and a PR with lymphocytosis (PR-L) in 18 (12.3%). In an ITT analysis, 9 of the 146 patients (6.2%) obtained a flow-cytometric uMRD at one or more time points, three at the EOCT, and six during the follow-up.When the analysis was restricted to the 33 patients with CR, the rate of patients with uMRD was 27.3% (9/33) 12.1%) it persisted for 6, 52, 52, and 54 months.Three of the nine patients with uMRD by flow-cytometry showed no residual disease in the PB also by ASO-PCR at one or more time points. Ten (6.9%) patients showed stable disease, one progressed (0.6%) while eight (5.5%) discontinued the ibrutinib and rituximab combination (adverse event, 7; second malignancy, 1). ",
"section_name": "Response to Treatment",
"section_num": "3.1."
},
{
"section_content": "Ten (6.9%) patients developed disease progression (CLL progression, 9; Richter syndrome, 1) with a 48-month PFS of 77% (95% CI 70.2-85.0)(Figure 1A). In an ITT analysis, 9 of the 146 patients (6.2%) obtained a flow-cytometric uMRD at one or more time points, three at the EOCT, and six during the follow-up.When the analysis was restricted to the 33 patients with CR, the rate of patients with uMRD was 27.3% (9/33).While uMRD was transient in five patients, in four (4/146, 2.7%; 4/33 patients with CR, 12.1%) it persisted for 6, 52, 52, and 54 months.Three of the nine patients with uMRD by flow-cytometry showed no residual disease in the PB also by ASO-PCR at one or more time points. Ten (6.9%) patients showed stable disease, one progressed (0.6%) while eight (5.5%) discontinued the ibrutinib and rituximab combination (adverse event, 7; second malignancy, 1). ",
"section_name": "Survival Analysis",
"section_num": "3.2."
},
{
"section_content": "Ten (6.9%) patients developed disease progression (CLL progression, 9; Richter syndrome, 1) with a 48-month PFS of 77% (95% CI 70.2-85.0)(Figure 1A).In the multivariate analysis, age (≥70 vs. <70 years), CIRS (≥8 vs. <8), Binet stage (C vs. A/B), CrCl, ml/min (≥70 vs. <70), LDH (increased vs. normal), IGHV (unmutated vs. mutated), and del 11q (present vs. absent) did not show a significant impact on PFS, while B-symptoms and TP53 disruption emerged as the only independent factors associated with a significantly shorter PFS (Table 2).In the multivariate analysis, age (≥70 vs. <70 years), CIRS (≥8 vs. <8), Binet stage (C vs. A/B), CrCl, ml/min (≥70 vs. <70), LDH (increased vs. normal), IGHV (unmutated vs. mutated), and del 11q (present vs. absent) did not show a significant impact on PFS, while B-symptoms and TP53 disruption emerged as the only independent factors associated with a significantly shorter PFS (Table 2).Twelve patients died, seven because of an adverse event (AE; heart failure, 1; severe infection, 5; liver failure, 1), four due to a second malignancy, and one due to disease progression.The 48-month OS rate was 90% (95% CI 84.7-95.3; Figure 1B).High LDH levels (p = 0.03), B symptoms (p = 0.03), and TP53 disruption (p = 0.04) showed a significant impact on OS in the univariate analysis (Supplementary Table S1).However, none of these factors maintained significance in the multivariate analysis. ",
"section_name": "Survival Analysis",
"section_num": "3.2."
},
{
"section_content": "The type and severity of the AEs recorded in this study are described in Supplementary Table S2.Grade 3-4 granulocytopenia, recorded in 27% of patients, was the most common AE leading to dose reduction or transient treatment interruption.At the last follow-up, the daily dose of ibrutinib received by the 80 patients still on treatment was 420 mg in 62 (77.5%), 280 mg in 14 (17.5%), and 140 mg in 5 (5%).Grade ≥3 infections were diagnosed in 18% of patients, and included lower respiratory tract infections in 8%, with three cases of lethal SARS-CoV-2 pneumonia.Any grade cardiovascular AEs were recorded in 30% of patients and included atrial fibrillation in 16% (grade 3-4 atrial fibrillation, 6%).New-onset hypertension was experienced by 13% of patients.Any grade bleeding disorders were observed in 23% of patients.However, severe bleeding events were uncommon (5%) and included cerebral hemorrhage in three cases.Other AEs frequently reported were any grade myalgias and arthralgias (16%), diarrhea (14%), and skin rash (10%).A non-skin second malignancy was diagnosed in 13 (9%) patients (gastric, 3; lung, 2; bladder, 2; breast, 1; mesothelioma, 1; neuroendocrine, 1; thyroid, 1; bowel, 1; hepatic, 1).No new safety signals or unknown/unwitnessed deaths were recorded. ",
"section_name": "Adverse Events",
"section_num": "3.3."
},
{
"section_content": "The main reason leading to the permanent discontinuation of treatment was represented by an AE, recorded in 44 (30.1%) patients.Treatment discontinuations rates due to AEs were 17.8% at 12 months, 23.3% at 24 months, 26.0% at 36 months, and 29.1% at 48 months.The median age of patients who discontinued ibrutinib permanently due to an AE was 78 years (range 56.8-90.2). Cardiovascular disorders were a common AE, leading to treatment discontinuation (11% of the cases, including atrial fibrillation in 8%), followed by infections (8%), non-skin cancers (6%), and cerebral hemorrhage, 3% (Supplementary Table S2).In the multivariate analysis, the male gender was significantly and independently associated with a higher rate of treatment discontinuations due to AEs (HR: 0.46; p = 0.05; Supplementary Table S3).Two other factors showed a significant and independent impact on discontinuations caused by AEs, aged older than 70 years (HR: 5.43; p = 0.002), and treatment managed at centers that enrolled less than five patients (HR: 0.51, p = 0.04).Based on an age older than 70 years and less than five patients enrolled by the referral centers, we identified three groups of patients.In the low-risk group, which included patients with none of the above risk factors, the rate of discontinuations was 11.8%; in the intermediate-risk group that included patients with one of the two risk factors, the rate was 28.3%, while for patients of the high-risk group who showed both risk factors, the rate of discontinuations was 52.5% (p = 0.001; Figure 2). ",
"section_name": "Adverse Events Leading to Permanent Treatment Discontinuation",
"section_num": "3.4."
},
{
"section_content": "At the time of the last follow-up, 80 (55%) patients, including 31 patients in CR (31/80, 39%; 31/146, 21.2%), were still on ibrutinib, while 66 (45%) discontinued treatment (Supplementary Figure S1).The 48-month cumulative rates of treatment discontinuation due to disease progression, AEs, and second malignancies were 5.6%, 29.1%, and 6%, respectively (Table 3 and Figure 3). ",
"section_name": "Prognostic Impact of Treatment Discontinuation",
"section_num": "3.5."
},
{
"section_content": "At the time of the last follow-up, 80 (55%) patients, including 31 patients in CR (31/80, 39%; 31/146, 21.2%), were still on ibrutinib, while 66 (45%) discontinued treatment (Supplementary Figure S1).The 48-month cumulative rates of treatment discontinuation due to disease progression, AEs, and second malignancies were 5.6%, 29.1%, and 6%, respectively (Table 3 and Figure 3).The 12-month survival rates of patients who permanently discontinued treatment due to AE, second malignancy, and disease progression were 85% (95% CI: 74.6-96.9),41.7% (95% CI: 14.7-100.0),and 33% (95% CI: 11.0-98.1),respectively (p = 0.01; Figure 4).The 12-month survival rates of patients who permanently discontinued treatment due to AE, second malignancy, and disease progression were 85% (95% CI: 74.6-96.9),41.7% (95% CI: 14.7-100.0),and 33% (95% CI: 11.0-98.1),respectively (p = 0.01; Figure 4).The 24-month next treatment-free survival rate of patients who discontinued ibrutinib due to AE was 63% (Supplementary Figure S2). ",
"section_name": "Prognostic Impact of Treatment Discontinuation",
"section_num": "3.5."
},
{
"section_content": "We investigated the benefit and safety of a front-line treatment with the ibrutinib and rituximab combination in an unfit cohort of CLL patients, defined by a CIRS comorbidity score >6 and/or a reduced renal function.The results of this study confirm the efficacy of this schedule in 146 patients with CLL and with a median age of 73 years.Furthermore, 87% of patients achieved a response, which included a CR in 22.6% of the cases.Moreover, 6.2% of all patients and 27% of CR patients showed a response with uMRD by flowcytometry.Although the absence of a control arm limits the results of this study, the relatively high CRs and PFS rates we observed, 77% at 48 months, were consistent with those of other trials investigating the efficacy of ibrutinib-based treatments in the frontline setting [4][5][6][7][15][16][17][18][19][20][21][22][23].The modulation of molecules interacting with the microenvironment produced by the treatment may have favored the fast mobilization of CLL cells [24].The low rate of disease progressions observed in our study, 10%, further confirmed that the emergence of ibrutinib-resistant subclones is rare in the front-line setting [4][5][6][7]. In this study, PFS was not significantly influenced by IGHV mutational status.Moreover, del (11q) or the achievement of CR did not exert the same beneficial impact on The 24-month next treatment-free survival rate of patients who discontinued ibrutinib due to AE was 63% (Supplementary Figure S2). ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "We investigated the benefit and safety of a front-line treatment with the ibrutinib and rituximab combination in an unfit cohort of CLL patients, defined by a CIRS comorbidity score >6 and/or a reduced renal function.The results of this study confirm the efficacy of this schedule in 146 patients with CLL and with a median age of 73 years.Furthermore, 87% of patients achieved a response, which included a CR in 22.6% of the cases.Moreover, 6.2% of all patients and 27% of CR patients showed a response with uMRD by flow-cytometry.Although the absence of a control arm limits the results of this study, the relatively high CRs and PFS rates we observed, 77% at 48 months, were consistent with those of other trials investigating the efficacy of ibrutinib-based treatments in the front-line setting [4][5][6][7][15][16][17][18][19][20][21][22][23].The modulation of molecules interacting with the microenvironment produced by the treatment may have favored the fast mobilization of CLL cells [24].The low rate of disease progressions observed in our study, 10%, further confirmed that the emergence of ibrutinibresistant subclones is rare in the front-line setting [4][5][6][7]. Cancers 2022, 14, 207 10 of 14 In this study, PFS was not significantly influenced by IGHV mutational status.Moreover, del (11q) or the achievement of CR did not exert the same beneficial impact on PFS described in other studies [25,26].As observed in the Alliance trial [6], patients with TP53 showed an inferior PFS.That being said, the 48-month PFS of 65% was higher than observed in the past with chemoimmunotherapy in this subset of patients, and is in line with that of other studies with ibrutinib in patients carrying TP53 disruption [27,28].A significantly lower PFS was associated with the presence of B symptoms.This finding underlines the unfavorable impact of symptomatic disease.In the study by Woyach et al., patients treated with ibrutinib and rituximab showed a PFS similar to those who received single-agent ibrutinib [6].The PFS value we observed with ibrutinib and rituximab was not superior to that described in other studies with an ibrutinib single agent [4,23].This observation further questions the benefit of adding rituximab to ibrutinib. In this patient population, treatment discontinuations due to AE were frequent, with a 48-month cumulative rate of 29.1%.This was not an unexpected finding in patients already older and unfit at baseline.In two trials that included younger patients, the discontinuation rates due to AEs were 19.1% at 4 years and 21% at 5 years, respectively [8,22].In a retrospective analysis that included 616 patients with CLL, toxicities were also the most common reason for treatment discontinuation [10].Variable rates of ibrutinib discontinuations have also been reported in real-world studies [29][30][31][32][33].It is noteworthy that treatment discontinuations rates due to AEs were lower with fixed-duration venetoclax combined with obinutuzumab or rituximab [34,35]. An intriguing finding was the relatively high 12-month survival rate, 85%, and the 24-month next treatment-free survival, 63%, of patients who discontinued treatment due to AE.Similar favorable outcomes have also been described in other studies [7,9,10]. Atrial fibrillation is a well-known AE associated with the use of ibrutinib [30].The rate of any grade atrial fibrillation was 16%, similar to that of other studies that included older patients treated front-line with ibrutinib [4,6]. Atrial fibrillation was the reason for treatment discontinuation in 8% of patients, a higher rate than previously reported [5,6,22,36].The characteristics of our patient population may have influenced the discontinuations rates due to atrial fibrillation and also to infections, in 8%.The impact of ibrutinib on cellular immunity has been extensively investigated, with conflicting results.While pre-clinical data described multiple inhibitory effects of ibrutinib on the activity of natural killer cells and macrophages [37,38], recent data suggest that ibrutinib may induce an in vivo immune modulation, with a TH2/TH1 shift in the peripheral blood lymphocytes that is more pronounced in IGHV unmutated and CR patients [39]. A higher rate of discontinuations due to AEs was observed among male patients.To the best of our knowledge, a relationship between sex and treatment discontinuation due to AEs has not been reported in patients treated with ibrutinib.The higher incidence of atrial fibrillation described in males may have had an impact on the increased rate of discontinuations.Older age, over 70 years, and treatment managed at centers that enrolled less than five patients were also associated with an increased rate of discontinuations due to AEs.The presence of both risk factors was associated with a 52.5% discontinuation rate.Older age plays an important role in developing AEs leading to treatment discontinuation.Increasing age is a risk factor for cardiovascular disorders, and the incidence of most cancers also increases with age.Moreover, functions of the immune system decline with age predisposing infections.As previously suggested [40,41], close collaboration withardio-oncologists and infectious disease specialists should be considered in order to avoid treatment discontinuations due to the toxicities of the targeted agents. Long-term follow-up data from studies will allow for evaluating whether secondgeneration BTK inhibitors or a time-limited therapy with venetoclax could be preferable in unfit and older patients. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In conclusion, this study shows that the ibrutinib and rituximab combination is an effective front-line treatment with sustained disease control in more than half of unfit and elderly patients with CLL.However, our data highlights the high rate of treatment discontinuations due to AEs and suggests careful monitoring to prevent and manage AEs in this patient population. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following are available online at https://www.mdpi.com/article/10.3390/cancers14010207/s1. Figure S1: Patients dispositions.Figure S2: Next treatment-free survival for patients who discontinued ibrutinib due to an adverse event.Table S1: Impact of baseline factors on overall survival: univariate analysis.Table S2: Adverse events.Table S3: Impact of clinical and biologic characteristics of patients on treatment discontinuation due to adverse events: univariate and multivariate analysis. Author Contributions: F.R.M., F.P., A.P., A.C. (Antonio Cuneo), R.F.: conception, design of the work, analysis, interpretation of data; drafting the work, revising it critically for important intellectual content; 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; final approval of the version to be published. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Azienda Ospedaliera Universitaria Policlinico Umberto I, Rome; Italy; protocol code: Rif.3455; date of approval: 27 November 2014. Informed Consent Statement: Informed written consent was obtained from all subjects involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
}
] |
[
{
"section_content": "The authors thank the patients and their families and the investigators of the participating centers. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Data Availability Statement: Study data were collected and managed using REDCap (Research Electronic Data Capture; Supplementary Material) a web-based software platform designed to support data capture for research studies.The data presented in this study are available on request.Qualified researchers may request access to anonymized patient data and study documents.Details on sharing criteria and processes for requesting access to data can be required to [email protected]. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Conflicts of Interest: F.R.M.: research funding from Gilead, advisory board participation fees from AbbVie, Gilead, Janssen, AstraZeneca, Takeda, Roche; speakers bureau fees from Gilead, Janssen, and Abbvie.P.S.: advisory board participation fees from Jansenn, Abbvie Astrazeneca.M.M.: consultancy: Gilead; paid speech: Novartis, Amgen; sponsored meetings: Abbvie, Takeda, Pfeitzer.R.M. (Roberto Marasca): Janssen e Abbvie: honoraria and travel grant.Astrazeneca: honoraria.G.G.: Abbvie (Advisory Board, Speaker's Bureau), Astra-Zeneca (Advisory Board), Beigene (Advisory Board), Incyte (Advisory Board), Janssen (Advisory Board, Speaker's Bureau).M.C.: Janssen honoraria and research funding), Abbvie and AstraZeneca (honoraria).A.M.L.: Research grants from: Takeda, Servier, Roche, Celgene, Abbvie, Incyte, Janssen.Sanofi, Verastem, Novartis, Morphosys, GSK, Oncopeptides, Karyopharm, Onconova, Archigen, Pfizer, Fibrogen, Beigene.Consulting fees from Incyte.Speakers bureaus: Takeda, Servier, Celgene, Abbvie, BMS, Janssen.Support for travels: Takeda, Roche, Janssen, BMS.M.T.: advisory board participation fees from Janssen and, Astra Zeneca.G.M.: Janssen: Consulting, Honoraria, Advisory role.Incyte: Consulting, Honoraria, Advisory roleRoche: Consulting, Honoraria, Advisory role.P.L.Z.: Advisory board participation fees from ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Conflicts of Interest: F.R.M.: research funding from Gilead, advisory board participation fees from AbbVie, Gilead, Janssen, AstraZeneca, Takeda, Roche; speakers bureau fees from Gilead, Janssen, and Abbvie.P.S.: advisory board participation fees from Jansenn, Abbvie Astrazeneca.M.M.: consultancy: Gilead; paid speech: Novartis, Amgen; sponsored meetings: Abbvie, Takeda, Pfeitzer.R.M. (Roberto Marasca): Janssen e Abbvie: honoraria and travel grant.Astrazeneca: honoraria.G.G.: Abbvie (Advisory Board, Speaker's Bureau), Astra-Zeneca (Advisory Board), Beigene (Advisory Board), Incyte (Advisory Board), Janssen (Advisory Board, Speaker's Bureau).M.C.: Janssen honoraria and research funding), Abbvie and AstraZeneca (honoraria).A.M.L.: Research grants from: Takeda, Servier, Roche, Celgene, Abbvie, Incyte, Janssen.Sanofi, Verastem, Novartis, Morphosys, GSK, Oncopeptides, Karyopharm, Onconova, Archigen, Pfizer, Fibrogen, Beigene.Consulting fees from Incyte.Speakers bureaus: Takeda, Servier, Celgene, Abbvie, BMS, Janssen.Support for travels: Takeda, Roche, Janssen, BMS.M.T.: advisory board participation fees from Janssen and, Astra Zeneca.G.M.: Janssen: Consulting, Honoraria, Advisory role.Incyte: Consulting, Honoraria, Advisory roleRoche: Consulting, Honoraria, Advisory role.P.L.Z.: Advisory board participation fees from ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
}
] |
10.1038/s41416-020-01205-9
|
Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL).</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.</jats:p> </jats:sec>
|
[
{
"section_content": "The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation, as well as a target for therapeutics in cancer and other diseases. 1The two complexes that facilitate signal transduction in the mTOR pathway are mTORC1 and mTORC2.mTOR-activating mutations occur in diffuse large B cell lymphoma (DLBCL), 2 and elevated mTORC1 activity correlates with chemotherapy resistance and poor prognosis in pre-B acute lymphoblastic leukaemia (B-ALL). 3TORC1 substrates include S6 kinases (S6Ks) and eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs).Phosphorylation of 4E-BP releases its inhibition of eIF4E.5][6] Phosphorylation of 4E-BP1 correlates with high risk in B-ALL and chronic lymphocytic leukaemia, 3,7 while mTOR inactivation impairs B-ALL survival. 8,9everal mTORC1 inhibitors have been developed and investigated in the treatment of B-cell non-Hodgkin's lymphoma (NHL), yet each has caveats.Rapamycin and its analogues (rapalogs) are only partial inhibitors of mTORC1 that do not effectively suppress 4E-BP1 phosphorylation. 10Second generation mTOR kinase inhibitors (TOR-KI) act as ATP-competitive inhibitors and fully inhibit both mTORC1 and mTORC2. 11,12One candidate TOR-KI studied in our lab, MLN0128/TAK-228, 13 has entered a phase 2 clinical trial in B-ALL.TOR-KI have shown improved proapoptotic activity in preclinical studies 11,12 yet their therapeutic potential is limited by several factors including toxicity, 14 adaptive survival signalling 15 and mTOR resistance mutations. 16 promising alternative is to identify and target processes downstream of mTOR that are selectively required for cancer cell survival.5][6] Compared to normal cells, cancer cells are \"addicted\" to high levels of eIF4F activity, [4][5][6] as demonstrated using eIF4E heterozygous mice, which were healthy yet resistant to Ras-driven tumorigenesis. 17Various malignant cells rely on cap-dependent translation of specific mRNAs encoding many oncogenes, cell cycle regulators and prosurvival factors [4][5][6] including BCL2 family members (e.g.MCL-1 18 ).Hence, targeting cap-dependent translation downstream of mTOR could enhance efficacy of other proapoptotic therapies. Currently available eIF4F inhibitors have liabilities, including low potency, lack of selectivity or poor pharmacological properties. 5ere we use the novel small molecule inhibitor of eIF4G1, named SBI-756, to target the eIF4F translation initiation complex. 19SBI-756 is a small molecule that binds to eIF4G1 and prevents its interaction with eIF4E. 19In our previous study, SBI-756 inhibited melanoma in vitro and in vivo. 19enetoclax is a small molecule BH3 mimetic drug that selectively binds BCL2 and inhibits its prosurvival function. 20,21Since initial FDA approval of venetoclax for treatment of chronic lymphocytic leukaemia (CLL) patients with 17p chromosomal deletion, 22 additional combination regimens have been approved. 23,24Novel combinations are needed to improve responses in NHL, where single agent venetoclax has limited activity. 25Here we tested the efficacy of SBI-0640756 (SBI-756 hereafter) in NHL cell lines, alone and in combination with venetoclax.We find that SBI-756 synergises with venetoclax in DLBCL and mantle cell lymphoma (MCL) cells in vitro, and promotes tumour regression in vivo.SBI-756 at nanomolar concentrations disrupts the eIF4G1:eIF4E interaction in cells, reprogramming mRNA translation and sensitising to apoptosis.Lymphoma cells with natural or engineered loss of 4E-BP1 were resistant to TOR-KIs yet retained sensitivity to SBI-756.Mechanistic experiments showed that SBI-756 had a selective effect on translation efficiency of components of the translation machinery, leading to reduced protein synthesis rates.These results identify disruption of eIF4F assembly as a promising approach to enhance venetoclax efficacy in NHL. ",
"section_name": "BACKGROUND",
"section_num": null
},
{
"section_content": "",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "We performed PLA as described before. 26Briefly: 2×10e6 cells were treated for 4 h as indicated.Cells were washed with 1× phosphate buffered saline (PBS) (Corning, NY) and fixed with 4% paraformaldehyde (Thermo Fisher Scientific).CometSlides (Trevigen, Gaithersburg, MD) were coated with Poly-L-Lysine 0.1% solution (Sigma-Aldrich (SA), St. Louis, MO), and cells were allowed to adhere.We followed the protocol of Duolink PLA; 27 briefly: Cells were blocked using Duolink blocking solution, followed by probing with primary antibodies for eIF4G1 (Cell signaling Technologies, Danvers, MA, Cat.#2858, 1:200 dilution) and eIF4E (BD Biosciences, San Diego, CA, Cat.#610269, 2.5 µg/ml final).Next, cells were incubated with Duolink In Situ PLA Probe Anti-Rabbit PLUS (Cat.# DUO92002) and Duolink In Situ PLA Probe Anti-Mouse (Cat.# DUO92004) and allowed to ligate using ligation mix.Next, amplification and washes were performed as instructed and the slides were mounted using media containing DAPI.Slides were imaged using Leica TCS SP8 confocal microscope.Signal obtained was quantified using ImageJ software, and normalised to the number of cells per field (using DAPI nuclei staining).Images shown indicate the signal (Orange Duolink TM ) and nuclei for each field imaged, while graphs presented indicate ratio values of signal per cell in each field imaged. Mice strain and compounds administration in vivo Thirty-two NOD scid gamma (NSG) healthy immunodeficient mice (Jackson Laboratories, Bar Harbor, ME) were used for in vivo experiments (8 weeks old, 23 gram in average) after 7 days acclimation in animal facility.Animal studies were approved by the Institutional Animal Care and Use Committee at UC Irvine.Female NSG mice were injected subcutaneously (s.c.) with 1 × 10e6 OCI-LY1 cells/mouse.We anesthetised the mice (100 mg/kg ketamine-10 mg/kg xylazine) and cells were injected in total volume of 200 µl along with Matrigel (Corning) for providing a supportive environment for tumour development.Once tumour size reached 110 mm 3 volume, mice were randomised into treatment groups (n = 8) and treated daily (non-blinded way) for 5 days.Each mouse body weight was examined throughout the trial to identify potential toxicity or changes in dosing parameters.Also, tumour sizes were monitored daily and recorded.All mice were monitored for clinical signs of pain or distress during the procedures and daily during tumour measurements; no clinical signs were observed.At the end of five days of dosing, each mouse was weighed, sacrificed (according to IACUC guidelines, using CO 2 inhalation followed by cervical dislocation) and tumours were excised for analysis.Analysis of tumours included tumour size, weight and preparation of single-cell suspension without exclusion of data points.Cells extracted were fixed using 4% PFA and used for intracellular staining as well as PLA. Polysome profiling Cells were grown to ~70% confluence.Cycloheximide (0.1 mg/mL final concentration) was added to the medium for 5 min at 37 °C to arrest the ribosomes.The cells were washed twice with PBS containing 0.1 mg/mL cycloheximide, and then pelleted.The supernatant was removed, and the pellet was flash frozen.Cell pellets were lysed for 10 min on ice with 400 µL polysome extraction buffer (15 mM Tris-Cl, pH7.4,15 mM MgCl 2 , 0.3 M NaCl, 0.1 mg/mL cycloheximide, 0.1 mg/mL heparin, 1% Triton X-100).The lysates were cleared by centrifugation at 13,200 × g for 10 min.Equal RNA concentrations were layered onto 20-50% sucrose gradients.Gradients were sedimented at 151,263 × g for 103 min in a SW55 Ti rotor at 4 °C.An ISCO UA-6 (Teledyne, Thousand Oaks, CA) fraction collection system was used to collect 12 fractions, which were immediately mixed with 1 volume of 8 M guanidine HCl.RNA was precipitated from polysome fractions by ethanol precipitation and dissolved in 20 µL of H 2 O. Briefly, fractions were vortexed for 20 s. 600 µL of 100% ethanol was added, and fraction was vortexed again.Fractions were incubated overnight at -20 °C to allow for complete RNA precipitation.Fractions were centrifuged at 13,200 rpm for 30 min at 4 °C.The RNA pellet was washed with 75% ethanol.The pellet was resuspended in 400 µL 1× Tris-EDTA (pH 8.0).0.1 volumes of 3 M NaOAc (pH 5.3) and 2.5 volumes 100% ethanol were added, and fractions were incubated at -20 °C to precipitate RNA.Fractions were centrifuged at 13,200 rpm for 30 min at 4 °C.The RNA pellet was washed with 75% ethanol.RNA was resuspended in 20 µL H 2 O.Total RNA samples were isolated from cell lysates using Trizol per the manufacturer's instructions. ",
"section_name": "Duolink proximity ligation assay (PLA)",
"section_num": null
},
{
"section_content": "Fractions containing four or more ribosomes (considered welltranslated) were pooled and RNA quality was measured by a Bioanalyzer (Agilent Technologies).RNA-seq was carried out by the New York University School of Medicine Genome Technology Core using the Illumina HiSeq 4000 single read.To examine differences in transcription and translation, total mRNA and polysome mRNA were quantile-normalised separately.Statistical analysis was performed using RIVET. 28GO analysis was performed using the DAVID online tool. ",
"section_name": "RNA-seq and analysis",
"section_num": null
},
{
"section_content": "The number \"n\" of biological replicates for each experiment is indicated in the figure legends.Two-way ANOVA for multiple comparisons was performed where indicated while considering sample independence, variance equality and normality.ANOVA analysis while adjusting for multiple comparisons was performed for the in vivo experiment described to test for tumour growth.Student t-tests were applied to population means assuming equal variance (standard deviations within two-fold).The use of one-versus twosample tests, and paired versus unpaired comparisons, was justified by the experimental design as indicated in the Figure Legends. Additional standard and published methods are provided in the Supplementary Materials. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Constitutively active 4E-BP1 mutant sensitises DLBCL cells to venetoclax, similar to TOR-KI treatment mTOR inhibitors enhance killing of DLBCL cells by BH3 mimetics, such as venetoclax (ABT-199), ABT-263 or ABT-737. 29To evaluate the role of the 4E-BP/eIF4E axis in this sensitisation, we used a doxycycline (DOX)-inducible system to express wild-type 4E-BP1 or a constitutively active form in which all five serine/threonine phosphorylation sites were changed into alanine (5A mutant).Since 4E-BP1-5A cannot be phosphorylated by mTORC1, expression of this mutant prevents eIF4E from associating with eIF4G1 and other proteins to form the eIF4F complex. 30We generated OCI-LY1 DLBCL cells expressing the reverse tetracycline transactivator (rtTA) protein and either empty vector (EV), WT 4E-BP1 or 4E-BP1 mutant (5A).Addition of DOX induced expression of the mutant protein after 16 h (Fig. S1a).Next, we treated cells (±DOX) for 48 h with a range of venetoclax concentrations in combination with either vehicle (DMSO), or TOR-KI (MLN0128 100 nM).As expected, TOR-KI treatment sensitised to venetoclax as shown by reduced IC50 values (Fig. S1b).Notably, OCI-LY1 cells expressing the active 4E-BP1 (5A) and treated with vehicle (lane 5, Fig. S1a) were as sensitive to venetoclax as control cells (WT or EV) treated with TOR-KI (lanes 2 and 4 Fig.S1b).TOR-KI further increased sensitisation to venetoclax in cells expressing 4E-BP1 MUT.A similar sensitisation was observed in OCI-LY1 cells expressing 4E-BP1 5A and treated with navitoclax (ABT-263)-an inhibitor of BCL2, BCL2L1 (BCL-xL), and BCL-W (Fig. S1c,d).In summary, the ability of the 4E-BP1 mutant to phenocopy the effect of TOR-KI demonstrates that targeting the 4E-BP1/eIF4E arm of mTORC1 signalling is a promising approach for sensitisation of DLBCL cells to venetoclax treatment.SBI-756 prevents eIF4E:eIF4G1 association and reduces capdependent translation in lymphoma cells Next, we took a chemical approach to disrupting eIF4F.Previously, we showed that the cell-permeable compound SBI-756 binds to eIF4G1 and disrupts formation of the mRNA cap-binding complex in melanoma cells and in fibroblasts. 19To assess the effect of SBI-756 on eIF4F formation in lymphoma cells, we used a proximity ligation assay (PLA) to quantitate the interaction of eIF4E and eIF4G1 in situ.As expected, treatment of OCI-LY1 cells with the TOR-KI compound MLN0128 suppressed eIF4E:eIF4G1 association whereas rapamycin, a weak inhibitor of 4E-BP1 phosphorylation, 10,30 had no significant effect (Fig. 1a,b).SBI-756 reduced eIF4E:eIF4G1 interaction in a dose-dependent manner (Fig. 1a,b).Similar results were observed in OCI-LY8 cells (Fig. S2a,b).Quantification of eIF4E:eIF4G1 interaction indicated a significant reduction by 500 nM SBI-756 in OCI-LY1 (76%, Fig. 1b) and 250 nM in OCI-LY8 (83%, Fig. S2b). We used dual-luciferase reporter assays to test the ability of TOR-KI and SBI-756 to reduce cap-dependent and IRES-dependent translation.Following 16 h of treatment of OCI-LY1 and the MCL cell line Mino1, SBI-756 in the range of 100-500 nM selectively reduced cap-dependent luciferase expression (Fig. 1c).Similar results were observed in two additional DLBCL lines (OCI-LY8, SU-DHL6) and in the Maver1 MCL line (Fig. S2c).Likewise, MLN0128 significantly reduced accumulation of cap-dependent luciferase (Figs.1c andS2). ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "A potential advantage of selective eIF4F targeting is that this approach should preserve activity of mTOR, reducing on-target toxicities associated with mTOR inhibition.Compared to the TOR-KI compound Torin-1, SBI-756 (1 µM or lower concentration) did not inhibit phosphorylation of mTORC1 or mTORC2 substrates in melanoma cells. 19To test whether SBI-756 alters mTOR activity in lymphoma cells, we measured mTORC1 and mTORC2 substrate phosphorylation.As expected, MLN0128 significantly reduced phosphorylation of both mTORC1 (p-S6, p-4E-BP1) and mTORC2 (AKT) substrates, whereas rapamycin reduced only p-S6 (Fig. S3a,b).In contrast, treatment with SBI-756 did not alter phosphorylation of any mTOR substrates tested, indicating that the mTOR signalling pathway was not altered by SBI-756 treatment (Fig. S3a,b).Similar results were obtained in other DLBCL cell lines (Fig. S3c,d).These results support the conclusion that SBI-756 directly disrupts the eIF4F complex without altering activity of mTORC1 or mTORC2. ",
"section_name": "SBI-756 does not change mTOR substrate phosphorylation",
"section_num": null
},
{
"section_content": "To test the ability of SBI-756 to promote apoptosis and sensitise lymphoma cells to venetoclax, we evaluated seven GCB-DLBCL and five MCL cell lines (Figs. 1d, e andS4).We treated the cells with titrated concentrations of venetoclax as a single agent, or in combination with either MLN0128 or SBI-756 (Figs. 1d, e andS4a, b) and performed viability assays.Eight of the cell lines tested had reduced viability following 48 h treatment with venetoclax as a single agent.Ten cell lines showed reduced viability following SBI-756 treatment as a single agent.In five of the SBI-756-sensitive DLBCL lines (OCI-LY1, OCI-LY8, OCI-LY18, SU-DHL-4, SU-DHL-6) and four of the MCL lines (Mino, Jeko1, MAVER-1, CCMCL-1), the combination of SBI-756 and venetoclax caused more cell death than individual agents (Figs.1d, e andS4).In comparison, MLN0128 only sensitised to venetoclax in one cell line (OCI-LY1) (Fig. 1d) despite effectively suppressing mTOR signalling outputs in other cell lines such as OCI-LY8 (Fig. S3a,c).Supporting an apoptotic mechanism, the combination of SBI-756 with venetoclax induced caspase-dependent death, as demonstrated by rescue of viability in cells co-treated with the pan-caspase inhibitor QVD-OPH (Fig. S5a).Moreover, venetoclax treatment with or without SBI-756 led to cleavage of caspase-3 and PARP (Fig. S5b).Treatment with SBI-756 alone did not induce cleavage of caspase-3 or PARP (Fig. S5b). Next, we chose sensitive DLBCL and MCL cells to further evaluate synergy between venetoclax and SBI-756 (Figs. 1f, g and S4c).We measured viability in cells treated for 48 h with fixed ratios of venetoclax and SBI-756 and assessed synergy using the Chou-Talalay method. 31Indeed, SBI-756 synergised with venetoclax (combination index < 1) in both OCI-LY1 (DLBCL) and Mino1 (MCL) cells (Fig. 1f,g).Furthermore, SBI-756 was found to synergise with venetoclax in five more cell lines tested (Fig. S4c).SBI-756 did not reduce viability or sensitise to venetoclax in the OCI-LY7 DLBCL line and had minimal effect in a subline of OCI-LY1 cells that we selected for resistance to SBI-756 (Fig. S4a).In both these cell lines, SBI-756 treatment did not prevent the eIF4E: eIF4G1 interaction measured by PLA (Fig. S4d). Lymphoma cells lacking 4E-BP1 are resistant to TOR-KI yet remain sensitive to SBI-756 Many cancer cells exhibit a reduction of 4E-BP expression 32,33 or increase in eIF4E expression, 34,35 enabling cap-dependent translation even following mTORC1 inhibition.Indeed, the eIF4E/4E-BP ratio can predict efficacy of mTOR targeted therapies. 36Previously we reported that SBI-756 can fully suppress proliferation in 4E-BP1/4E-BP2 double knockout fibroblasts that are partially resistant to the TOR-KI compound Torin-1. 19To determine whether SBI-756induced DLBCL death is 4E-BP-dependent, we used CRISPR/Cas9 genome editing to generate clones of OCI-LY1 cells lacking 4E-BP1 (Fig. S6).We compared these 4E-BP1 knockout (KO) clones to OCI-LY1 cells expressing Cas9 and empty sgRNA vector (EV) in viability assays, using a range of venetoclax concentrations without or with MLN0128 or SBI-756.As in parental cells, 10 nM venetoclax caused ~10% death in the EV and 4E-BP1 KO lines (Fig. 2a-d); higher concentrations of venetoclax caused more death.MLN0128 and SBI-756 sensitised to venetoclax in EV cells (Figs. 2a,b) as in parental OCI-LY1 (Fig. 1d).Notably, OCI-LY1 cells lacking 4E-BP1 were completely resistant to MLN0128 yet remained sensitive to SBI-756 alone or in combination with venetoclax (Figs.2c, d andS6).Similarly, SBI-756 reduced eIF4E:eIF4G1 interaction among all cells, whereas treatment with MLN0128 reduced interaction only in control cells containing 4E-BP1 (Figs.2e,f).Our findings indicate that in cells lacking 4E-BP1 expression, SBI-756 retains its effect and prevents eIF4F formation, thus sensitising those cells to BCL2 inhibition. To further support this conclusion, we used VAL cells, a DLBCL line lacking 4E-BP1 32 (Fig. S3d).Consistent with previous observations, 32 MLN0128 at concentrations up to 3 µM did not affect VAL cell viability (Fig. 2g) or eIF4E:eIF4G interaction (Fig. 2h).In contrast, SBI-756 reduced viability of VAL cells (Fig. 2g) and disrupted the eIF4E:eIF4G interaction (Fig. 2h).VAL cells were completely insensitive to venetoclax, with or without SBI-756.Nevertheless, these data confirm that prevention of eIF4F complex formation is achievable using SBI-756, even among cells lacking 4E-BP1 (thus insensitive to TOR-KI). ",
"section_name": "SBI-756 synergises with venetoclax to increase apoptosis",
"section_num": null
},
{
"section_content": "We assessed whether sensitisation of DLBCL to venetoclax treatment by cotargeting eIF4F could be recapitulated in vivo.We injected NSG mice with OCI-LY1 (s.c.) and once palpable tumours were established, treated with vehicle, venetoclax, SBI-756 or their combination for 5 consecutive days (Fig. 3a).There was no significant change in body weight among the different groups (Fig. S7a), indicating that the treatments were well tolerated.Both venetoclax and SBI-756 significantly slowed tumour growth when administered as single agents (Figs.3b,S7b,c).Notably, venetoclax and SBI-756 combination caused tumour regression and significantly reduced tumour volume (7/7 mice) when compared to SBI-756 (1/8 mice) or venetoclax (2/8 mice) as single agents or vehicle alone (0/8 mice).Together these results indicate that a synergistic relationship between venetoclax and SBI-756 occurs not only in vitro but also in vivo. ",
"section_name": "SBI-756 is effective and well tolerated in vivo",
"section_num": null
},
{
"section_content": "To assess whether SBI-756 treatment prevents eIF4E:eIF4G1 interaction in vivo, lymphoma tumours excised from euthanised mice were dissociated into single cells and subjected to PLA.Analysis of the samples obtained showed a reduction in eIF4E: eIF4G1 interaction among tumours treated with SBI-756 as a single agent or in combination with venetoclax, compared to vehicle or venetoclax alone (Fig. 3c,d).Additionally, we performed intracellular staining to measure phosphorylation levels of mTOR substrates, S6 kinase and 4E-BP1, among the samples extracted from the tumours to test for alteration in mTOR kinase activity.No significant changes were observed in mTOR substrate phosphorylation in mice treated with SBI-756 and/or venetoclax in vivo (Fig. S7d,e).These results suggest that SBI-756 potentiates venetoclax efficacy in vivo by preventing eIF4E:eIF4G1 interaction without affecting mTOR activity.SBI-756 has both direct and indirect effects on mRNA translation Chemical inhibition of mTORC1 or eIF4F in cancer cells selectively suppresses translation of mRNAs with specific features in the 5′ untranslated region (UTR). 37These eIF4F-sensitive mRNAs include several that encode prosurvival proteins, including MCL-1, BCL-xL and survivin. 18To determine whether SBI-756 affects expression of these factors in DLBCL cells, we measured expression of candidate proteins by western blot.In cells growing asynchronously, 4-h treatment with MLN0128 or SBI-756 did not change expression of these candidate proteins (data not shown).In a previous study we found that serum starvation of DLBCL cells, followed by readdition of serum without or with mTOR inhibitors, revealed consistent changes in protein expression. 32Taking this approach, we observed modest and variably reduced expression (~2-fold) of MCL-1, BCL-xL and survivin among MLN0128 and SBI-756 treated cells (Fig. S8a,b).Expression of eIF4E or eIF4G1 were also reduced following SBI-756 or MLN0128 treatment (Fig. S8a,b).For each of these targets (MCL-1, BCL-XL, survivin, eIF4E or eIF4G1), the abundance of mRNA was not significantly changed in cells treated with SBI-756 or MLN0128 (with the exception of survivin transcripts elevated in cells treated with 500 nM SBI-756) (Fig. S8c). To gain a broad, unbiased view of how SBI-756 affects mRNA translation efficiency, we treated OCI-LY1 cells with vehicle (DMSO) or SBI-756 (250 nM) for 4 h, and isolated RNA from heavy and light polysome fractions, as well as total cellular RNA.The 4-h treatment did not cause detectable changes in RNA distribution from heavy to light polysomes (Fig. 4a) and did not change rates of overall protein synthesis (Fig. 4b).However, comparison of mRNAs associated with heavy polysomes to total mRNAs showed that SBI-756 selectively changed the translation efficiency of 538 mRNAs; 13 showed differences in both translation and transcription (Fig. 4c,d,Supp.Table 1).Only 24 genes showed changes in transcription alone (Fig. 4c,d), supporting the conclusion that SBI-756 is an on-target and selective inhibitor of mRNA translation. Of the 538 mRNAs with selective change in translation, the majority (385) had reduced translation efficiency (Fig. 4c) (Supp.Table 1).Gene ontology (GO) analysis showed a highly significant enrichment for genes involved in mRNA translation.The most enriched biological processes for the downregulated genes (Fig. 4e top) include translation initiation, translation, rRNA processing and ribosomal small subunit assembly.Likewise, top molecular functions associated with the downregulated genes include structural constituent of the ribosome, RNA binding and poly(A) RNA binding (Fig. 4f).Among the translationally downregulated biological processes were negative regulators of apoptosis (Fig. 4e).This family contained 10 genes (Supplementary Table 2) yet did not include the candidates mentioned above (MCL-1, BCL-xL, survivin).Among the translationally upregulated mRNAs, only one was found to contain an IRES motif (TP53). The dramatic reduction in translation of mRNAs encoding ribosomal proteins and regulators at 4 h suggested that SBI-756 might indirectly reduce overall translation efficiency over time.Indeed, 16-h treatment with SBI-756 reduced rates of total protein synthesis in parental OCI-LY1 cells (Fig. 5a) but not in the SBI-756resistant subline of OCI-LY1 cells (Fig. 5b).MLN0128 or SBI-756 treatment did not reduce protein synthesis in the OCI-LY7 cell line (Fig. 5c), in which these agents did not have cytotoxic activity (Fig. S4a).Nevertheless, SBI-756 did reduce protein synthesis in OCI-LY8 and SU-DHL-6 cell lines (Fig. 5d,e).This reduced protein synthesis could account for the sensitisation to venetoclax observed among these cells (Fig. S4a).Considering that SBI-756 reduced protein synthesis in OCI-LY1 cells at 16 h but not 4 h, we assessed cell survival at these timepoints as well as 24 h and 48 h.Sensitisation to venetoclax cytotoxicity was first evident 16 h following SBI-756 treatment (Fig. 5f), and was significantly greater than combination with TOR-KI.SBI-756 is not cytotoxic to human CD4+T cells, CD8+T cells, NK cells or monocytes Lastly, we evaluated whether SBI-756 cytotoxic effects are selective for transformed lymphoma cells versus normal lymphocytes.We cultured PBMCs from healthy donors for 48 h with SBI-756 or MLN0128 alone, or in combination with venetoclax.Compared to vehicle treated cells, we observed a significant reduction in cell viability only among CD19 + B cells following MLN0128 or SBI-756 treatment (Fig. 6a).The effect of SBI-756 on B cell viability was dose dependent (Fig. S9a).Venetoclax alone greatly reduced viability of B cells, with partial effects on CD4 + and CD8 + T cells and natural killer cells as published before. 38However, there was no further effect when venetoclax was combined with either MLN0128 or (Fig. 6a).We also tested purified lymphocytes from mice, which are more readily available in quantities needed for correlation of functional and biochemical readouts.Similarly, MLN0128 and SBI-756 were selectively cytotoxic to mouse B cells but not mouse T cells cultured in supportive cytokines (Fig. 6b).The cytotoxic effect of SBI-756 in B cells at 48 h (Fig. 6b) correlated with suppression of protein synthesis selectively in B cells, measured after 16-h treatment (Fig. 6c). ",
"section_name": "SBI-756 has a pharmacodynamic effect in vivo",
"section_num": null
},
{
"section_content": "Venetoclax is a BCL2-specific inhibitor whose use is expanding in hematologic malignancies. 20,21However, blood cancer cells frequently engage distinct mechanisms that can maintain survival following BCL2 inhibition.Therefore, responses to venetoclax are broader and more durable when the drug is combined with other agents that promote cell death through distinct mechanisms. 39In DLBCL, venetoclax combined with the standard of care (R-CHOP) is more effective than venetoclax monotherapy and components of the CHOP regimen can increase sensitivity to venetoclax in vitro. 40,41Incorporation of additional targeted agents has potential to further improve responses.Here, report experiments showing great potential for sensitisation of NHL cells to venetoclax via combination with SBI-756, a potent inhibitor of capdependent translation that is active in cells in the 100-500 nM range.SBI-756 synergised with venetoclax to induce apoptosis in NHL cells while not interfering with mTOR signalling.SBI-756 prevented eIF4E:eIF4G1 interaction among sensitive cells (OCI-LY1 and OCI-LY8), but not among resistant cells (OCI-LY7 or OCI-LY1 induced to be resistant to SBI-756).After 4-h of treatment, SBI-756 also reduced translation efficiency of mRNAs encoding ribosomal proteins and rRNA processing factors.Consistent with reduced ribosome biogenesis, SBI-756 reduced overall protein synthesis after 16 h of treatment.Additionally, in DLBCL cell lines lacking 4E-BPs (naturally or genetically edited), SBI-756 retained ability to inhibit eIF4F formation and sensitise to venetoclax, whereas the TOR-KI compound MLN0128 lacked activity in this setting.SBI-756 synergy with venetoclax in vitro was recapitulated in vivo, with the combination reducing tumour progression that correlated with prevention of eIF4E-eIF4G1 interaction.Treatment with SBI-756 was tolerable in mice, and the compound selectively reduced survival of B cells in cultures of human PBMCs and murine lymphocytes.Together, these findings urge further investigation of eIF4F disruption for sensitisation to venetoclax and possibly other BH3 mimetics.In this regard, an initial experiment to sensitise DLBCL cell lines to an MCL-1 inhibitor (S63845) using SBI-756 (Fig. S9b) yielded a similar response as the venetoclax combination. PI3K/mTOR signalling pathway activation has been correlated with poor prognosis and resistance to chemotherapy. 3Thus, this pathway remains an investigational target for cancer therapeutics, including rapalogs or TOR-KIs such as MLN0128/TAK-228. 11However, rapalogs have limited anticancer activity and the therapeutic window for TOR-KIs remains to be established.Targeting individual pathways downstream of mTOR might hold the key to developing better tolerated and more effective anticancer interventions.5][6] Genetic inhibition of eIF4F via inducible expression of the 4E-BP1 mutant sensitised to venetoclax in OCI-LY1 cells, an effect enhanced by the TOR-KI compound MLN0128 that reactivates endogenous 4E-BP1.Our studies of the eIF4G-binding compound SBI-756 in human lymphoma cell line models provide proof-of-concept for eIF4F disruption by a small molecule at sub-µM concentration.Further studies using pharmacologically optimised inhibitors of eIF4G1, or specific knockouts of eIF4G1 could clarify the importance of this component in lymphomagenesis or lymphocyte differentiation.In a distinct approach, rocaglate compounds targeting the eIF4A helicase can also enhance venetoclax cytotoxicity in lymphoma cells. 42n most of the DLBCL and MCL cell lines tested, 48-h treatment with SBI-756 alone had cytotoxic effects as measured by staining with Annexin V and propidium iodide.In OCI-LY1 and OCI-LY8 cells, this cytotoxic effect was prevented by co-incubation with a pan-caspase inhibitor.However, SBI-756 alone did not cause detectable cleavage of caspase-3 or PARP after 16 h, a time point where these markers were readily induced by venetoclax.It is possible that SBI-756 treatment alone triggers apoptosis at a later time point.Of note, two of the sensitive cell lines (OCI-LY1 and OCI-LY8) express mutant p53, 43 suggesting that the mechanism of apoptosis is p53-independent. Notably, the data suggest that SBI-756 is not cytotoxic to essential cellular mediators of immune function and immunotherapy efficacy (T cells and NK cells).A potential advantage for tolerability is that SBI-756 treatment (at concentrations of up to 500 nM) does not interfere with mTOR substrate phosphorylation while disrupting eIF4F complex assembly downstream of mTORC1.We did observe that higher concentrations of SBI-756 (>500 nM) do cause inhibition of the mTOR pathway; thus, future efforts should aim to optimise the selectivity of SBI-756 and similar agents.Another advantage of SBI-756 compared to TOR-KI treatment is its ability to suppress cap-dependent translation among cells lacking 4E-BP1, a finding relevant to tumours with reduced ratio of 4E-BPs to eIF4E.We used cells that naturally lack 4E-BP1. 32or were genome edited to lack 4E-BP1 to show that these cells remained sensitive to SBI-756 yet lacked TOR-KI sensitivity.Further experiments are needed to determine whether cells with high eIF4E or eIF4G1 retain sensitivity to SBI-756.These results suggest that B lineage cells have a unique dependence on the mTORC1/eIF4F axis for survival. Polysome profiling showed that SBI-756 treatment in lymphoma cells alters translation efficiency of 538 genes, transcription of 24 genes, while 13 genes are regulated both transcriptionally and translationally.GO analysis of the translationally downregulated genes identified several groups related to mRNA translation, including structural constituent of the ribosome, ribosomal biogenesis, rRNA binding and translational elongation (Fig. S10).Together, those functions indicate two main effects of SBI-756 treatment: first, direct suppression of translation efficiency of a key subset of cellular mRNAs; second, indirect inhibition of the translation machinery that is needed to sustain protein synthesis.The two effects together (direct and indirect) reprogram mRNA translation in a way that promotes lymphoma cell death and sensitises to BCL2 inhibition.Supporting the correlation of protein synthesis inhibition and cell death, the OCI-LY7 cell line was resistant to SBI-756 cytotoxicity and did not show reduced protein synthesis rates.Moreover, protein synthesis was not reduced among OCI-LY1 cells selected for SBI-756 resistance.Interestingly, the selective cytotoxic effect of SBI-756 on B cells versus T cells correlated with suppression of global protein synthesis. The polysome profiling dataset does not definitively establish acute effects of SBI-756 on translation efficiency of survival factors, such as ones reported previously to be eIF4F-dependent.Western blotting experiments suggested that SBI-756 reduces protein amounts of MCL-1 and survivin, whose expression is known to be sensitive to changes in cap-dependent translation. 44This reduction of prosurvival factors may block back-up mechanisms that the cells might use to adjust to BCL2 inhibition. 45Another possibility is that the broad reprogramming of translation produces subtle changes that alter the overall balance of prosurvival and proapoptotic proteins over time, tipping the balance towards cell death. 4,46Reducing ribosome production using inhibitors of ribosomal DNA transcription likewise promotes lymphoma cell death. 475][6] Natural products and synthetic small molecules have been identified with various mechanisms including: (i) interfering with eIF4E binding to the m 7 -GTP cap (for example, 4EGI-1 48 ); (ii) inhibiting MNK kinases that phosphorylate eIF4E (CGP57380; 49 eFT508 50 ); (iii) disrupting the function of eIF4A helicase (Silvestrol 51 and synthetic rocaglates 42,52 ); (iv) inhibiting translational elongation (homoharringtonine 53 ).Many of the compounds have low selectivity, weak potency or cell penetrance, poor pharmacological properties or are difficult to synthesise.In the case of 4EGI-1, activity requires 4E-BP1 expression. 48SBI-756 is a prototype of a novel class that binds to eIF4G and disrupts association of this scaffolding protein with eIF4E, independent of 4E-BP1.Active in cells in the mid-nanomolar range and in mouse models, this compound provides proof of concept for suppressing eIF4F function via blockade of key protein:protein interactions in the complex.Our data indicate that eIF4F-disrupting molecules like SBI-756 have great potential to sensitise lymphoma cells to venetoclax.Together with our previous finding that SBI-756 can circumvent resistance to BRAF inhibitors in melanoma, these results support further combination studies of SBI-756 with other targeted agents. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "We thank Dr. Selina Chen-Kiang and Dr. Maurizio Di Liberto for providing MCL cell lines, Dr. Bert Semler for providing us the dual-luciferase reporter construct, Dr. Grant MacGregor for his assistance with performance of dual-luciferase assays and Dr. Adeela Syed for her assistance with microscopy imaging.We also want to thank Dr. Changchun Xiao and his lab for their assistance with polysome fractionation and data analysis, and Dr. Ivan Topisirovic for helpful comments on the manuscript, and Joshua Thao and Madeleine Duong for research contributions. ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "Funding information This study was supported by: National Institutes of Health grant R01-CA158383 (to D.A.F.), R01-CA202021 (to Z.A.R.), R01-CA178509 (to R.J.S.), American Society for Hematology Bridge grant ASH-5557789 (to D.A.F.), Breast Cancer Research Fund grant BCRF-16-143 (to R.J.S.), Melanoma Research Alliance award 509524 (to Z.A.R.), UC Irvine Chao Family Comprehensive Cancer Center pilot grant from the 2018 Anti-Cancer Challenge (to D.A.F.), and by Cancer Center Support Grant P30-CA062203 to UC Irvine (PI: Van Etten).L.H. and H.C. were supported by National Institutes of Health grant T32 AI 060573.B.W. was supported by a National Institutes of Health grant T32 CA9161. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Ethics approval and consent to participate Animal studies were approved by the Institutional Animal Care and Use Committee (protocol AUP-18-050) at UC Irvine (accredited by American Association for Accreditation of Laboratory Animal Care).No human subjects research was performed. Data availability All data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author on reasonable request.All RNA-seq data files along with their associated metadata have been deposited in the GEO database under the accession code GSE159906. The authors declare no competing interests. Supplementary information is available for this paper at https://doi.org/10.1038/s41416-020-01205-9. Note This work is published under the standard license to publish agreement.After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0). 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
},
{
"section_content": "Ethics approval and consent to participate Animal studies were approved by the Institutional Animal Care and Use Committee (protocol AUP-18-050) at UC Irvine (accredited by American Association for Accreditation of Laboratory Animal Care).No human subjects research was performed. Data availability All data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author on reasonable request.All RNA-seq data files along with their associated metadata have been deposited in the GEO database under the accession code GSE159906. ",
"section_name": "ADDITIONAL INFORMATION",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. Supplementary information is available for this paper at https://doi.org/10.1038/s41416-020-01205-9. Note This work is published under the standard license to publish agreement.After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0). 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
}
] |
10.3390/life13081666
|
New Treatment Horizons in Uveal and Cutaneous Melanoma
|
<jats:p>Melanoma is a complex and heterogeneous malignant tumor with distinct genetic characteristics and therapeutic challenges in both cutaneous melanoma (CM) and uveal melanoma (UM). This review explores the underlying molecular features and genetic alterations in these melanoma subtypes, highlighting the importance of employing specific model systems tailored to their unique profiles for the development of targeted therapies. Over the past decade, significant progress has been made in unraveling the molecular and genetic characteristics of CM and UM, leading to notable advancements in treatment options. Genetic mutations in the mitogen-activated protein kinase (MAPK) pathway drive CM, while UM is characterized by mutations in genes like GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Chromosomal aberrations, including monosomy 3 in UM and monosomy 10 in CM, play significant roles in tumorigenesis. Immune cell infiltration differs between CM and UM, impacting prognosis. Therapeutic advancements targeting these genetic alterations, including oncolytic viruses and immunotherapies, have shown promise in preclinical and clinical studies. Oncolytic viruses selectively infect malignant cells, inducing oncolysis and activating antitumor immune responses. Talimogene laherparepvec (T-VEC) is an FDA-approved oncolytic virus for CM treatment, and other oncolytic viruses, such as coxsackieviruses and HF-10, are being investigated. Furthermore, combining oncolytic viruses with immunotherapies, such as CAR-T cell therapy, holds great potential. Understanding the intrinsic molecular features of melanoma and their role in shaping novel therapeutic approaches provides insights into targeted interventions and paves the way for more effective treatments for CM and UM.</jats:p>
|
[
{
"section_content": "Many hypotheses deliberate why cells with similar embryonic origins and biological functions (i.e., melanin production) undergo diverse tumor transformation pathways.Significant knowledge concerning the features of cutaneous and uveal melanoma has been acquired in the last decade, including molecular and genetic characteristics, primary tumor treatment, and metastatic disease approach. Life 2023, 13, 1666 2 of 17 Genetic alterations play a crucial role in the development and progression of uveal and cutaneous melanoma and these features have led to significant advancements in treatment options for these aggressive forms of cancer.Thus, more preclinical studies and testing of the active substances of newly emerging therapies should utilize specific model systems for uveal and cutaneous melanoma due to their unique genetic characteristics [1]. This review delineates the intrinsic molecular characteristics of melanoma and demonstrates how these features provide a basis for new therapies such as viral oncolysis and immunotherapies, as standalone treatments or in conjunction with each other. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "CM and UM are genetically distinct tumors.Defects in proteins involved in the mitogen-activated protein kinase (MAPK) pathway are found in most CM cases.This is a crucial intracellular signaling system that plays a role in cell growth, division, and survival [2].Although multiple mechanisms can activate the MAPK pathway ontogenically, the most prevalent is a constitutively activated mutant BRAF kinase.BRAF kinase mutations are encountered in 40-60% of the cases of CM.BRAF-mutated melanoma has different clinical characteristics and is associated with a more aggressive bioactivity than BRAF wild-type (WT) melanoma.BRAF-mutated melanoma may be connected to a significantly shorter life expectancy and poor prognostic indicators; however, this is currently under investigation [3][4][5].Mutated NRAS is the second most frequent MAPK pathway aberration in CM, occurring in 15-30% of patients [6,7].CM with mutations in the stem cell factor receptor tyrosine kinase gene (KIT) is an uncommon subtype that occurs in around 20% of mucosal, acral, and chronically sun-damaged areas [8].The findings that a mutation in BRAF kinase causes many of the CM has led to the development of the selective inhibitors of the BRAF V600-mutated kinase (vemurafenib, dabrafenib, and encorafenib) as well as inhibitors of the downstream MEK kinase (trametinib, cobimetinib, and binimetinib).Although individuals with the BRAF V600E or V600K mutation have a high response rate to BRAF treatment, most patients will develop acquired resistance.When compared to BRAF monotherapy, a combination of BRAF and MEK inhibitors seems to be more effective in preventing acquired resistance [9]. GNA11, GNAQ, BAP1, EIF1AX, and SF3B1 are typically the affected genes in UM [2].BAP1 is a tumor-suppressor gene on chromosome 3, which is mutated in 47% of those with UM.Inactivating mutations in the isolated viable BAP1 gene cause [10] UM in BAP1 germline mutants, which is like the common loss of chromosome 3 seen in high-risk sporadic disorders [11].Patients with UM have an 11% higher risk of secondary malignancies, including cutaneous melanoma, compared to the general population, which can be related to germline BAP1 mutations [12].GNA11 or GNAQ mutations occur in more than 90% of UM and these mutations stimulate signaling between G-protein-coupled receptors and downstream effectors, as well as upregulate MAPK pathway signaling [13].In most uveal melanomas, these alterations are strictly exclusive and are recognized as an early event in the pathogenesis of UM.GNAQ and GNA11 mutations were not correlated with a worse prognosis or the development of metastatic tumors [14,15].Primary UM has been separated into four clinically meaningful molecular categories, each with a distinct metastatic rate and prognosis [13].Tumors belonging to classes 1A and 1B have distinct melanocyte phenotypes, implying mutations in either EIF1AX or SF3B1.with an alteration of chromosome 3, Class 1A presumes a lower metastatic rate than 1B.Class 2 UM is distinguished by chromosome 3 monosomy, BAP1 expression abnormalities, and ubiquitous DNA methylation, and is associated with a high metastatic risk.Based on chromosome 8q copy number mutations, RNA expression patterns, and cellular pathway activity profiles, classes 2A and 2B were defined [16].When compared to Class 2A, Class 2B has an increased metastatic rate [17].Since most UM are generated by mutations in GNAQ or GNA11, studies have been directed into treatments that target the downstream effectors of these pathways such as MEK, Akt, and protein kinase C (PKC).Unfortunately, the findings were unsatisfactory, with response rates averaging less than 10% [18].Epigenetic dysregulation Life 2023, 13, 1666 3 of 17 could be a successful innovative target in UM.Somatic mutations throughout the tumor suppressor gene BAP1 were associated with metastatic behavior, as previously disclosed.The deletion of BAP1 gene appears to make UM cell lines more susceptible to histone deacetylase (HDAC) inhibitor therapy.Both in vivo and in vitro, HDAC causes a G1 cell cycle arrest with an enhanced cyclin D1, a decreased cell proliferation, growth inhibition, and apoptosis activation in UM [19,20]. Given the fact that the relationship between histone acetylation and deacetylation is altered in various types of cancers, therapy with HDAC inhibitors may be effective for both UM and CM.This balance determines the extent of histone acetylation and thus represents a crucial factor in gene expression regulation [21].While acetylation by histone acetyltransferases (HAT) is correlated to gene transcription, histone deacetylation by HDAC is involved in gene suppression.HDAC inhibition was found to suppress cell growth and division [22]. ",
"section_name": "Genetic Profiles",
"section_num": "2."
},
{
"section_content": "The deletion of one of the two copies of chromosome 3 (monosomy 3) is the most common chromosomal abnormality encountered in UM.Monosomy 3 is found in about 50% of all cases [23] and appears to be a chromosomal aberration unique to UM, as it is rarely seen in CM or other forms of cancer [24].Several studies have demonstrated a significant relationship between monosomy 3 and the onset of metastatic disease [25].Monosomy 3 is also associated with epithelioid cytology, closed vascular patterns, a large tumor diameter, and ciliary body involvement in clinical and histopathological examinations [26].In addition, monosomy 3 is considered to appear early in the tumorigenesis process because it frequently occurs in conjunction with all other known chromosomal alterations [27]. A gain of 8q (+8q) is identified in around 40% of UM cases and is a major independent prognostic marker for poor survival [25].It is usually observed in conjunction with monosomy 3, either as +8q or as isodisomy 8q, and this combination has been strongly correlated with metastatic tumors [25,28].Chromosome 3 and 8 aberrations are more common in ciliary body UM, whereas alterations on the long arm of chromosome 8 are more common in choroid UM [25][26][27].While chromosome 8q aberrations were shown to be associated with a large tumor diameter in one study [29], a univariate analysis revealed no significant relationship between the gain of 8q and the metastatic phenotype.A gain of 8q is also frequently detected in various copy numbers in UM, suggesting that it is a late event following the initiation of monosomy 3 [30]. Although chromosome 8q gain is encountered in 25% of CM, the concomitant occurrence of monosomy 3 and 8q gain, as observed in UM, is uncommon in CM [31,32]. The loss of 1p36 in combination with monosomy 3 has been proven to be a relevant prognostic factor: these aberrations occurring together have shown a stronger correlation with poor survival than monosomy 3 or the loss of 1p36 alone (although-1p36 alone is not of prognostic value) [33].The frequent deleted sites on chromosome 1 were identified to range between 1p34.3 and 36.2 [34]. Chromosome 6 changes occur in both UM and CM, but they have a lower prognostic value as compared to monosomy 3 or a gain of 8q in UM [24,25].Among all these alterations, the gain of DNA material on the short arm of chromosome 6 (+6p) has been identified in 25-29% of UM and is associated with spindle cell morphology and a lower probability of evolution to metastatic disease [24,27,35].The concomitant occurrence of +6p and -3 is an exceptional event.The deletion of DNA material on the long arm of chromosome 6 (-6q), occurring in 25-38% of UM, might be another late event in the carcinogenesis process and is associated with a worse prognosis [24,26,35,36].Other chromosome abnormalities, such as loss of 9p, loss of chromosome 10, loss of 11q23-q25, and gain of chromosomes 7 and 10, have been rarely described [25,26,36], but their involvement in the tumorigenesis process and metastatic disease development in UM has yet to be determined. In comparison to UM, CM exhibits a more complex karyotype.Monosomy 10 is the most common chromosomal abnormality in CM individuals.Monosomy 10 is identified in 60% of CM cases, which appears to be considerably more prevalent as compared to UM, where monosomy 10 is documented in only 27% of the cases.Other chromosomal abnormalities associated with CM include the loss of 1p, 4, 5, 6q, 9p, 11q, 12q, 14, 15, 16, 17p, 21, and chromosome 22 and a gain of DNA material on chromosomes 1q, 7, 18, and 20 [24]. Rearrangements of the distal region of chromosome 1's short arm, resulting in a loss or gain of 1p, have been documented in 28% and 33% of CM.Because they include the NRAS and AKT3 genes, some areas of chromosome 1 are of relevance.NRAS is found on chromosome 1p13 and has been demonstrated to be triggered by a mutation in 15-25% of CM [37].NRAS is thought to be involved as well in the MAP-kinase pathway.The MAP-kinase pathway is initiated by the activation of the NRAS gene, ultimately leading to cellular proliferation.NRAS is binding and activates the lipid kinase phosphoinositide-3 kinase (PI3K), which promotes the AKT pathway activation and prevents apoptosis [38].In 40-67% of CM, a direct activating mutation of the AKT3 gene on 1q44 is identified [39].PTEN loss can lead to the selective activation of AKT in CM [40], while the overexpression of AKT3 makes cells less sensitive to apoptotic stimuli.NRAS mutations are quite uncommon in UM, according to many studies [41,42]. In total, 66% of CM displayed chromosome 6 abnormalities, with +6p occurring in 24% and -6q observed in 42% [24].The 6q10-q27 region presents, by far, the most rearrangements because of the deletion, translocation, or development of an isochromosome of its short arm.The segment on the short arm of chromosome 6 that is often altered spans 6p21 to 6p25 and is characterized by DNA material gain.Both +6p and +6q are prevalent in UM, as previously stated.However, the prognostic value of these abnormalities is less significant than in CM [24,25]. The gain of DNA material on both arms of chromosome 7 is reported in 36% of CM.Somatic mutations in the 7q34 region, where the BRAF gene is found, are the most frequently reported.Activating mutations in BRAF are detected in up to 60-70% of CM [43].The BRAF gene encodes a kinase that is essential in the MAP-kinase pathway and is thought to be involved in the constitutive activation of the pathway and cell proliferation when mutated.More than 90% of all BRAF mutations appear to be caused by a single alteration (p.V600E) [44].The identical mutation is also identified in 80% of benign naevi, suggesting that it might be a precondition to melanoma genesis [45] and implicated in later phases of tumor growth [45].BRAF mutations are missing in UM pathology [41,46].However, a small study has identified BRAF mutations in 48% of iris melanoma [46].Chromosome 9 abnormalities exhibiting deletions of the short arm, -9p10-24 (37% of CM), or the long arm, +9q22-34 (15% of CM) have been reported.CDKN2A, which is located on 9p21, is one of the well-studied genes in CM.The two encoding tumor suppressor genes p16 and p14 are inhibited by inactivating mutations or deletions.Both genes were previously associated with significant CM susceptibility and were found in 30-80% of familial CM cases [47].On the contrary, these mutations are infrequently identified in sporadic CM or UM [48]. ",
"section_name": "Chromosomal Aberrations",
"section_num": "3."
},
{
"section_content": "Various malignancy types feature different distributions of immune cells.Over the last few decades, the adaptive immune response's function in tumor growth has garnered considerable attention in CM.The presence of CD3+CD8+ lymphocytes in both the tumor and the stroma, specifically activated (HLA-DR expressing) CD8+ T cells, was associated with disease-specific survival in primary CM [49].Moreover, the role of macrophages has also been evaluated.The macrophages that support an appropriate antitumor response (M1) and the macrophages that stimulate tumor development (M2) are two primary subtypes of macrophages.The M1-recruited macrophages transition to the M2 phenotype early in the development of CM, increasing tumor proliferation and metastasis.In contrast to CM, a significant degree of immune cell infiltration of UM, such as lymphocytes and macrophages, is correlated with a poor prognosis [50].Increased lymphocytes in tumor cells may be causing an increase in inflammatory mediators' synthesis, resulting in a tumor-promoting inflammatory environment.Phagocytic activity, tissue remodeling, tumor development, and angiogenesis are promoted by M2 phenotype macrophages [51]. UM does not display any ultraviolet mutation signature [52]. ",
"section_name": "Immune System Implications",
"section_num": "4."
},
{
"section_content": "The clinical assessment of suspicious lesions is a crucial component of CM and UM diagnoses.Dermatologists use clinical evaluation to diagnose CM and reserve excisional biopsy for tumors with uncertain etiology.The iris melanoma can be detected earlier, at a routine slit-lamp evaluation, or noticed by the patient themselves due to the iris color heterogeneity.On the contrary, both choroidal and ciliary body melanomas are diagnosed much later at a funduscopic examination with dilated pupils completed by an ultrasound evaluation as they can be asymptomatic for a long period.Tumor growth can cause in such cases heterogenous manifestations, ranging from mild visual field abnormalities to central visual loss, due to accompanying retinal detachment or massive extraocular tumor extension [53]. Early signs of melanoma presence and growth are easily omitted by the patient.Nevertheless, the tumor thickness and diameter at the time of diagnosis are considered to have a major impact on the overall survival rate in both tumors.As a result, practitioners must remain focused on the early diagnoses of CM and UM.Early detection resulted in an average CM thickness decrease of 0.76 mm at the time of diagnosis, associated with a 90% overall 10-year survival rate [54].UM with a diameter of less than 4 mm have an 84% 5-year survival rate.Medium-sized UM (4-8 mm in diameter) has a 5-year survival rate of 68%, while large UM (≥8 mm in diameter) has a 5-year survival rate of 47% [55].Patients with metastatic disease from both CM and UM have a poor life expectancy of 2-7 months [56,57]. ",
"section_name": "Diagnostic",
"section_num": "5."
},
{
"section_content": "CM and UM require very different therapeutic approaches [58].An excisional biopsy of the main tumor is recommended as the primary treatment for CM.Wide local excision is performed, with the requirement of different negative margins depending on the tumor thickness [59].Additionally, the use of Mohs micrographic surgery (MMS), a specialized surgical excision technique which promotes tissue sparing and provides optimal margin control through a complete evaluation of both the peripheral and deep margin for the treatment of CM, has been gaining the same widespread attention as standard wide local excision.Even though there is growing evidence demonstrating similar or improved cure rates when compared to standard excisional biopsy, MMS is not recommended for the routine surgical management of CM [60,61]. Regarding the radical surgical treatment of the UM enucleation of the eye, which was once considered the gold standard in the treatment of intraocular tumors, is still recommended in cases of large UM, tumors that are unresponsive or recurrent to conservative treatments, and total visual loss due to severe complications [62,63]. Over time, the treatment of primary UM has constantly improved and different radiation techniques such as brachytherapy, proton beam radiotherapy, and stereotactic radiation therapy using CyberKnife, Gamma Knife, or a linear accelerator have successfully replaced enucleation in selected cases [51,63,64].Currently, conservative treatment using a radioactive plaque temporarily sutured on the sclera adjacent to the tumor is one of the most effective and preferred methods for controlling medium-sized UM [65].Also, eyes with extensive orbital tumor development are now treated more conservatively, avoiding orbital exenteration with a combination of the surgical enucleation of the eye and local radiation therapy [66]. Other conservative treatment methods, less widespread and with more limited indications, include watchful waiting in the case of inactive small uveal lesions, direct laser photocoagulation-which has been abandoned in many centers due to the modest tumor control and the increased rate of recurrence-transpupillary thermotherapy for the treatment of small UM, and photodynamic therapy, which has been approved by the FDA for the selective treatment of choroidal neovascularization secondary to various conditions [67]. The recent past has witnessed unprecedented clinical progress in the treatment of advanced CM with multiple therapeutic options and a longer, more durable survival in this type of cancer [68].Adjuvant and neoadjuvant therapy have evolved to benefit individuals who are most at risk of disease recurrence following surgical excision.In both the adjuvant and neoadjuvant settings, as CM has historically been considered radioresistant, early antitumor chemotherapeutic and biochemical agents are giving way to novel immune therapies, mutation-specific targeted therapies, and oncolytic vaccinations, which are revolutionizing the treatment of CM.The combination of these systemic medications with surgical treatment has been found to improve overall survival.In the adjuvant treatment of advanced melanoma, targeted treatments are considered first-line therapy.These monoclonal antibodies specifically target mutant proteins in the MAPK pathway induced by common BRAF and MEK gene mutations that result in uncontrolled cell proliferation [69]. Other noteworthy therapies include immunotherapeutic drugs used to treat CM, which target checkpoint molecules that are bypassed by the common mutations detected in melanoma cells.Cytotoxic T-lymphocyte antigen 4 (CTLA4) inhibitors (ipilimumab), programmed cell death-1 (PD-1) pathway inhibitors (pembrolizumab, nivolumab), and high-dose interferon-alpha-2b (IFN-alpha-2b; HDI) are several of the most promising types [70][71][72]. Advances in immune checkpoint therapy have led to an improvement in overall survival for patients with advanced melanoma, and combining immunotherapeutic agents with different mechanisms of action may also enhance efficacy [73]. Oncolytic virotherapy (OV) is a new approach in cancer therapy that uses native or modified viruses that selectively infect malignant cells.There are several mechanisms described: virus-mediated lysis, viruses that promote immunogenic cell death, inflammatory response, and localized cytokine production or the disruption of tumor vessels, potentially facilitating immune cell migration into the tumor microenvironment (TME) (for example, HSV-1).Talimogene laherparepvec (T-VEC) is the first OV approved for melanoma treatment, a first-class recombinant type 1HSV [74]. Other oncolytic viruses such as coxsackieviruses have also been shown to have promising potential as effective oncolytic therapies for melanoma, as well as reovirus, rigvir, poxvirus, or adenovirus [75,76]. Also, recent studies have shown the utility of recombining OV and CAR-T (chimeric antigen receptor) cell therapy in mice.The data highlight that the stimulation of this combination could provide synergistic interactions in mouse melanoma tumor model [77]. HF10 is a non-neuroinvasive HSV-1 virus with naturally occurring deletions and insertions that decrease the potential in non-tumor cells while allowing for active proliferation in tumor cells.A phase I clinical trial in patients with refractory superficial cancers and melanoma was conducted at the University of Pittsburgh in the United States.This trial evaluated the tolerability and efficacy of HF10 therapy in 26 patients, including HSV seropositive and seronegative patients, with refractory superficial cancers and melanoma.The reduction in tumor size in some patients ranged from 30 to 61%.Interestingly, one patient showed a pathological complete response after 4 months of treatment [78]. A new approach is to enhance the efficiency of OVs by genetically encoding one or more tumor-associated antigens and neoantigens (TAAs) into the OV genome.Another strategy to enhance OV is the coating of OVs with specifically designed tumor epitope peptides.For example, the intratumoral administration of adenoviruses coated with modified tumor epitope peptides (PeptiCRAd) induces systemic anticancer immunity in mouse and humanized mouse cancer models of melanoma [79,80]. OVs possess the capability of selectively inducing oncolysis, as well as drawing in immune system cells, activating them, and consequently instigating both innate and adaptive antitumor responses, with minimal systemic effects [81].The viruses can generate \"danger signals\" that create a less immune-tolerant tumor microenvironment, act as carriers for the expression of inflammatory and immunomodulatory cytokines, and present antigens associated with the tumors.The effectiveness of treatments utilizing OVs was first demonstrated through the application of a genetically engineered herpes simplex virus known as talimogene laherparepvec (T-VEC), in the management of CM [82]. The herpes simplex virus presents an appealing prospect for an OV in melanoma due to its large genome containing several non-essential genes that can be eliminated to reduce pathogenicity, thereby enabling the insertion of genes of interest [83]. Talimogene laherparepvec (T-VEC) is the first and still stands as the sole oncolytic virus authorized by the FDA for the management of CM, including in stages III and IV melanoma [84]. Positive outcomes were observed in phases I, II, and III clinical trials involving the administration of T-VEC for the management of melanoma [82].A randomized clinical trial represented the first instance of demonstrating the advantageous therapeutic potential of OV for individuals with advanced or unresectable CM [83]. While its use as a neoadjuvant therapy is emerging, further data are necessary to establish its efficacy.Oncolytic viruses are expected to have long-term application in CM treatment, with T-VEC specifically predicted to maintain its function in managing patients with easily reachable cutaneous lesions, both for local containment and to induce a synergistically antitumor immune response, as part of combination therapies. Currently, several new viral vectors, such as coxsackieviruses, HF-10, adenovirus, reovirus, echovirus, and Newcastle disease virus, are being actively developed and investigated for their effectiveness in targeting CM, with varying degrees of success [84]. In an in vitro investigation involving HF10, it was discovered that an infection with this virus and a strain of HSV-1 that has undergone spontaneous mutation, resulting in the deletion of certain viral genes, had significant cytolytic effects on murine and human melanoma tumor cells.In that same study, HF10 was administered intratumorally to immunocompetent mice with advanced melanomas.The findings showed a decrease in tumor growth in both injected and non-injected tumors, indicating direct oncolysis as well as the induction of a systemic antitumor immune response [85,86]. In preclinical investigations, coxsackievirus A21 displayed oncolytic activity in melanoma cells while maintaining low viral pathogenicity and tolerability [82].The clinical assessment of CVA21, a commercial variant of coxsackievirus A21, was carried out in phases I and II on patients with advanced and unresectable melanoma who were treated with an intratumoral administration of the virus for 15 weeks.The outcomes demonstrated that the treatment was generally well-tolerated, with low-grade reactions, and resulted in complete therapeutic responses and an acceptable safety profile [87]. In a phase II trial, the oncolytic properties of a derived OV reovirus were examined in patients with metastatic melanoma who received the therapy through intravenous injections.All the patients tolerated the injections well, and in a few cases, viral replication was noticeable when analyzing post-treatment biopsy samples.However, the research did not obtain objectively identified responses or achieve its primary efficacy objective.Despite this, the trial data support using reovirus in combination with other therapies for treating malignant melanoma [88]. Currently, several oncolytic viruses are being tested as well for the treatment of uveal melanoma.The oncolytic potential of the present ECHO-7 virus strain has been observed in several malignancies, including melanoma cell lines [89].Therefore, the experiments were extended to examine the effect on the cell viability of cytolytic ECHO-7 virus strain on UM cell lines, and out of the seven cell lines tested, only 3 UM cell lines were able to be successfully propagated in the laboratory [90]. It is also noteworthy that phase I clinical trials have evaluated the use of oncolytic adenovirus ICOVIR-5 for the treatment of cutaneous and uveal melanoma.Although the virus was able to reach the tumor, a single injection did not result in any observed effects Life 2023, 13, 1666 8 of 17 on tumor regression.This suggests that a systematic administration of the virus over a longer duration should be investigated [91,92]. Taking into consideration the success that HSV already has as a treatment for CM, the oncolytic potential has also been tested in 3D uveal melanoma cell spheroids.According to the results, the HSV-1 virus exhibits oncolytic potential in certain melanoma cell lines, but it also stimulates the growth of other melanoma cells (Figure 1).be successfully propagated in the laboratory [90]. It is also noteworthy that phase I clinical trials have evaluated the u adenovirus ICOVIR-5 for the treatment of cutaneous and uveal melanoma virus was able to reach the tumor, a single injection did not result in any ob on tumor regression.This suggests that a systematic administration of th longer duration should be investigated [91,92]. Taking into consideration the success that HSV already has as a trea the oncolytic potential has also been tested in 3D uveal melanoma cell sphe ing to the results, the HSV-1 virus exhibits oncolytic potential in certain m lines, but it also stimulates the growth of other melanoma cells (Figure 1). ",
"section_name": "Primary Tumor Treatment",
"section_num": "6."
},
{
"section_content": "Both tumors display a strong tendency to metastasize, although the mode of spreading is different [93].The development of metastatic disease is a significant predictor of the clinical course and survival in both CM and UM [2]. CM tends to spread mainly via the lymphatic system; however, hematogenous dissemination has been reported as well.Although all organs can be implicated, the skin (13-38%), the lungs (18-36%), distant lymph nodes (5-34%), distant subcutaneous tissues (32%), the brain (2-20%), and the bones (4-17%) are the most prevalent sites for distant CM metastases.Approximately 14-20% of people will develop liver metastases [94]. The uveal tract is devoid of any lymphatics, so ocular melanoma spreads mainly via the bloodstream (hematogenous).The liver is the most predominant metastatic region (80-90% of cases), followed by the lung (24%) and the bones (16%) [95].Regardless of enucleation or eye-sparing radiation therapies, 45% of UM patients die from metastatic disease [87].This has led to hypotheses about the presence of micrometastasis in the early stages of the disease, misdiagnosed for years before becoming clinically detectable macrometastasis [96].The duration of the state of dormancy, as well as the triggers for metastatic development, are still unknown.Tumors as small as 1.0 mm do metastasize, highlighting the necessity for highly specific and sensitive prognostic markers to determine which patients are at risk of developing metastasis.Early metastatic disease and shorter survival are mainly associated with the age of over 60 years, a maximum basal tumor diameter over 18 mm, an epithelioid tumor cell type, and closed vascular patterns [97,98]. Regarding CM, an increasing Breslow tumor thicknesses, invasion level, an older age, male gender, head/neck or trunk primary tumor anatomic site, the number of metastatic lymph nodes, and ulceration on histopathological examination are independent significant prognostic factors of early metastasis.The TNM-staging system summarizes these aspects into a single staging system.This system is based on the tumor stage at the time of diagnosis, which has been proven to be the most significant prognostic factor in CM and is now commonly utilized for prognosis and clinical decisions.Integrin expression levels of circulating plasma exosomes isolated from CM and UM, for instance, are a prognostic factor for determining future metastatic sites.Furthermore, chemokine receptors are expressed in a wide range of malignancies, and their ligands are present in the organs with the highest frequency of metastasis.Chemokine receptors may have an impact on overall survival in patients and could be used as a therapy target [99]. Overexpression of c-Met, a hepatocyte growth factor (HGF) receptor, is linked to tumor growth and metastasis in CM.In vitro, suppressing HGF-induced c-Met proliferation inhibited the migration and invasion of melanoma cell lines [100].In UM, c-Met accelerates tumor growth and facilitates tumor invasion.The presence of c-Met in primary UM increases the possibility of hepatic metastasis development.Cabozantinib is a tyrosine kinase inhibitor that targets the receptors for MET, AXL, and VEGF (vascular endothelial growth factor).It inhibits HGF-induced migration and invasion in CM cells, and it has been demonstrated to inhibit liver metastasis development in a UM xenograft model [101][102][103]. Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone that stimulates tissue growth and has been associated with the development of a variety of tumors, including CM.The blood IGF-1 level served as a prospective prognostic biomarker for metastatic tumor development in both CM and UM.Remarkably, whereas metastatic UM patients reported lower IGF-1 serum concentrations than healthy controls, metastatic CM patients exhibited greater IGF-1 serum levels [104,105].In UM, an increased insulin-like growth factor 1 receptor (IGF-1R) expression was detected in liver metastasis and was correlated with mortality in the context of metastatic disease.In both CM and UM, the IGF/IGF-1R axis has been a target for novel therapy combinations.Considering that IGF plays a role in both primary and acquired treatment resistance, IGF-targeting medications have been administered in combination with other therapeutic approaches in CM [106,107]. In various forms of cancer, hypoxia-inducible factor (HIF) plays a significant role in carcinogenesis and metastatic dissemination.It plays a crucial function in the development of skin melanoma from melanocytes.HIF activity is upregulated in melanoma even at normal oxygen levels, boosting tumor cell infiltration into surrounding tissues and preserving adequate blood supply.FBXO22 has recently been suggested as a potential new therapy approach for CM since it is thought to modulate HIF expression.In UM, it was reported that a relative hypoxic activity identified the subtypes, regardless of chromosome 3 status [49,108]. There exists a demand for further treatment options for metastatic melanoma beyond FDA-approved anti-PD1 antibodies, which are only sanctioned for adjuvant therapy in stage III or resected stage IV melanoma [90]. The evaluation of the immune system's role in tumorigenesis in the latest research resulted in the TIL (tumor-infiltrating lymphocyte) clinical success of adoptive cell therapy (ACT) in the treatment of CM.Due to its revolutionary status, TIL treatment has been applied to various malignancies, including UM, although its efficacy has been documented mainly in metastatic cutaneous melanoma [109][110][111][112]. Treatment with chimeric antigen receptor (CAR) T cells is another cell-based therapy that might be attractive.Both the United States and the European Union have approved two CAR-T cell models that target CD19 in hematological malignancies.c-Met is a target antigen in one of the pilot trials actively recruiting melanoma patients now.Considering that c-Met is associated with both CM and UM, this could be a promising therapeutic approach for both types of melanomas [2]. The FDA and EMA have recently approved tebentafusp for the treatment of patients with metastatic UM and HLA-A*02:01 positivity, which is encountered in approximately 45% of the Caucasian population.The bispecific fusion protein targets gp100 through a T cell receptor (TCR) binding domain and a CD3 T-cell engaging domain.In pivotal clinical studies, the drug demonstrated a 1-year overall survival in 73% of treated patients versus 59% in the control group and a progression-free survival of 31% versus 19% in the control group at 6 months [113,114]. ",
"section_name": "Metastatic Disease",
"section_num": "7."
},
{
"section_content": "CM presents significant challenges in its management, as traditional chemotherapy exhibits a limited response rate.Immunotherapies have shown promise, but there is a pressing need for more effective treatments.Molecularly targeted therapies, including FDA-approved tyrosine kinase inhibitors (TKIs), have demonstrated initial efficacy [115].However, prolonged use often leads to acquired resistance through genetic mutations and alternative mechanisms in melanoma tumors, undermining their long-term effectiveness.Efforts to enhance treatment options focus on understanding drug resistance mechanisms, particularly \"phenotype switching\", and investigating novel combination therapies that target both fast-and slow-proliferating cells [116].Overcoming drug resistance is crucial to improve outcomes and prolong survival for melanoma patients, urging the continued exploration of innovative approaches and new therapeutic targets (Table 1) [117]. ",
"section_name": "Future Directions",
"section_num": "8."
},
{
"section_content": "Nrf2 transcription factor implicated in the altered redox homeostasis, which led to changes in genes related to melanoma progression, suggesting its involvement in phenotype switching [125][126][127] MITF transcription factor as a key regulator of melanoma cell proliferation, survival, and invasion [128,129] IDO enzyme that mediates the conversion of tryptophan to kynurenine, leading to immune suppression in the tumor microenvironment [130][131][132] MDM2 overexpression can lead to the inactivation of p53, promoting tumor growth and decreasing survival [133][134][135] Among these promising biomarkers, nicotinamide N-methyltransferase (NNMT) has emerged as a key player in various malignancies, including skin cancer.Its role in catalyzing the N-methylation of nicotinamide and involvement in homeostasis and detoxification processes suggest its potential as a therapeutic target for melanoma treatment [118][119][120][121]. Paraoxonase-2 (PON2) is another notable biomarker, with significant upregulation observed in melanomas compared to control nevi.PON2 expression correlates with important prognostic parameters, indicating its potential as a prognostic biomarker and its involvement in melanoma cell resistance to chemotherapy [122][123][124].Moreover, the transcription factor Nrf2 has been linked to an altered redox homeostasis in melanomas, influencing phenotype switching.While directly targeting NRF2 may require a combinatorial approach, its downstream antioxidant gene products could be explored as more effective therapeutic targets [125][126][127]. Additionally, other biomarkers such as MITF (microphthalmia-associated transcription factor), IDO (indoleamine 2,3-dioxygenase), and MDM2 (mouse double minute 2 homolog) have shown promise in regulating melanoma cell behavior, offering potential avenues for targeted therapeutic strategies.As research progresses, the identification and validation of these biomarkers could revolutionize melanoma management, leading to improved patient outcomes and personalized treatment approaches [128][129][130][131][132][133][134][135]. ",
"section_name": "Therapeutic Target Effectory Function Evidence",
"section_num": null
},
{
"section_content": "CM and UM are distinct tumor types with significant differences in tumorigenesis, genetic alterations, metastatic dissemination pathways, and responses to therapies.Although both malignancies commonly involve abnormalities in certain chromosomal regions, the rates at which these occur differ.CM frequently exhibits abnormalities on chromosomes 1, 6, 7, 9, 10, 14, 16, and 21, while UM is characterized by chromosomal aberrations on chromosomes 1, 3, and 8. Monosomy 3, the gain of 8q, monosomy 3, and the loss of 1p36 are all significant factors associated with a poor prognosis. In CM, approximately 80% of patients have mutations in the BRAF, NRAS, or NF1 genes, leading to the deregulation of the ERK pathway.On the other hand, UM frequently harbors activating mutations in GNAQ/11 (83%).UM also exhibits recurrent genetic mutations in BAP1, which plays a role in cell cycle regulation, cell identity, genome integrity, and metastasis development. Both types of tumors have a propensity to spread, with UM mainly spreading hematogenously and showing a strong liver tropism.In contrast, CM metastasis affects the lungs, liver, bones, and brain at nearly equal rates. Significant advancements have been made in the treatment of advanced CM, leading to improved life quality and overall survival.Primary UM diagnosis and therapies have also seen significant improvements in recent decades, and the recent approval of tebentafusp has increased life expectancy in HLA-A*02:01-positive patients with metastatic disease.Genetic, clinical, and histopathological markers have shown promise in predicting prognosis and guiding patient-tailored therapies. ",
"section_name": "Conclusions",
"section_num": "9."
}
] |
[
{
"section_content": "The study of NNMT, PON2, Nrf2, MITF, IDO, and MDM2 as potential biomarkers in melanoma holds great promise for improving diagnosis and advancing targeted therapies.Further research and exploration are needed to fully understand their roles and interactions, paving the way for novel treatments in the battle against melanoma. Despite these advances, no efficacious treatment has been found for metastatic melanoma.Therefore, a further exploration of mutations associated with tumor growth, proliferation, metastasis, and survival is necessary to better understand their pathogeneses and metastatic mechanisms and develop more potent treatments. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Funding: This research received no external funding. Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Funding: This research received no external funding. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Data Availability Statement: All information provided in this review is documented by relevant references. The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Data Availability Statement: All information provided in this review is documented by relevant references. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.3390/diagnostics14050469
|
PrecisionLymphoNet: Advancing Malignant Lymphoma Diagnosis via Ensemble Transfer Learning with CNNs
|
<jats:p>Malignant lymphoma, which impacts the lymphatic system, presents diverse challenges in accurate diagnosis due to its varied subtypes—chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). Lymphoma is a form of cancer that begins in the lymphatic system, impacting lymphocytes, which are a specific type of white blood cell. This research addresses these challenges by proposing ensemble and non-ensemble transfer learning models employing pre-trained weights from VGG16, VGG19, DenseNet201, InceptionV3, and Xception. For the ensemble technique, this paper adopts a stack-based ensemble approach. It is a two-level classification approach and best suited for accuracy improvement. Testing on a multiclass dataset of CLL, FL, and MCL reveals exceptional diagnostic accuracy, with DenseNet201, InceptionV3, and Xception exceeding 90% accuracy. The proposed ensemble model, leveraging InceptionV3 and Xception, achieves an outstanding 99% accuracy over 300 epochs, surpassing previous prediction methods. This study demonstrates the feasibility and efficiency of the proposed approach, showcasing its potential in real-world medical applications for precise lymphoma diagnosis.</jats:p>
|
[
{
"section_content": "Lymphoma, a form of hematological disorder, arises due to uncontrolled proliferation of lymphocytes, a subset of leukocytes.The lymphocytes, which are found in the blood and lymphatic tissues of the human body, have a crucial role in protecting the individual from various diseases.The lymphatic system comprises lymph nodes and lymphatic vessels responsible for draining fluid from bodily tissues and redirecting it to the circulatory system.Additionally, these structures aid in the removal of impaired, foreign, or aged cells.There are two types of lymphocytes, namely T and B. Both T and B lymphocytes reside in the lymph nodes.T cells have the ability to identify new antigens and transport them out of the body, while B lymphocytes produce antibodies.Lymphoma can be caused by any of these cells individually or in combination.As lymphocytes develop and form a mature lymph node, foreign lymphocytes will exceed the normal cells within the node.The symptoms and signs of the disease can vary depending on where the cancer originates, which body regions are affected, and the specific type of lymphoma [1]. Lymphoma typically presents itself in two distinct forms, namely Hodgkin lymphoma and non-Hodgkin lymphoma, as shown in Figure 1.The primary difference between these two variations of lymphatic cancer lies in the specific type of lymphocyte that is affected.While both Hodgkin lymphoma and non-Hodgkin lymphoma originate from B cells, only the latter is affected.Hodgkin lymphoma frequently originates in the upper regions of the body, such as the neck, chest, or armpits, whereas non-Hodgkin lymphoma can initiate in any lymph node throughout the body.Hence, there is a crucial need for diagnoses using automated techniques to overcome the existing challenges in malignant lymphoma [2]. phoma and non-Hodgkin lymphoma, as shown in Figure 1.The primary difference tween these two variations of lymphatic cancer lies in the specific type of lymphocyte t is affected.While both Hodgkin lymphoma and non-Hodgkin lymphoma originate fr B cells, only the latter is affected.Hodgkin lymphoma frequently originates in the up regions of the body, such as the neck, chest, or armpits, whereas non-Hodgkin lymp ma can initiate in any lymph node throughout the body.Hence, there is a crucial need diagnoses using automated techniques to overcome the existing challenges in malign lymphoma [2].Malignant lymphoma classification adopts several deep learning networks, wh have played a significant role in recent years in analyzing Whole Side Images (WSIs) pathological tissues of lymph nodes.This study focused on geometric, texture, a morphological analyses with feature-associated clinical and cytogenetic data [3]. The process of manually detecting malignant lymphoma is unfeasible.Poten solutions could be found using artificial intelligence models.Classification, segmen tion, detection, and prediction are some of the applications of artificial intelligence m els.Malignant lymphoma detection is recently popular and related works have been haustively analyzed and discussed [4]. This paper focuses on the objective of developing systems that are capable of p dicting the specific type of malignant lymphoma based on histopathological samples t have been stained with Hematoxylin/Eosin (H + E). The primary contributions of this paper are as follows: • Introduces a transfer learning CNN model comprising convolutional layers, pool layers, and a fully connected layer tailored for multi-classification; • A novel ensemble architecture, incorporating InceptionV3 and Xception, is p posed to enhance accuracy in lymphoma diagnosis, achieving an impressive 9 accuracy on multi-cancer datasets; Extensive testing is conducted on multi-class datasets from diverse sources, feat ing chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle c lymphoma (MCL).This ensures the robustness and generalization of the propos models; • Features are extracted via convolutional layers, employing image scaling prep cessing, data augmentation, and spatial dimensionality reduction.T non-ensemble model, particularly compatible with Xception, outperforms ot models with 97% accuracy and minimal validation loss on multiple cancer datase The remaining sections of this work are as follows: Section 2 provides a summary various relevant studies pertaining to the classification of malignant lymphoma.Sectio Malignant lymphoma classification adopts several deep learning networks, which have played a significant role in recent years in analyzing Whole Side Images (WSIs) of pathological tissues of lymph nodes.This study focused on geometric, texture, and morphological analyses with feature-associated clinical and cytogenetic data [3]. The process of manually detecting malignant lymphoma is unfeasible.Potential solutions could be found using artificial intelligence models.Classification, segmentation, detection, and prediction are some of the applications of artificial intelligence models.Malignant lymphoma detection is recently popular and related works have been exhaustively analyzed and discussed [4]. This paper focuses on the objective of developing systems that are capable of predicting the specific type of malignant lymphoma based on histopathological samples that have been stained with Hematoxylin/Eosin (H + E). The primary contributions of this paper are as follows: • Introduces a transfer learning CNN model comprising convolutional layers, pooling layers, and a fully connected layer tailored for multi-classification; • A novel ensemble architecture, incorporating InceptionV3 and Xception, is proposed to enhance accuracy in lymphoma diagnosis, achieving an impressive 99% accuracy on multi-cancer datasets; Extensive testing is conducted on multi-class datasets from diverse sources, featuring chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL).This ensures the robustness and generalization of the proposed models; ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Features are extracted via convolutional layers, employing image scaling preprocessing, data augmentation, and spatial dimensionality reduction.The non-ensemble model, particularly compatible with Xception, outperforms other models with 97% accuracy and minimal validation loss on multiple cancer datasets. The remaining sections of this work are as follows: Section 2 provides a summary of various relevant studies pertaining to the classification of malignant lymphoma.Section 3 describes the system's architecture, specifically focusing on the pre-trained weights of VGG16, VGG19, DenseNet201, Inceptionv3, and Xception.Section 4 provides a malignant lymphoma image data description for the proposed architecture.Section 5 illustrates the proposed Non-Ensemble and Ensemble Transfer Learning architectures for malignant lymphoma classification.Section 6 discusses the performance evaluation metrics for training and testing the proposed system.Section 7 elaborates the experimental setup of the training and testing environment.Section 8 analyzes the results derived from the proposed system.Section 9 compares the performance of the proposed ensemble model with prior works.Lastly, Section 10 concludes the proposed system's limitation and future work. ",
"section_name": "•",
"section_num": null
},
{
"section_content": "This section provides an overview of several prior investigations that are pertinent to the identification of malignant lymphoma.All researchers intended to attain favorable outcomes via the implementation of distinct methodologies. ",
"section_name": "Related Works",
"section_num": "2."
},
{
"section_content": "Capobianco et al. [1] proposed an ensemble model to find the Total Metabolic Tumor Volume (TMTV) calculated from F-labelled fluoro-2-deoxyglucose.The computed results showed that the model TMTV obtained 85% classification accuracy, 80% sensitivity, and 88% specificity in detecting lymphoma.Patil et al. [2] addressed the overlapping of blood cell image classification using Canonical Correlation Analysis (CCA).Several deep learning models were combined to perform the prediction of overlapping blood cell classification.A CNN was merged with many other deep learning models and computed the validation accuracy.The blood cells are mainly in two categories.Granular cell is the first category.The subcategories of granular cell are neutrophil, eosinophil, and basophil.Non-granular cell is the second category.Its subtypes are monocyte and lymphocyte.The combined deep learning models of CNN, VGG16, RNN, and LSTM obtained 89% accuracy.A CNN, InceptionV3, RNN, and LSTM combination achieved 91% accuracy.A CNN, ResNet50, RNN, and LSTM combination approach obtained 93% accuracy.A CNN, Xception, RNN, and LSTM combination obtained 95% accuracy.Tambe et al. [5] explored the automated morphometric analysis of cancer diagnosis using a deep learning technique.This method classified subtypes of lymphoma as chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma and achieved an accuracy of 97.33%.Steinbuss, G et al. [6] demonstrated that an EfficientNetB3 deep learning model is suitable for the classification of tumor-free lymph nodes and tumor lymph cells of CLL and DLBCL.The EfficientNetB3 secured 95.56% accuracy on classification above lymph categories.El Achi et al. [7] proposed the prediction and diagnosis of lymphoma using CNN modeling.The CNN modeling is used to build diagnostic models into four types, namely benign lymph nodes, diffuse large B-cell lymphoma, Burkitt lymphoma, and lymphoma small cells.The obtained validation accuracy of the CNN modeling on diagnosis of the above five categories was 95%.Shrot et al. [8] distinguished different types of brain tumors via an experimental study of 141 patients (41 glioblastomas, 38 metastatic tumors, 50 meningioma, and 12 primary CNS lymphomas) using basic and advanced MRI sequencing and obtained better accuracy than other existing methods.Miyoshi et al. [9] determined malignant lymphoma from histopathological images by using an ensemble approach.The ensemble model combines prediction results of each three deep learning models and an averaging approach is used to predict the final malignant lymphoma.Sibille et al. [10] evaluated lung cancer and lymphoma using a deep convolutional neural network (CNN) which classified the 18F-FDG PET/CT images into cancer patients or not.Gaidano et al. [11] developed an immune phenotypic prediction model which consisted of multiple decision tree approaches for detecting B-cell non-Hodgkin lymphoma in blood cell images.Three different decision trees are built from the features present in the dataset.Since the selected decisions are very suitable for lymphoma detection for model 2, model 2 obtained 92% accuracy, model 1 obtained 87%, model 3 obtained 89%, and model 4 obtained 87%.The author demonstrates the strong discriminating power of MIB1 and Bcl2, whose integration in the predictive model significantly increases the performance of the algorithm.The method measured the potential utility of some nonconforming markers in the B-NHL classification.The FC markers do not qualify as positive or negative under fixed thresholds, but rather they are correlated with different B-NHLs depending on their expression level.Ijaz et al. [12] presented a cervical cancer prediction model for the early detection of cervical cancer using Random Forest classifier models such as iForest-SMOTETomek and iForest-SMOTE approaches.This model outperformed when compared to other existing approaches. ",
"section_name": "Machine Learning Methods for Malignant Lymphoma Classification",
"section_num": "2.1."
},
{
"section_content": "Zhao et al. [13] distinguished diseased samples from healthy samples using a CNN model.They classified seven subtypes of adult B-cell tumors such as chronic lymphocytic leukemia, marginal zone lymphoma, mantle cell lymphoma, prolymphocytic leukemia, follicular lymphoma, hairy cell leukemia, and lymphoma.The reliability of the classification was 70% of appeals with 95% confidence.Sheng, B., Zhou et al. [14] used a large number of blood cell datasets, which contained lymphoma cells, lymphocytes, blast cells, and an annotation file of each image file.The authors determined the final model by testing the performance of a combination of different training methods and networks on this dataset and testing its performance against a brand-new dataset.The final results of the test found that the lymphoma detection rate was greater than 95%.Lippi et al. [15] developed a multi-version learning model using support vector machine with texture features.The result showed the detection of Hodgkin lymphoma was more than 90% accurate.Zhang et al. [16] presented the classification of NHL subtypes based on the fusion of transfer learning (TL) to detect non-Hodgkin lymphoma and its subtypes from digital pathological images.For feature extraction, a Principal Component Analysis (PCA) approach was used. Rajpurohit et al. [17] diagnosed acute lymphoblastic leukemia blood cancer.The detection of this type of cancer is performed manually by looking at a patient's blood sample under a microscope and performing a variety of tests.The authors used blood images and applied various classifiers such as CNN, FNN, SVM, and KNN to automate the above-mentioned manual work.Brancati et al. [18] presented a deep learning approach with specific parameters for cancer detection and classification.They used fusion Net encoder for image segmentation and the reconstruction approach was adapted for cancer detection and histological image classification.They conducted a comparison with conventional approaches.Biccler et al. [19] presented the predictive performance of prognostic scores in various types of malignant lymphoma and plotted the obtained score results. Srinivasu et al. [20] proposed a deep-learning-based MobileNet V2 and Long Short Term Memory (LSTM) model for detecting skin disease from an image of the region of interest at an early stage, assisting physicians in predicting skin conditions efficiently and minimizing further complications. ",
"section_name": "Deep Learning Methods for Malignant Lymphoma Classification",
"section_num": "2.2."
},
{
"section_content": "We analyzed a pre-trained weight transfer learning model for malignant lymphoma classification.The findings achieved in this study are quite promising and the performance values are represented in Table 1. ",
"section_name": "Transfer Learning Methods for Malignant Lymphoma Classification",
"section_num": "2.3."
},
{
"section_content": "Lymphomas can be categorized into two primary groups.These groups consist of non-Hodgkin lymphoma and Hodgkin lymphoma.The non-Hodgkin type encompasses three key subgroups and is more malignant compared to the Hodgkin type.These subgroups include chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and follicular lymphoma (FL).The most dangerous and persistent form of leukemia within this group is chronic lymphocytic leukemia (CLL).The proposed system utilized Convolution Neural Network (CNN) neural network algorithms to train the models, make predictions, compare the results, and determine the most accurate outcome. ",
"section_name": "Materials and Methods",
"section_num": "3."
},
{
"section_content": "The Visual Geometry Group VGG-16 is comprised of a total of 16 layers.Among these layers, there are 3 fully linked layers and 13 convolutional layers.The max-pooling layers within the network possess a filter size of 2 × 2, with a stride of 2 pixels.On the other hand, each individual convolutional layer has a filter size of 3 × 3, with a stride of 1 pixel.This network is designed to receive an RGB image with an input size of 224 × 224 pixels.The output layer of the network consists of 1000 units, each corresponding to one of the 1000 Image Net classes.The VGG-16 architecture effectively utilizes the combination of depth and tiny filters (3 × 3) in its convolutional layers, enabling it to capture complex features within images [29]. ",
"section_name": "Visual Geometry Group16 (VGG16)",
"section_num": "3.1."
},
{
"section_content": "The Visual Geometry Group VGG-19 consists of a total of 19 layers, comprising 16 convolutional layers and 3 fully linked layers.The additional convolutional layers in VGG-19 are specifically designed to capture more complex features present in the input images, thereby improving its accuracy for image recognition.Similar to the VGG-16, the VGG-19 consists of 2 × 2 max-pooling layers.These layers have a stride of 2 pixels and small 3 × 3 filters in all convolutional layers.The stride of these filters is set to 1 pixel.The final layer of the network consists of 1000 units, aligning with the 1000 ImageNet classes and the input to the network is RGB image with a size of 224 × 224 pixels [30]. ",
"section_name": "Visual Geometry Group19 (VGG19)",
"section_num": "3.2."
},
{
"section_content": "DenseNet-201 primarily addresses disappearing gradients in deep neural networks via the implementation of feed-forward networks linking each layer to all other layers.This dense connectivity approach decreases the required parameters and enables the reuse of features across layers, thus improving the performance of the model.DenseNet-201 comprises 201 levels, which consist of multiple dense blocks that are connected by transition layers.Each dense block consists of a series of convolutional layers with a predetermined number of filters, followed by a bottleneck layer that reduces the number of channels.The input for each layer in a dense block is the concatenated feature maps from all previous layers.This transition layer includes a batch normalization layer, a 1 × 1 convolution layer for dimensionality reduction, and a max-pooling layer [31].The input for DenseNet-201 consists of an RGB image which has a dimension of 224 × 224 pixels.The output layer of DenseNet-201 is composed of 1000 units that correspond to the 1000 classes in the ImageNet dataset. ",
"section_name": "DenseNet201",
"section_num": "3.3."
},
{
"section_content": "The primary objective of Inception v3 is to augment the precision and effectiveness of the Inception architecture via the implementation of significant modifications.Notably, one of the most prominent modifications is the integration of batch normalization, which decreases the internal covariate shift and expedites the process of training.Furthermore, Inception v3 adopts factorized 7 × 7 convolutions rather than 7 × 7 convolutions to minimize the quantity of parameters within the network.Inception v3 comprises a pooling layer and multiple parallel convolutional layers having diverse filter sizes of 1 × 1, 3 × 3, and 5 × 5.The outputs of these parallel layers are merged and provided to the subsequent layer.To enhance the network's ability to learn more distinctive features, auxiliary classifiers are integrated into the model and placed into the middle of the network.The input to the Inception v3 network is an RGB image with a size of 299 × 299 pixels, which is significantly larger than the original Inception design's input size.The output layer of the network consists of 1000 units, corresponding to the 1000 classes in the ImageNet dataset [32]. ",
"section_name": "Inception v3",
"section_num": "3.4."
},
{
"section_content": "Xception uses depth-wise separable convolutions as a standard alternative for the basic convolutional layers found in the Inception architecture.A depth-wise separable convolution is a two-step convolution process that first implements a spatial convolution on each input channel separately, and subsequently performs a point-wise convolution to combine the outputs of the spatial convolutions.This approach increases the power of the convolutional layers with a smaller number of parameters and computations.To optimize the effectiveness of the model, Xception combines both skipping connections and residual connections in addition to multiple depth-wise separable convolutional layers.The input to the Xception network is an RGB image with dimensions of 299 × 299 pixels, while the output layer comprises 1000 units that map to the 1000 classes in the ImageNet dataset [33]. ",
"section_name": "Xception",
"section_num": "3.5."
},
{
"section_content": "Lymphoma has become the seventh most common cancer expected to occur and the ninth most common cause of cancer death in both males and females.However, pathological diagnosis as the main diagnostic method is time-consuming, expensive, and error-prone.Most of the researchers use the lymphopath database of the cancer research institute.The analysis is performed with multiple datasets such as ImageNet, PASCAL VOC, and MS COCO Dataset kaggle multicancer WSI images.The researchers use microscopic blood image datasets with samples of leukocytes and modified by their type's lymphoma.Lymphoma has three categories such as CLL (chronic lymphocytic leukemia), FL (follicular lymphoma), and MCL (mantle cell lymphoma).CLL is one of the bone marrow and blood cancer types.The bone marrow contains a soft tissue from which blood cells are generated.FL is the second type of cancer in the lymph nodes, bone marrow, and organs.FL is naturally indolent and its cancer cell development is slow in this category.The MCL cancer type starts with white blood cells in lymph nodes. This system evaluated histopathological images of the multiple datasets of malignant lymphoma.The non-ensemble model of pre-trained network will be fine-tuned using benchmarked datasets.There are two different datasets used in this paper.Both the datasets are downloaded from the kaggle repository.The first dataset is downloaded from the URL https://www.kaggle.com/datasets/andrewmvd/malignant-lymphomaclassification(accessed on 12 December 2023) and the dataset size is 374.In this work, a total 334 TIF-formatted samples are used for training and 40 samples are used for testing the framework.Within the 334-training dataset are 109 samples of CLL, 124 samples of FL, and 109 samples of Myelofibrosis of MCL [34].The test data are split into 12 samples of CLL, 15 samples of FL, and 13 samples of MCL.The convolutional neural network is initialized with RGB images of size 240 × 240 pixels.Finally, the most effectively trained models, the InceptionV3 and Xception models, are combined as an ensemble architecture for diagnosing lymphoma.The second dataset used in this paper consists of 15,000 images and the URL is https://www.kaggle.com/datasets/obulisainaren/multi-cancer(accessed on 12 December 2023).The dataset contains three equal parts of 5000 for CLL, 5000 for FL, and 5000 for MCL.The test data are split into 1029 samples of CLL, 962 samples of FL, and 1009 samples of MCL.All dataset images were in the JPEG file format with a size of 512 × 512 pixels.The dataset employed in this investigation is shown in Figure 2. ",
"section_name": "Dataset Description",
"section_num": "4."
},
{
"section_content": "The non-ensemble model is trained using VGG16, VGG19, DenseNet201, Incep-tionv3, and Xception as shown in Figure 3.A Convolutional Neural Network (CNN) is a type of neural network architecture used for tasks such as image classification, object detection, and other computer vision applications.CNNs are specifically designed to identify patterns of input images via the utilization of trained convolutional layers.The CLL, FL, and MCL image datasets are imported from Kaggle and multiple platforms and fed as an input block.Several pre-processing methodologies such as data augmentation, scaling, image formatting, and RGB conversion are used to create and train the model.Initially, color inversion is performed on the picture dataset using RGB conversion.Subsequently, image formatting was undertaken, where the file extension of the image dataset was modified from JPEG to TIFF.Furthermore, image scaling is done to the size of 224 × 224 pixels.Finally, data augmentation is performed using flipping, rotating, cropping, and padding of the image [35].The Convolutional Neural Network (CNN) comprises several layers, such as con- ",
"section_name": "Proposed Non-Ensemble and Stacked Ensemble Transfer Learning Architecture for Malignant Lymphoma Classification",
"section_num": "5."
},
{
"section_content": "The non-ensemble model is trained using VGG16, VGG19, DenseNet201, Inceptionv3, and Xception as shown in Figure 3. ",
"section_name": "Proposed Non-Ensemble and Stacked Ensemble Transfer Learning Architecture for Malignant Lymphoma Classification",
"section_num": "5."
},
{
"section_content": "The non-ensemble model is trained using VGG16, VGG19, DenseNet201, Incep-tionv3, and Xception as shown in Figure 3.A Convolutional Neural Network (CNN) is a type of neural network architecture used for tasks such as image classification, object detection, and other computer vision applications.CNNs are specifically designed to identify patterns of input images via the utilization of trained convolutional layers.The CLL, FL, and MCL image datasets are imported from Kaggle and multiple platforms and fed as an input block.Several pre-processing methodologies such as data augmentation, scaling, image formatting, and RGB conversion are used to create and train the model.Initially, color inversion is performed on the picture dataset using RGB conversion.Subsequently, image formatting was undertaken, where the file extension of the image dataset was modified from JPEG to TIFF.Furthermore, image scaling is done to the size of 224 × 224 pixels.Finally, data augmentation is performed using flipping, rotating, cropping, and padding of the image [35].The Convolutional Neural Network (CNN) comprises several layers, such as con- A Convolutional Neural Network (CNN) is a type of neural network architecture used for tasks such as image classification, object detection, and other computer vision applications.CNNs are specifically designed to identify patterns of input images via the utilization of trained convolutional layers.The CLL, FL, and MCL image datasets are imported from Kaggle and multiple platforms and fed as an input block.Several preprocessing methodologies such as data augmentation, scaling, image formatting, and RGB conversion are used to create and train the model.Initially, color inversion is performed on the picture dataset using RGB conversion.Subsequently, image formatting was undertaken, where the file extension of the image dataset was modified from JPEG to TIFF.Furthermore, image scaling is done to the size of 224 × 224 pixels.Finally, data augmentation is performed using flipping, rotating, cropping, and padding of the image [35].The Convolutional Neural Network (CNN) comprises several layers, such as convolutional layers, pooling layers, and fully connected layers.In a typical CNN, the first few layers are convolutional layers that extract features from the input images.These layers employ multiple filters to the input data, and the output of each filter is a feature map that represents the presence of a specific element in the data.Pooling layers are often utilized after convolutional layers to simplify the spatial dimensionality of feature maps.The most widely used type of pooling is max pooling, which reduces the size of the feature map by selecting the highest value within each spatial region.The resulting data are transformed into a compressed form and transmitted to one or more completely interconnected layers, which execute the categorization, following a series of repeated convolutional and pooling stages.The Softmax activation function is applied to the outcome of the final fully interconnected layer to obtain the probability distribution across all potential classes.The initial input to the Convolutional Neural Network (CNN) consists of an image represented as a matrix of pixel values.Subsequently, multiple convolutional layers are applied to this image.Each convolutional layer uses a set of trainable filters on the input image, resulting in a set of feature maps.These feature maps represent the activation of the filters at various spots across the input image.To add non-linearity and to increase the efficacy of the model, each feature map undergoes a non-linear activation function, such as Rectified Linear Unit (ReLU).Following this, the feature maps are subjected to pooling layers that down-sample them by selecting the highest or average value within each spot of the map.This process reduces the spatial dimensionality of the feature maps and improves the model's robustness against minor input variations. The output of the final pooling layer is then flattened and applied through one or more fully connected layers for classification.Typically, a SoftMax layer is employed as the top layer, generating a probability distribution for all possible classes.The class with the highest probability is chosen as the prediction.To determine the optimal values for the filter weights and biases, the model is trained using a dataset of labelled images and a stochastic gradient descent optimization algorithm.For testing the non-ensemble model, 342 TIF-formatted samples are used for training and 40 samples are used for testing the framework.The test data are split into 12 samples of CLL, 15 samples of FL, and 13 samples of MCL.Inceptionv3 and Xception attain maximum efficiency when compared to other pre-trained models.To improve the accuracy more than one algorithm is essential.Thus the proposed approach has used Inceptionv3 and Xception advanced deep neural network models to enhance the prediction accuracy.Figure 4a shows a step-by-step approach for stacking the ensemble approach to classify lymphoma cells. The stacked ensemble model is trained and tested using a second dataset.The multiclass images are divided into three equal parts of 5000 for CLL, 5000 for FL, and 5000 for MCL.The 15,000 samples are used for training and 3000 samples are used for testing the model.The test data are split into 1029 samples of CLL, 962 samples of FL, and 1009 samples of MCL.All dataset images were in the JPEG file format with a size of 512 × 512 pixels.The advanced deep learning models such as Xception and InceptionV3 are the best suited models for image classification.Thus, the proposed method deployed these two models as the level-0 classifier models.These two advanced neural network architectures are trained using training samples and generate a new dataset for the second-level classification. (a) The stacked ensemble model is trained and tested using a second dataset.The multi-class images are divided into three equal parts of 5000 for CLL, 5000 for FL, and 5000 for MCL.The 15,000 samples are used for training and 3000 samples are used for testing the model.The test data are split into 1029 samples of CLL, 962 samples of FL, and 1009 samples of MCL.All dataset images were in the JPEG file format with a size of 512 × 512 pixels.The advanced deep learning models such as Xception and InceptionV3 are the best suited models for image classification.Thus, the proposed method deployed these two models as the level-0 classifier models.These two advanced neural network architectures are trained using training samples and generate a new dataset for the second-level classification. ",
"section_name": "Proposed Non-Ensemble and Stacked Ensemble Transfer Learning Architecture for Malignant Lymphoma Classification",
"section_num": "5."
},
{
"section_content": "The Mean Absolute Error (MAE) shown in Equation ( 1) is a widely used metric for assessing the efficacy of a predictive model.It quantifies the average absolute difference between the predicted values and the actual values of the target variable. ",
"section_name": "Evaluation Metrics for Proposed Models",
"section_num": "6."
},
{
"section_content": "",
"section_name": "InceptionV3 Xception",
"section_num": null
},
{
"section_content": "",
"section_name": "Generate",
"section_num": null
},
{
"section_content": "The Mean Absolute Error (MAE) shown in Equation ( 1) is a widely used metric for assessing the efficacy of a predictive model.It quantifies the average absolute difference between the predicted values and the actual values of the target variable. The Mean Squared Error (MSE), as depicted in Equation (2), quantifies the average of the squared difference between the predicted values and the original values of the target variable. The Mean Absolute Percentage Error (MAPE), as shown in Equation (3), is used as a quantitative measure for evaluating the accuracy of a forecasting model.This measure is derived from computing the mean percentage difference between the actual values and the predicted values. Accuracy, precision, recall, and F1 score are performance metrics commonly used in classification problems to evaluate the performance of a model.The measure of accuracy is derived from the ratio of correctly classified points to the total number of points, as expressed in Equation ( 4). where TP is True Positive, TN is True Negative, FP is False Positive, and FN is False Negative.Precision refers to the fraction of correctly categorized instances out of the overall classified instances, as shown in Equation (5). The recall or sensitivity can be defined as the ratio of correctly classified instances to the total number of instances classified, as shown in Equation ( 6). ",
"section_name": "Evaluation Metrics for Proposed Models",
"section_num": "6."
},
{
"section_content": "The F1 score can be defined as the harmonic mean of precision and recall, as expressed in Equation (7). Specificity measures the number of instances of true negatives that are correctly identified by the model, as shown in Equation (8). In the context of classification models, True Positives (TPs) refer to the instances that are truly positive and have been accurately classified as positive by the model.False Positives (FPs), on the other hand, denote the instances that are actually negative but have been erroneously classified as positive by the model.Similarly, True Negatives (TNs) represent the instances that are genuinely negative and have been correctly classified as negative by the model.Lastly, False Negatives (FNs) pertain to the instances that are truly positive but have been wrongly classified as negative by the model [36]. ",
"section_name": "Sensitivity or Recall",
"section_num": null
},
{
"section_content": "The Kaggle Framework was used to train the experiment using an Intel i9-12900 2.4 GZ 30 MB 16 Cores 64 W CPU (Intel, Santa Clara, CA, USA) and NVIDA RTX A2000 Graphics system (NVIDA, Santa Clara, CA, USA).The proposed stacked ensemble method is implemented in the Kaggle notebook. ",
"section_name": "Experimental Setup",
"section_num": "7."
},
{
"section_content": "In this particular section, our primary focus is on the multiple source datasets employed throughout the training and testing phases of five different CNN models such as VGG16, VGG19, DenseNet201, Inceptionv3, and Xception.An ensemble architecture is proposed to increase accuracy using InceptionV3 and Xception.Training and testing are performed for the ensemble architecture using a multi-cancer lymphoma Kaggle dataset.Subsequently, we discuss the outcomes of the proposed ensemble learning model on the mentioned CLL, FL, and MCL datasets.The pre-trained models are trained and tested at a learning rate of 0.001.The proposed model has used the kaggle notebook for the implementation.The CLL, FL, and MCL datasets are available in individual directories.The directories are loaded into the kaggle user environment [37].To assign target values for each image to train the deep learning models, python library label.index(foldername)and other predefined python library functions are used. ",
"section_name": "Performance Analysis and Discussion",
"section_num": "8."
},
{
"section_content": "The VGG16 model is sequentially composed of 16 deep convolutional layers.The model shown in Figure 5 is developed with the input, functional, and output layers.The pooling layer is a fixed operation with no weighting factor [38]. ",
"section_name": "Performance Evaluation of VGG16 Model",
"section_num": "8.1."
},
{
"section_content": "The VGG16 model is sequentially composed of 16 deep convolutional layers.The model shown in Figure 5 is developed with the input, functional, and output layers.The pooling layer is a fixed operation with no weighting factor [38].The validation loss is greater than the training loss.Thus, the model is over fitted.The capability of the VGG16 MAE value is 0.0281, the MSE value is 0.0045, and the MAPE value is 0.3310 for the test data [39]. The confusion matrix shows that 15 samples are correctly predicted out of the total 40.Thus, overall accuracy is 38%.The macro average precision is the simple arithmetic average of the precision of all the class and the value obtained is 0.12.The weighted average precision obtained is 0.14.The higher F1 score suggests better model performance and the FL class has a maximum of 0.55.The F1 scores of all classes are balanced between precision and recall as shown in Table 2.The confusion matrix shows that 15 samples are correctly predicted out of the total 40.Thus, overall accuracy is 38%.The macro average precision is the simple arithmetic average of the precision of all the class and the value obtained is 0.12.The weighted average precision obtained is 0.14.The higher F1 score suggests better model performance and the FL class has a maximum of 0.55.The F1 scores of all classes are balanced between precision and recall as shown in Table 2. ",
"section_name": "Performance Evaluation of VGG16 Model",
"section_num": "8.1."
},
{
"section_content": "The VGG-19 model has a convolutional neural network of 19 deep layers.The model developed is shown in Figure 7 and includes the input, functional, and output layers.The pooling layer is a fixed operation with no weighting factor [40]. Figure 8 shows the experimental results obtained using the VGG19 model.The number of epochs is set to 50.In VGG19, the training accuracy is gradually increased from 30% to 55%.The increasing and decreasing validation accuracy attains a maximum of 55% on the 48th epoch.The validation loss is around 9 in the 1st epoch and instantly increases to 16.The increasing and decreasing validation loss attains a minimum value at epoch 45.The ",
"section_name": "Performance Evaluation of VGG19 Model",
"section_num": "8.2."
},
{
"section_content": "The VGG-19 model has a convolutional neural network of 19 deep layers.The model developed is shown in Figure 7 and includes the input, functional, and output layers.The pooling layer is a fixed operation with no weighting factor [40]. Figure 8 shows the experimental results obtained using the VGG19 model.The number of epochs is set to 50.In VGG19, the training accuracy is gradually increased from 30% to 55%.The increasing and decreasing validation accuracy attains a maximum of 55% on the 48th epoch.The validation loss is around 9 in the 1st epoch and instantly increases to 16.The increasing and decreasing validation loss attains a minimum value at epoch 45.The validation loss is greater than the training loss.Thus, the model is over fitted.The capability of the VGG19 MAE value is 0.30, the MSE value is 0.15, and the MAPE value is 1.2 for the test data [41].The confusion matrix shows that 20 samples are correctly predicted out of a total 40.Thus, overall accuracy is 50%.The macro average precision of all the multiple classes is 0.33.The weighted average precision obtained is 0.34.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 0.67.The F1 scores of all classes are balanced between precision and recall as shown in Table 3. for the test data [41].The confusion matrix shows that 20 samples are correctly predicted out of a total 40.Thus, overall accuracy is 50%.The macro average precision of all the multiple classes is 0.33.The weighted average precision obtained is 0.34.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 0.67.The F1 scores of all classes are balanced between precision and recall as shown in Table 3. ",
"section_name": "Performance Evaluation of VGG19 Model",
"section_num": "8.2."
},
{
"section_content": "DenseNet201 is a convolutional neural network consisting of 201 deep layers.The model is developed with the input, functional, and output layers as shown in Figure 9.The pooling layer is a fixed operation with no weighting factor [42]. Figure 10 shows the experimental results obtained using the DenseNet201 transfer model.The number of epochs is set to 50.The model shows that training accuracy is gradually increased from 30% to 90%.The increasing and decreasing validation accuracy attains a maximum 97% on the 13th epoch.The validation loss is around 1.2 in the 1st epoch and varies between 0.1 and 1.2.The validation loss attains a minimum value of 0.1 at epoch 50.The model shows a better fit where the training loss and validation loss both decrease and stabilize at a specific point [43].The capability of the DenseNet201 MAE value is 0.02, the MSE value is 0.01, and the MAPE value is 0.01 for the test data.The confusion matrix shows that 37 samples are correctly predicted out of a total 40.Thus, overall accuracy is 93%.The macro average precision of all the multiple classes and the weighted average precision attain 0.92 and 0.93 as shown in Table 4.The higher F1 scores among the mul- The macro average precision of all the multiple classes and the weighted average precision attain 0.92 and 0.93 as shown in Table 4.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 0.93.The F1 scores of all classes are balanced between precision and recall [44]. ",
"section_name": "Performance Evaluation of DenseNet201 Model",
"section_num": "8.3."
},
{
"section_content": "Inceptionv3 is a deep-learning convolutional neural network image classification model [35].The model is developed with the input, functional, and output layers as shown in Figure 11.The pooling layer is a fixed operation with no weighting factor [45]. Figure 12 shows the experimental results obtained using the Inceptionv3 transfer model.The number of epochs is set to 50.The model shows that training accuracy is gradually increased from 35% to 80%.The validation accuracy is slightly higher than the training accuracy for all epochs and attains a maximum 90% at the 50th epoch. model [35].The model is developed with the input, functional, and output layers as shown in Figure 11.The pooling layer is a fixed operation with no weighting factor [45]. Figure 12 shows the experimental results obtained using the Inceptionv3 transfer model.The number of epochs is set to 50.The model shows that training accuracy is gradually increased from 35% to 80%.The validation accuracy is slightly higher than the training accuracy for all epochs and attains a maximum 90% at the 50th epoch.The validation loss is around 1.1 in the 1st epoch and decreases to 0.1 at the 50th epoch.The model shows good fit where the training loss and validation loss both decrease and stabilize at a specific point.The capability of the Inceptionv3 MAE value is 0.01, the MSE value is 0.01, and the MAPE value is 0.01 for the test data [46]. The confusion matrix shows that 36 samples are correctly predicted out of a total 40.Thus, overall accuracy is 90%.The macro average precision of all the multiple classes and the weighted average precision attain 0.90 and 0.91 as shown in Table 5.The higher F1 The validation loss is around 1.1 in the 1st epoch and decreases to 0.1 at the 50th epoch.The model shows good fit where the training loss and validation loss both decrease and stabilize at a specific point.The capability of the Inceptionv3 MAE value is 0.01, the MSE value is 0.01, and the MAPE value is 0.01 for the test data [46]. The confusion matrix shows that 36 samples are correctly predicted out of a total 40.Thus, overall accuracy is 90%.The macro average precision of all the multiple classes and the weighted average precision attain 0.90 and 0.91 as shown in Table 5.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 0.97.The F1 scores of all classes are balanced between precision and recall [47]. ",
"section_name": "Performance Evaluation of Inceptionv3 Model",
"section_num": "8.4."
},
{
"section_content": "Xception is a deep-learning convolutional neural network of 71 layers.The model is developed with the input, functional, and output layers as shown in Figure 13.The pooling layer is a fixed operation with no weighting factor.Figure 14 shows the experimental results obtained using the Xception transfer model.The number of epochs is set to 50.The model shows that training accuracy is gradually increased from 40% to 80%.The validation accuracy is slightly higher than the training accuracy for all epochs and attains a maximum 97% at the 50th epoch.The validation loss is around 1.4 in the 1st epoch and decreases to 0.1 at the 50th epoch.The model shows good fit where the training loss and validation loss both decrease and stabilize at a specific point [48].The model performance is improved where training loss is slightly higher than the validation loss.The capability of the Xception MAE value is 0.01, the MSE value is 0.01, and the MAPE The pooling layer is a fixed operation with no weighting factor.Figure 14 shows the experimental results obtained using the Xception transfer model.The number of epochs is set to 50.The model shows that training accuracy is gradually increased from 40% to 80%.The validation accuracy is slightly higher than the training accuracy for all epochs and attains a maximum 97% at the 50th epoch.The validation loss is around 1.4 in the 1st epoch and decreases to 0.1 at the 50th epoch.The model shows good fit where the training loss and validation loss both decrease and stabilize at a specific point [48].The model performance is improved where training loss is slightly higher than the validation loss.The capability of the Xception MAE value is 0.01, the MSE value is 0.01, and the MAPE value is 0.01 for the test data.The confusion matrix shows that 39 samples are correctly predicted out of a total 40.Thus, overall accuracy is 97%.The macro average precision of all the multiple classes and the weighted average precision values are 0.97 and 0.98 as shown in Table 6.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 1.0.The F1 scores of all classes are balanced between precision and recall.The confusion matrix shows that 39 samples are correctly predicted out of a total 40.Thus, overall accuracy is 97%.The macro average precision of all the multiple classes and the weighted average precision values are 0.97 and 0.98 as shown in Table 6.The higher F1 scores among the multiple classes improve the model performance and the FL class has a maximum of 1.0.The F1 scores of all classes are balanced between precision and recall.The ensemble architecture of InceptionV3 and Xception is developed to diagnose lymphoma cells using a new multi-cancer kaggle dataset which consists of 15,000 multiclass images.There are three types of ensemble techniques, namely bagging, boosting, and stacking.Each ensemble technique has its own merits and demerits; the stacking approach is a two-level classification technique and improves accuracy.Thus, the proposed system adopts a stacking-based ensemble approach to classifying lymphoma cancer cells. There are two levels of classification approach followed in the stacking method.On the first level, base or weak learners are used to predict the probabilities of each class.The predicted probabilities are then fed into the second-level classifier or Meta classifier to predict the final results (target value such as CLL, FL, and MCL).The proposed approach is adopted advanced deep network architectures as the base level classifiers such as Inception v3 and Xception.The base level classifiers then generate a matrix which contains the predicted probabilities of CLL, FL, and MCL images.The proposed approach used a CNN model for the Meta classifier.The generated matrix is then inputted to the Meta classifier or CNN model to be trained.Finally, the CNN can predict the lymphoma cancer categories such as CLL, FL, and MCL of the test set.The dataset contains three equal parts of 5000 for CLL, 5000 for FL, and 5000 for MCL.The 20 percent of 3000 test samples is split into 1029 samples of CLL, 962 samples of MCL, and 1009 samples of FL.All dataset images were in the JPEG file format with a size of 512 × 512 pixels.These two advanced deeplearning models produce the predicted probabilities of each class as the output.For each image, there are three probability values outputted by each level-0 classifier.The proposed method uses two deep-learning models such as Xception and Inceptionv3 in the level-0.So, for each image, six predicted probabilities are generated.In this way, for all the training images, predicted probabilities are generated that create a new dataset with six columns of predicted probabilities.A sample of the generated new dataset is shown in Figure 15. The output of the level-0 classifiers is given as the input for the level-1 classifier.For the Meta classifier, the CNN is used and the input for the CNN is the new dataset, which is generated by level-0 classifiers. ",
"section_name": "Performance Evaluation of Xception Model",
"section_num": "8.5."
},
{
"section_content": "The ensemble architecture of InceptionV3 and Xception is developed to diagnose lymphoma cells using a new multi-cancer kaggle dataset which consists of 15,000 multi-class images.There are three types of ensemble techniques, namely bagging, boosting, and stacking.Each ensemble technique has its own merits and demerits; the stacking approach is a two-level classification technique and improves accuracy.Thus, the proposed system adopts a stacking-based ensemble approach to classifying lymphoma cancer cells. There are two levels of classification approach followed in the stacking method.On the first level, base or weak learners are used to predict the probabilities of each class.The predicted probabilities are then fed into the second-level classifier or Meta classifier to predict the final results (target value such as CLL, FL, and MCL).The proposed approach is adopted advanced deep network architectures as the base level classifiers such as Inception v3 and Xception.The base level classifiers then generate a matrix which contains the predicted probabilities of CLL, FL, and MCL images.The proposed approach used a CNN model for the Meta classifier.The generated matrix is then inputted to the Meta classifier or CNN model to be trained.Finally, the CNN can predict the lymphoma cancer categories such as CLL, FL, and MCL of the test set.The dataset contains three equal parts of 5000 for CLL, 5000 for FL, and 5000 for MCL.The 20 percent of 3000 test samples is split into 1029 samples of CLL, 962 samples of MCL, and 1009 samples of FL.All dataset images were in the JPEG file format with a size of 512 × 512 pixels.These two advanced deep-learning models produce the predicted probabilities of each class as the output.For each image, there are three probability values outputted by each level-0 classifier.The proposed method uses two deep-learning models such as Xception and In-ceptionv3 in the level-0.So, for each image, six predicted probabilities are generated.In this way, for all the training images, predicted probabilities are generated that create a new dataset with six columns of predicted probabilities.A sample of the generated new dataset is shown in Figure 15.The output of the level-0 classifiers is given as the input for the level-1 classifier.For the Meta classifier, the CNN is used and the input for the CNN is the new dataset, which is generated by level-0 classifiers.The confusion matrix shows that 2997 samples are correctly predicted out of a total 3000.The proposed stacked ensemble method prediction of CLL, FL, and MCL classes is explained in this section.For the CLL category, the correctly predicted test samples are 1028 out of 1029.For the MCL category, 960 test samples are correctly classified out of 962.For FL, all the test samples are correctly predicted and there are no incorrect samples.Thus, overall accuracy is 99%, which is shown in Table 7a.The higher F1 scores among the multiple classes improve the model performance.The F1 scores of all classes are balanced between precision and recall.A high sensitivity shows that the model is correctly identifying most of the positive results and the high value of specificity shows a higher value of true negatives.In Table 7b, the obtained results of sensitivity and specificity are shown.The confusion matrix shows that 2997 samples are correctly predicted out of a t 3000.The proposed stacked ensemble method prediction of CLL, FL, and MCL classe explained in this section.For the CLL category, the correctly predicted test samples 1028 out of 1029.For the MCL category, 960 test samples are correctly classified ou 962.For FL, all the test samples are correctly predicted and there are no incorrect sa ples.Thus, overall accuracy is 99%, which is shown in Table 7(a).The higher F1 sco among the multiple classes improve the model performance.The F1 scores of all clas are balanced between precision and recall.A high sensitivity shows that the mode correctly identifying most of the positive results and the high value of specificity show higher value of true negatives.In Table 7(b), the obtained results of sensitivity and sp ificity are shown. ",
"section_name": "Performance Evaluation of Proposed Ensemble of Inceptionv3 and Xception Models",
"section_num": "8.6."
},
{
"section_content": "This section presents an approach for classifying the diagnosis of malignant lymphoma by applying non-ensemble models such as VGG16, VGG19, DenseNet201, Inceptionv3, and Xception.A stacked ensemble model (Inceptionv3, Xception, and CNN) is developed to improve the accuracy rate.The accuracy rate, precision, sensitivity, and F1 score are the indexes used to test and evaluate the performance of these models.The comparative experimental results are summarized in Table 8.The average accuracy rate of this multi-classification lymphoma is the final performance evaluation index [49].The accuracy of the Xception pre-trained network is higher than that of the other pre-trained networks, showing that Xception is a better classification non-ensemble model in the lymphoma multi-class images dataset.The proposed ensemble model had a higher testing accuracy of 99% greater than the efficient non-ensemble Xception model.It also attained the highest score for precision, recall, F1 score, and sensitivity metrics of 99% consistently compared with non-ensemble methods. ",
"section_name": "Comparative Analysis of Non-ensemble and Proposed Ensemble Models",
"section_num": "9."
},
{
"section_content": "By comparing the performance of the proposed system to that of the previous systems, it is made evident that the proposed stacked ensemble model proposed in this study exhibits superior performance across all metrics.Hamdi et al. [21] developed a model for identifying the critical features for diagnosing WSI images of malignant lymphomas.The composite model combining the features of MobileNet-VGG16, VGG16-AlexNet, and MobileNet-AlexNet was developed using XGBoost and decision tree networks.The average accuracy of the proposed model was 96.2%.The performance measures of sensitivity, specificity, and precision are 96.5%,97.8%, and 96.77%, respectively.Al-Mekhlafi et al. [50] proposed a two-hybrid systems model that utilized the FFNN classifier to classify images of malignant lymphomas from two datasets.In both malignant lymphoma datasets, the ResNet-50 + SVM network exhibits superior performance compared to the DenseNet-121 + SVM network.The overall accuracy rate of the model was 98.4 and the other performance measures such as sensitivity, specificity, and precision were 98.2, 98.4, and 98.5, respectively. ",
"section_name": "Comparative Analysis of Proposed Ensemble Model with Prior Models",
"section_num": null
},
{
"section_content": "The diagnosis of malignant lymphoma cells faces numerous challenges in distinguishing different classes, particularly during the early stages.Artificial intelligence supports physicians in distinguishing the classes of malignant lymphoma.In our work, the malignant lymphoma multi-class image datasets from various sources are trained using five pre-trained methodologies for diagnosing malignant lymphoma.The non-ensemble Convolutional Neural Network model is used to train the learning model with pre-trained weights VGG16, VGG19, DenseNet 201, InveptionV3, and Xception.The trained model is tested with the sample dataset and the predicted results match the trained data.DenseNet201, Inceptionv3, and Xception attain greater than 90% accuracy.These models are a good fit with minimum MAE, MSE, MAPE, and validation loss.The accuracies of VGG16 and VGG19 are very low and these are found to be overfitted models.Among the non-ensemble models, the Xception network outperformed all other models with 97% validation accuracy and minimum validation loss.Further, to improve the accuracy of the model, an ensemble architecture is proposed by using two efficient architectures, the Inceptionv3 and Xception models.The stacked ensemble model is trained and tested using the multi-cancer kag-gle WSI image dataset.The stacked ensemble model exhibits higher testing accuracy of 99% greater than the Xception model.The proposed model has demonstrated that, it has achieved greater performance measures for precision, recall, F1 score, and sensitivity of 99%.There are still some misclassifications in our models.Further research is needed to improve the model's performance using difficult cases and training using a multi-center database.The limitation of the proposed stacked ensemble technique is that no image feature extraction techniques are used to extract significant features that help the stacking model to make the classification output faster.The second limitation of the proposed model is that, the proposed method considered CLL, MCL, and FL categories of lymphoma for diagnosis.The sub categories of CLL, MCL, and FL is not considered in the presented work.The third limitation is that memory insufficiency was raised due to 15,000 Numpy array generation.The images are converted into Numpy array for the classification models for training and testing purposes.To overcome this problem, an image resizing approach is applied in the preprocessing step.Without this image resizing process, the proposed model and non-ensemble model outputs could be poor.In future work, a federated learning approach will be deployed to make the prediction from different computer systems.This feature will allow the system to take more input samples for the training and testing phases. ",
"section_name": "Conclusions and Scope for Future Research",
"section_num": "10."
}
] |
[
{
"section_content": "The multi cancer kaggle dataset of malignant Lymphoma images used in this study donloaded from the https://www.kaggle.com/datasets/andrewmvd/malignantlymphoma-classification& https://www.kaggle.com/datasets/obulisainaren/multi-cancer(accessed on 12 December 2023). The authors declare no conflicts of interest. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "The multi cancer kaggle dataset of malignant Lymphoma images used in this study donloaded from the https://www.kaggle.com/datasets/andrewmvd/malignantlymphoma-classification& https://www.kaggle.com/datasets/obulisainaren/multi-cancer(accessed on 12 December 2023). ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflicts of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The dataset used in this study was collected from publicly available multi cancer kaggle dataset.There are no potentially identifying marks/Features and no patient identifiers in the images or accompanying text.Therefore it does not require consent for publication. Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
}
] |
10.1038/s41408-021-00520-5
|
Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression
|
<jats:title>Abstract</jats:title><jats:p><jats:italic>BIRC3</jats:italic> is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to <jats:italic>BIRC3</jats:italic> biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or <jats:italic>BIRC3</jats:italic> mutations, modeling monoallelic and biallelic <jats:italic>BIRC3</jats:italic> loss. Our results reveal that monoallelic <jats:italic>BIRC3</jats:italic> deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic <jats:italic>BIRC3</jats:italic> loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including <jats:italic>BIRC3</jats:italic> within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that <jats:italic>BIRC3</jats:italic> mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring <jats:italic>BIRC3</jats:italic> deletion and mutation.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3deletion (del(11q)) are characterized by the presence of bulky lymphadenopathy, rapid disease progression and short time to first treatment (TTFT) and overall survival (OS) [1][2][3][4], even in early stage Binet A CLL cases [5].The size of this deletion is heterogeneous, it can cover a region greater than 20 Mb in most of the patients, involving the loss of over a hundred genes [6].The minimal deleted region almost always includes ATM, a putative CLL driver gene and one of the key components of the DNA damage response signaling [7,8].Another gene that has been hypothesized to also contribute to the pathobiology of del(11q) is BIRC3, which is located in the chromosomal band 11q22.2 and is entirely deleted in approximately 80% of del(11q) cases [9].In addition, it has been shown that BIRC3 disruption through truncating mutations occurs recurrently in CLL, ranging from frequencies of 3-5% in untreated cohorts to a two-fold higher incidence in relapsed/refractory CLL patients [10][11][12][13].Interestingly, BIRC3 mutations can appear in the remaining allele of approximately 10% of del(11q) patients with BIRC3 monoallelic loss, resulting in a biallelic BIRC3 inactivation [10,12,14].Recent studies have shown that biallelic inactivation of BIRC3 is an independent prognostic marker of inferior TTFT and OS in CLL [14,15].However, the clinical significance of BIRC3 monoallelic mutations or deletion remains uncertain.Some studies have provided evidence of the clinical impact of BIRC3 monoallelic mutations whereas others have not [10,12,14,[16][17][18][19][20].Moreover, BIRC3 mutations have also been found to be enriched in fludarabine relapsed/refractory CLL cases in some cohorts [10,18], although the mechanistic insights by which BIRC3 mutations could contribute to fludarabine resistance have not been elucidated. Biologically, BIRC3 is known to have a role as a negative regulator of the non-canonical NF-κB signaling [21].This pathway, alongside with the canonical NF-κB signaling, plays a key role on CLL pathogenesis, evolution and therapy response [22].The noncanonical signaling is initiated by tumor necrosis factor (TNF) signals engaging B-cell activation factor receptor (BAFFR), CD40, lymphotoxin β-receptor (LTβR) or receptor activator for NF-κB (RANK) among others.In the absence of a stimulus, this pathway is kept inactive by the BIRC3-mediated ubiquitination and proteasomal degradation of NF-κB-inducing kinase (NIK).Upon receptor stimulation, BIRC3 is recruited to the active receptor complex and NIK is stabilized in the cytoplasm, promoting IKKα activation which in turn phosphorylates p100, leading to the proteasomal degradation of its C-terminus and the translocation of p52-RelB heterodimers into the nucleus to initiate NF-κBdependent transcription [23].In CLL, BIRC3 mutations usually result in the loss of the E3 ubiquitin ligase domain essential for NIK targeting for proteasomal degradation, constitutively activating the non-canonical NF-κB signaling in a ligand-independent manner [10].Nevertheless, the most frequent BIRC3 alteration in CLL is monoallelic deletion of the entire gene through del(11q), being the functional consequences of this type of BIRC3 monoallelic loss unexplored.In addition, it is unclear how biallelic BIRC3 defects through del(11q) and BIRC3 mutation in the remaining allele could contribute to a NF-κB-dependent acceleration of CLL progression. The implementation of novel genomic editing technologies into the study of CLL has opened exciting possibilities to interrogate the functional effects of multiple driver genetic alterations as well as how some of these events cooperate to drive CLL progression and therapy response [24][25][26][27].In this study, we used the CRISPR/ Cas9 system to generate isogenic CLL-derived cell lines harboring del(11q) and/or BIRC3 mutations in the remaining allele.We show that monoallelic BIRC3 loss through del(11q) is enough to promote NIK-mediated non-canonical NF-κB signaling via p52-RelB nuclear translocation.Ex vivo experiments in primary del(11q) CLL cases revealed that del(11q) patients encompassing BIRC3 within the deleted region had higher NIK levels as well as p52-RelB activity, which correlated with BCL2 overexpression.In addition, BIRC3 lossof-function mutations in del(11q) cells resulted in a higher activation of the non-canonical NF-κB signaling cascade, ultimately leading to increased clonal advantage in vitro and acceleration of leukemic progression in an in vivo xenograft model.Thus, our study provides novel biological insights about the role of BIRC3 deletion and mutation in CLL evolution and progression. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "HG3 and MEC1 Cas9-expressing cell lines (HG3-Cas9 and MEC1-Cas9) were previously generated and tested for Cas9 activity [27].Singleguide RNAs (sgRNAs) targeting BIRC3 (exons 2 or 7) were designed using the online CRISPR tool (http://crispr.mit.edu/),based on the predicted on-target efficiency and the lowest off-target effects.In addition, a sgRNA designed not to target the human genome was used as a negative control.SgRNAs targeting BIRC3 were cloned into pLKO5.sgRNA.EFS.GFP (Addgene_#57822) plasmids and control sgRNAs were cloned in pLKO5.sgRNA.EFS.tRFP(Addgene_#57823).Sequences of the selected sgRNAs are detailed in Supplementary Table S1.The procedures and sgRNAs used for the generation of del(11q) and TP53 mutations in HG3 cells were previously described [27,28].pLKO5 vectors carrying the desired sgRNAs were transduced into HG3-Cas9 and MEC1-Cas9 cells and single-cell flow-sorted clones were expanded and screened.At least three different clones harboring loss-of-function mutations were chosen for each CRISPR-generated cell line to perform functional studies. ",
"section_name": "METHODS CRISPR/Cas9-mediated engineering of CLL cell lines",
"section_num": null
},
{
"section_content": "Viably cryopreserved peripheral blood mononuclear cells (PBMCs) from 22 CLL patients were used in the ex vivo studies.PBMCs were isolated by Ficoll-Paque Plus density gradient media (GE Healthcare, Life Sciences) and a complete immunophenotypic analysis was performed in all samples by flow cytometry.Only samples with a CD19 + /CD5 + fraction greater than 85% were included in the study.Supplementary Table S2 summarizes the main biological characteristics of CLL patients.The research was conducted in accordance with the Declaration of Helsinki and prior approval by the Bioethics Committee from our institution.Written informed consent was obtained from all patients. Next-generation sequencing (NGS) data from the primary CLLs used in the ex vivo experiments are detailed in Supplementary Table S3 and Supplementary Fig. S1.A custom NGS panel was applied and analyzed as previously reported [27,29].Full details about NGS procedure and analysis can be found in Supplementary Information. ",
"section_name": "Primary CLL samples",
"section_num": null
},
{
"section_content": "Canonical (p65/RelA, NF-κB1 p50, c-Rel) and non-canonical (NF-κB2 p52, RelB) NF-κB activity of nuclear extracts of HG3 and MEC1 clones and lysates from primary CLL samples was measured using the NF-κB Transcription Factor Assay Kit (Colorimetric) (Abcam, ab207216) following manufacturer's instructions.Briefly, an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilized onto a 96-well plate.Active NF-κB subunits present on the nuclear extracts specifically bind this oligonucleotide and p65, p50, c-Rel, p52, or RelB subunits are recognized by using specific primary antibodies accessible only when NF-κB is activated and bound to its target DNA. Ex vivo co-culture conditions HS-5 stromal cells were seeded 24 h prior to the ex vivo experiments at a concentration of 7.5 × 10 4 cells/well in a 6-well plate.On the following day, primary CLL cells were viably thawed and resuspended in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS, 1% penicillin/ streptomycin and 1.5 μg/mL CpG ODN (Sigma-Aldrich) plus 50 ng/mL IL-2 (Peprotech) and subsequently seeded onto the HS-5 cell layer at a coculture ratio of 100:1 (7.5 × 10 6 CLL cells /well) [30].CLL cells were carefully detached and lysed 24 h after co-culture and proteins were subjected to NF-κB activity assays and/or immunoblot.CpG stimulation was chosen in order not to involve receptors directly implicated in non-canonical NF-κB activation such as CD40 or BAFFR. ",
"section_name": "NF-κB family members activity ELISA",
"section_num": null
},
{
"section_content": "Animal studies were conducted in accordance with the Spanish and European Union guidelines for animal experimentation (RD53/2013, Directive-2010/63/UE, respectively) and received prior approval from the Bioethics Committee of our institution. For intravenous xenograft experiments, 20 four-to-five-week-old female NSG mice were used for injection of HG3 cells harboring del(11q) and/or BIRC3 mutations (n = 5/each group).3 × 10 6 cells were resuspended in 100 μL of RPMI media and injected into the tail vein of the mice.14 days after cell injection, mice were culled and spleens were subjected to FACS and immunohistochemistry analyses.For FACS analysis, spleens were lysed with erythrocyte lysis buffer, and the remaining cells were then washed twice in PBS.Samples were stained with fluorophore-conjugated antibodies against mouse-CD45 (PerCP-Cy5.5, BD Biosciences) and human-CD45 (hCD45) (CF Blue, Immunostep).Data were obtained on a FACSAria flow cytometer and analyzed with FlowJo software.Full details about subcutaneous xenografts in the Supplementary Information. ",
"section_name": "Xenograft experiments",
"section_num": null
},
{
"section_content": "Statistical analyses were carried out using GraphPad Prism software v6 (GraphPad Software).Otherwise specified, data are summarized as the mean ± standard deviation (SD).Student's t test, Mann-Whitney, ANOVA, or Kruskal-Wallis tests were used to determine statistical significance.Pvalues lower than 0.05 were considered statistically significant.At least three independent clones per condition were used in the functional studies. ",
"section_name": "Statistics",
"section_num": null
},
{
"section_content": "Supplementary Methods section includes detailed protocols of cell lines, culture conditions, drugs and reagents, NGS, FISH, subcellular fractionation and western blot, viability, apoptosis, and cell cycle analyses, in vitro clonal competition assays, subcutaneous in vivo xenografts and immunohistochemistry. ",
"section_name": "Supplementary methods",
"section_num": null
},
{
"section_content": "CRISPR/Cas9-mediated generation of isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations In order to understand how monoallelic or biallelic BIRC3 loss contributes to the pathobiology of del(11q) CLL, we used the CRISPR/Cas9-editing technology to model these alterations in an in vitro system.For this purpose, we selected HG3 and MEC1 CLLderived cell lines.HG3 is diploid for chromosome 11 and has wildtype (WT) BIRC3 gene.sgRNAs targeting chromosomal bands 11q22.1 and 11q23.3 were introduced in Cas9-expressing HG3 cells, generating an isogenic HG3 CLL cell line harboring a ∼17 Mb monoallelic del(11q) (HG3-del(11q)) encompassing BIRC3 gene among others [27].sgRNAs specifically targeting BIRC3 (exon 2) were then introduced in HG3-del(11q) cells in order to induce BIRC3 truncating mutations (BIRC3 MUT ) in the remaining WT allele, generating HG3-del(11q) BIRC3 MUT isogenic cell lines (Fig. 1A), mimicking the BIRC3 biallelic loss through del(11q) and mutation observed in high-risk CLL patients.Furthermore, we also generated HG3 cell lines harboring only BIRC3 mutation (HG3 BIRC3 MUT ) following the same strategy (Fig. 1A).BIRC3 mutations were generated in a monoallelic or a biallelic fashion either in BIRC3 exon 2 or exon 7, having as a consequence the truncation of the BIR or CARD BIRC3 protein domains, respectively (Fig. 1B, left panel), emulating the type of BIRC3 mutations mainly detected in CLL [12,16].BIRC3 protein expression was evaluated by western blot in all the generated clones, showing that BIRC3 levels were absent in cells harboring truncating mutations in the exon 2 (BIR domain) or detecting a truncated form of BIRC3 in those clones harboring exon 7 mutations (CARD domain) (Fig. 1B; right panel). In parallel, we used the MEC1 cell line as a model to study the BIRC3 allelic dose effects in CLL.Parental MEC1 cells harbor a monoallelic BIRC3 deletion (MEC1 BIRC3 DEL/WT ) as indicated by NGS copy number analysis (Supplementary Fig. S1).We also introduced sgRNAs targeting BIRC3 (exon 2) following the previous approach, generating MEC1 cell lines harboring biallelic BIRC3 loss through deletion and mutation (MEC1 BIRC3 DEL/MUT ) (Supplementary Fig. S2).BIRC3 loss through del(11q) promotes p52-RelB nuclear translocation and activation of the non-canonical NF-κB signaling downstream targets Considering the role of BIRC3 in the NF-κB signaling [21,31], we assessed the impact of monoallelic and biallelic BIRC3 loss through del(11q) and/or mutation in this pathway using our CRISPR/Cas9engineered cell lines.We first analyzed the nuclear DNA-binding activity of the main NF-κB transcription factors implicated in both the canonical and non-canonical pathway.Regarding the canonical signaling, we did not observe significant changes in the activity of p65 and c-Rel.However, HG3-del(11q) BIRC3 MUT cells showed a significant increase of p50 nuclear activity in comparison to HG3 WT cells (Fig. 2A).Conversely, monoallelic BIRC3 loss in HG3-del(11q) cells resulted in a marked increase of p52 and RelB activity, being this effect further enhanced in HG3-del(11q) BIRC3 MUT cells (Fig. 2A).These results were also confirmed in HG3 in all BIRC3 MUT clones (Supplementary Fig. S3a), confirming that either truncating mutations in the BIR or CARD domains have the same functional consequence on the non-canonical NF-κB signaling.Additional characterization of proteins involved in the non-canonical NF-κB signaling by western blot revealed that monoallelic, and to a greater extent, biallelic BIRC3 loss resulted NIK cellular stabilization and increased levels of phosphorylated IKKα/β and NF-κB2, in line with a higher p52 and RelB accumulation in the nucleus (Fig. 2B; Supplementary Fig. S3b).Furthermore, we corroborated an increase of p50 nuclear levels in the nucleus of HG3-del(11q) BIRC3 MUT cells (Fig. 2B; Supplementary Fig. S3b). To validate these results in an independent CLL cell line, NF-κB activity as well as NIK and p52 levels were analyzed in MEC1 cells.As expected, MEC1 BIRC3 DEL/MUT clones likewise presented higher p52, RelB and p50 activity rates, NIK stabilization and accumulation of p52 in the nucleus than MEC1 BIRC3 DEL/WT cells (Supplementary Fig. S3c,d). Given that activation of the non-canonical signaling has been shown to upregulate some anti-apoptotic proteins such as BCL2 and BCL-xL [32,33], we next assessed the protein levels of such these targets in our CRISPR/Cas9-edited cell lines in order to determine the impact of monoallelic or biallelic BIRC3 loss in the regulation of these proteins expression.Interestingly, HG3-del(11q) BIRC3 MUT CLL cell lines showed higher levels of BCL2 and BCL-xL, alongside to reduced levels of proapoptotic protein BAX, whereas no changes were observed regarding MCL1 or pro-apoptotic family members such as BIM, BAK, and NOXA (Fig. 2C; Supplementary Fig. S4a).To test whether these increased levels of anti-apoptotic proteins were the result of BIRC3-mediated non-canonical signaling activation, cells were treated with a NIK small molecule inhibitor (NIK SMI1) [34], showing that NIK-dependent inhibition of p100/p52 processing translated into downregulation of BCL2 and BCL-xL protein expression in HG3-del(11q) BIRC3 MUT cells (Supplementary Fig. S4b). ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "In order to validate whether the results obtained in our CRISPR/ Cas9-generated models could resemble the actual biology of BIRC3-deleted del(11q) CLL patients, we tested the DNAbinding activity of non-canonical NF-κB transcription factors in a cohort of 22 CLL cases (n = 11 BIRC3 WT ; n = 11 BIRC3deleted through del(11q) or mutation) (Supplementary Table S2) in the absence or presence of stromal + CpG + IL-2 stimulation.Remarkably, stimulated BIRC3-deleted CLL cells showed higher p52 activity than BIRC3 WT cases (Fig. 3A; left panel), in line with the results observed in HG3-del(11q) cells.To a lesser extent, BIRC3-deleted cases also showed a trend of higher RelB activity than BIRC3 WT cells (Fig. 3A, left panel).In addition, focusing on the subgroup of patients harboring del (11q), we could observe a significant correlation between the percentage of BIRC3-deleted cells and p52 activity (Fig. 3A, right panel), further evidencing the NF-κB-related effect of BIRC3 monoallelic loss in del(11q) cases. Next, we performed western blot analyses in a homogenous cohort of del(11q) samples including or not including BIRC3 within the deleted region (n = 4, del(11q)/BIRC3 deleted; n = 3, del(11q)/BIRC3 undeleted).Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, resulting in a marked NF-κB2 p52 processing, which was virtually absent in del (11q)/BIRC3 undeleted cases (Fig. 3B).Indeed, there was a clear correlation between NIK and p52 levels in these patients (Fig. 3C, upper panel).Of note, del(11q)/BIRC3 deleted cases also showed increased levels of RelB and a reduction of NF-κB1 p105 precursor levels, although we did not observe differences in p50 protein expression between these groups (Fig. 3B).In addition, among anti-apoptotic BCL2 family members, del(11q)/ BIRC3 deleted cases showed higher BCL2 protein expression than del(11q)/BIRC3 undeleted cases (Fig. 3B), which correlated to the amount of p52 levels in these patients (Fig. 3C, lower panel). ",
"section_name": "BIRC3-deleted primary del(11q) CLL cells show enhanced noncanonical NF-κB activity which correlates with high BCL2 levels",
"section_num": null
},
{
"section_content": "Considering the effects of BIRC3 loss in the upregulation of some anti-apoptotic family members, we next evaluated the response of the isogenic CRISPR/Cas9 HG3 clones to selective BCL2, BCL-xL or MCL1 inhibition.BCL2 inhibition with venetoclax (ABT-199) highlighted a higher sensitivity of HG3-del(11q) BIRC3 MUT cells than HG3 WT cells (Fig. 4A), in line with the observed non-canonical NF-κB-dependent BCL2 upregulation of these cell lines.In addition, HG3-del(11q) and HG3-del(11q) BIRC3 MUT cells were also more sensitive to BCL-xL inhibition by A1331852 than HG3 WT cells (Fig. 4A).Contrarily, monoallelic or biallelic BIRC3 loss in HG3 cells did not seem to influence the response to MCL1 inhibition by S63845 (Fig. 4A), consistently with our observations regarding MCL1 protein levels.Furthermore, we also tested the response of these cell lines to the BTK inhibitor ibrutinib, showing that HG3-del (11q) BIRC3 MUT cells were slightly less sensitive in comparison to HG3 WT cells (Supplementary Fig. S5a). Moreover, since BIRC3 disruption in CLL patients has been associated with fludarabine refractoriness even in TP53 wild-type CLLs [10], we tested HG3-del(11q) clones (with or without BIRC3 disruption) for evidence of resistance to fludarabine treatment.HG3 TP53 MUT clones, also generated by CRISPR/Cas9, were used as positive controls for fludarabine resistance.Interestingly, after 72 h of fludarabine treatment, only TP53 MUT clones presented marked resistance (by MTT assay) whereas HG3-del(11q) BIRC3 MUT clones showed the same sensitivity as HG3 WT cells (Fig. 4B, left panel).Longer drug exposures were also tested, and we found no significant resistance of BIRC3 disrupted clones (Fig. 4B, right panel).Further support for fludarabine treatment-induced apoptosis in BIRC3-deficient clones was observed through the appearance of a sub-G 0 peak in cell cycle profiles and annexin studies (Supplementary Fig. S5b,c). ",
"section_name": "Biallelic BIRC3 loss confers sensitivity to BCL2 and BCL-xL inhibition in vitro",
"section_num": null
},
{
"section_content": "We next hypothesized that the effects of BIRC3 loss in the NF-κB signaling and apoptosis may have an impact on CLL evolution and progression.For this purpose, proliferation assays were performed to characterize the consequences of the CRISPR/Cas9-generated alterations in CLL expansion.We noted that HG3-del(11q) BIRC3 MUT cells showed enhanced viability and growth than HG3del(11q) and HG3 WT cells (Fig. 5A, left panel; Supplementary Fig. S6a).In addition, cell cycle analyses of these clones revealed that HG3-del(11q) BIRC3 MUT cells had a higher proportion of cells transitioning through S-phase (Supplementary Fig. S6b).To test whether this effect on proliferation could be attributed to BIRC3 loss, MTT and growth assays were carried out in HG3 BIRC3 MUT cells without del(11q), and these indeed confirmed the higher proliferation rates of BIRC3-deficient cells (Fig. 5A, right panel; Supplementary Fig. S6a).Moreover, HG3 WT cells treated with the BIRC2/BIRC3 inhibitor birinapant also displayed increased growth (Supplementary Fig. S6c) as well as MEC1 BIRC3 DEL/MUT cells in comparison to MEC1 BIRC3 DEL/WT (Supplementary Fig. S6d).We also determined that this enhanced proliferation rate was driven by enhanced BIRC3-mediated non-canonical NF-κB signaling activation, since NIK inhibition by SMI1 was able to reduce viability of HG3-del(11q) and HG3-del(11q) BIRC3 MUT cells (Fig. 5B). In order to evaluate how BIRC3 deletion and/or mutation could contribute to CLL clonal dynamics, we next carried out in vitro clonal competition experiments by mixing RFP-or GFP-tagged CRISPR/Cas9-edited cells at a ratio 1:1 and tracked clonal evolution overtime by flow cytometry.In the first experiment, clonal competition was assessed to investigate how BIRC3 mutation could confer a clonal advantage of del(11q) cells.Notably, HG3-del (11q) BIRC3 MUT cells progressively outgrew HG3-del(11q) cells overtime (Fig. 5C).In a second experiment, we evaluated the clonal competition between HG3 WT and HG3 BIRC3 MUT cells, showing that HG3 BIRC3 MUT cells were able to outcompete HG3 WT cells (Fig. 5C). ",
"section_name": "Biallelic BIRC3 loss in del(11q) CLL cells favors clonal advantage in vitro",
"section_num": null
},
{
"section_content": "To confirm the effects of BIRC3 loss in a physiological context in vivo, we individually injected the monoallelic and biallelic BIRC3-deleted CRISPR/Cas9-edited cell lines intravenously into NSG mice, observing that mice xenografted with HG3 BIRC3 MUT and HG3-del(11q) BIRC3 MUT cells showed an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3 WT and HG3-del(11q) cells, respectively, by flow cytometry (Fig. 6A).By immunohistochemistry, spleens collected from HG3 BIRC3 MUT and HG3-del(11q) BIRC3 MUT intravenous xenografted cells propagating in vivo also showed evidence of NF-κB2 activation (Fig. 6B).S2.B Whole-cell lysates from stimulated CLL primary samples harboring del(11q) not involving BIRC3 (del(11q)/BIRC3-undeleted) (ID-21, ID-07, ID-11) and del(11q) involving BIRC3 (del(11q)/BIRC3 deleted) (ID-04, ID-01, ID-10, ID-19) primary CLL samples were analyzed by immunoblotting for BIRC3, NIK, NF-κB2 (p100/p52), RelB, NF-κB1 (p105/p50), BCL2, BCL-xL and MCL1 proteins.β-actin was used as loading control.C Correlation between p52 protein levels and NIK (left panel) or BCL2 (right panel) protein levels from the patients analyzed by immunoblot. In addition, to validate this effect in the proliferation, HG3 WT and HG3 BIRC3 MUT cells were injected subcutaneously in the flank of NSG mice and tumor growth was monitored for 17 days.HG3 BIRC3 MUT cells generated larger tumors than HG3 WT cells (Supplementary Fig. S7a).Besides, tumors collected from HG3 BIRC3 MUT engrafted mice showed higher levels of p52 expression than those from HG3 WT mice (Supplementary Fig. S7b). ",
"section_name": "Biallelic BIRC3 loss in del(11q) CLL cells accelerates leukemic progression in in vivo xenografts",
"section_num": null
},
{
"section_content": "Del(11q) is one of the most frequent cytogenetic abnormalities occurring in CLL patients [4,35,36], yet, the functional consequences of the haploinsufficiency of the vast majority of genes comprised within this region remains largely unknown.Here, we undertook a CRISPR/Cas9-based genome editing approach to characterize novel biological implications of monoallelic and biallelic BIRC3 loss in del(11q) CLL.In this way, our work presents in vitro, ex vivo, and in vivo evidence of how BIRC3-deletion and/or mutation in the remaining allele of del (11q) cells contributes to NF-κB signaling activation, CLL progression, and therapy response. Our data indicate that monoallelic BIRC3 deletion contributes to the pathobiology of del(11q) by a NIK-dependent triggering of the non-canonical NF-κB signaling, resulting in enhanced p52-RelB nuclear translocation and activation (Supplementary Fig. S8).This effect appears to be allelic dose-dependent since biallelic BIRC3 loss resulted in higher activation rates (Fig. 2).Our results obtained in isogenic CLL-derived cell lines provide a more comprehensive landscape of the role of each CLL BIRC3-related alterations in the non-canonical NF-κB pathway, complementing previous findings hinted in a panel of lymphoid-related cell lines [18].In addition, we were also able to address the biological differences between del(11q) CLL patients including or not BIRC3 within their deleted region, showing that, in response to stromal stimulation and TLR ligation by CpG, del(11q)/BIRC3-deleted cases present marked levels of stabilized NIK and p52 activity.Indeed, further evidence of hyperactive non-canonical signaling was found in Blymphocytes from mice lacking cIap1/cIap2 (Birc2/Birc3) [37] and in B-cells treated with BIRC2/BIRC3 inhibitors [21], as well as in our isogenic del(11q) CLL cell lines (Fig. 2).Furthermore, a previous report observed that CLL cells with low BIRC3 mRNA expression presented activation of the canonical NF-κB signaling in the presence of BAFF or CD40L stimulation [31].Interestingly, we did find partial evidence of canonical NF-κB activation by increased nuclear p50 activity in HG3-del(11q) BIRC3 MUT cells, which has also been shown to contribute in the pathogenesis of Eμ-TCL1 model of CLL [38].This enhanced p50 activity could be in line with the high phosphorylation levels of IKKβ, a member of the IκB-kinase (IKK) complex, implicated in canonical NF-κB activation [39].Altogether, these results suggest that, in the presence of the CLL microenvironment, BIRC3 loss displays a dual role on both canonical and non-canonical NF-κB signaling activation.The recent introduction of the selective BCL2 inhibitor venetoclax into the CLL treatment scheme has led to effective remissions for relapsed/refractory CLL patients, especially when combined with anti-CD20 antibodies [40,41].Nevertheless, little is still known regarding which genetic alterations may predict for better venetoclax responses in CLL.We show that enhanced noncanonical NF-κB activity in BIRC3-deleted cells results in BCL2 overexpression, making isogenic del(11q) BIRC3 MUT cells more sensitive to venetoclax treatment.These observations are limited due to the use of CLL cell lines which do not display the same venetoclax sensitivity as primary CLL cells [42].However, we also observe a correlation between BCL2 levels and the percentage of BIRC3-deleted cells in del(11q) cases, as well as between p52 and BCL2 levels, suggesting that BIRC3-deleted cases may potentially benefit from venetoclax-based regimes.In fact, recent data from the CLL14 trial suggest that del(11q) or BIRC3 MUT patients significantly favor from venetoclax plus obinutuzumab in comparison to the chlorambucil plus obinutuzumab treatment arm [43].In addition, we show that NIK pharmacological inhibition can counteract BIRC3-mediated non-canonical NF-κB signaling and anti-apoptotic protein overexpression, making it an attractive candidate for combinatorial therapy with venetoclax [44].Contrarily, del(11q) BIRC3 MUT cells did not selectively benefit from ibrutinib treatment, in agreement with previous reports indicating that BTK inhibition does not suppress non-canonical NF-κB signaling activity [18,45].Furthermore, we also assessed the treatment implications of BIRC3 deletion and/or mutation in response to fludarabine, given that these alterations have been associated with fludarabine relapse in some, but not all, cohorts [10,12,18].Nonetheless, neither isogenic HG3-del(11q) BIRC3 MUT nor BIRC3 MUT cells show evidence of fludarabine resistance, whereas isogenic HG3 TP53 MUT cells present marked resistance in the same conditions (Fig. 4B).These results indicate that BIRC3 alterations may not be enough to generate fludarabine resistance per se, as opposed to TP53 alterations.Further investigation is required to decipher whether extrinsic factors such as the CLL microenvironment as well as the concurrence with other genetic alterations would play a critical role in fludarabine resistance of BIRC3 mutated CLL cells. Although there is still controversy regarding clinical impact of BIRC3 mutations, recent reports have highlighted the negative predictive impact on TTFT and OS of biallelic BIRC3 loss through del(11q) and mutation in the remaining allele [14,15].Our work biologically demonstrates that biallelic BIRC3 loss promotes CLL proliferation, clonal evolution and progression in vitro and in vivo.These results are further supported by the notion that mice lacking cIap1/cIap2 show an uncontrolled accumulation of B-cells in vivo [37].Interestingly, we did not observe the enhanced proliferation of isogenic cell lines harboring monoallelic BIRC3 loss, suggesting that BIRC3 mutations may only have a clinical impact in patients with a previous del(11q) background.These data therefore reinforce the notion that biallelic BIRC3 inactivation should be considered as a high-risk CLL entity. In summary, this work displays a comprehensive biological analysis of the impact of monoallelic and biallelic BIRC3 lesions in del(11q) CLL patients by combining in vitro, ex vivo, and xenograft models.We show that monoallelic BIRC3 deletion activates NF-κB signaling in del(11q) CLL cells, contributing to the pathobiology of this high-risk cytogenetic alteration.We also demonstrate that BIRC3 mutation in the remaining allele of del(11q) CLL cells confers clonal advantage which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL.Moreover, cells harboring these alterations could be therapeutically targeted with BCL2 inhibitors.Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) \"Una manera de hacer Europa\", \"Consejería de Educación, Junta de Castilla y León\" (SA271P18), \"Proyectos de Investigación del SACYL\", Spain GRS 2062/A/19, GRS 1847/A/18, GRS1653/A17,\"Fundación Memoria Don Samuel Solórzano Barruso\" (FS/23-2018), by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Universidad de Salamanca (Programa XIII), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233) and SYNtherapy \"Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia\" (ERAPERMED2018-275); ISCIII (AC18/00093), cofunded by ERDF/ESF, \"Investing in your future\".M.Q.Á. and A.E.R.V. are supported with a research grant by FEHH (\"Fundación Española de Hematología y Hemoterapia\"); M.H.S. holds a Sara Borrell postdoctoral contract (CD19/00222) from the Instituto de Salud Carlos III (ISCIII).C.P.C. was supported by an \"Ayuda predoctoral en Oncología\" (AECC) and is a recipient of a PFIS grant (FI19/00191) from Instituto de Salud Carlos III; PFIS grant and Sara Borrell postdoctoral contrat are co-founded by Fondo Social Europeo (FSE) \"El Fondo Social Europeo invierte en tu futuro\"; J.L.O. and R.B.S. are supported by a grant from the University of Salamanca (\"Contrato postdoctoral programa II\").We thank Irene Rodríguez, Sandra Santos, Sara González, Cristina Miguel, Almudena Martín-Martín, Teresa Prieto, Mª Ángeles Ramos, Filomena Corral, Mª Almudena Martín, Ana Díaz, Ana Simón, María del Pozo, Isabel M Isidro, Vanesa Gutiérrez, Sandra Pujante and Mª Ángeles Hernández from the Cancer Research Center of Salamanca, Spain, for their technical support.We are grateful to Ángel Prieto, Ana I García and Sara Armenteros, María Luz Sánchez and María Carmen Macías from the Microscopy Unit, Cytometry Unit and Molecular Pathology Unit, respectively, from the Cancer Research Center of Salamanca for their technical assistance.We thank Luis Muñoz and all the members from the Animal Experimentation Research Center from the University of Salamanca.The authors are grateful to C.J. Wu and E. ten Hacken (Dana-Farber Cancer Institute) for constructive and valuable discussion on this project. ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s41408-021-00520-5. Correspondence and requests for materials should be addressed to J.-M.H-R. 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": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-021-00520-5. Correspondence and requests for materials should be addressed to J.-M.H-R. 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/s41467-022-35161-0
|
Engineering receptors in the secretory pathway for orthogonal signalling control
|
<jats:title>Abstract</jats:title><jats:p>Synthetic receptors targeted to the secretory pathway often fail to exhibit the expected activity due to post-translational modifications (PTMs) and/or improper folding. Here, we engineered synthetic receptors that reside in the cytoplasm, inside the endoplasmic reticulum (ER), or on the plasma membrane through orientation adjustment of the receptor parts and by elimination of dysfunctional PTMs sites. The cytoplasmic receptors consist of split-TEVp domains that reconstitute an active protease through chemically-induced dimerization (CID) that is triggered by rapamycin, abscisic acid, or gibberellin. Inside the ER, however, some of these receptors were non-functional, but their activity was restored by mutagenesis of cysteine and asparagine, residues that are typically associated with PTMs. Finally, we engineered <jats:underline>o</jats:underline>rthogonal <jats:underline>c</jats:underline>hemically <jats:underline>a</jats:underline>ctivated cell-surface <jats:underline>r</jats:underline>eceptors (OCARs) consisting of the Notch1 transmembrane domain fused to cytoplasmic tTA and extracellular CID domains. Mutagenesis of cysteine residues in CID domains afforded functional OCARs which enabled fine-tuning of orthogonal signalling in mammalian cells.</jats:p>
|
[
{
"section_content": "Naturally existing receptors are powerful tools in bioengineering settings, but their application is restricted to specific ligands [1][2][3] .Furthermore, receptors for many clinically important ligands and disease biomarkers have not yet been identified, and in some cases may not exist.Thus, there is a need for artificial receptors that are capable of detecting and responding to user-defined signal inputs [4][5][6][7] .In addition to their unique recognition capabilities, such customized receptors can be molecularly coupled to cellular signaling circuits, and indeed, have been extensively utilized in cell-based therapy, diagnostics, and basic research 8 .An example of a successful clinical application of synthetic receptors is the introduction of FDA-approved chimeric antigen receptor (CAR)-T cells 9 .These genetically engineered T cells are equipped with an artificial receptor that enables selective recognition of certain onco-markers presented on malignant cells, facilitating the effective elimination of the target tumors. Another limitation in the application of natural receptors is that their signaling mechanisms rely on endogenous signaling pathways.These pathways are highly interconnected biochemical reaction networks, and many of them actually share the same components.Therefore, some activatory signaling inputs, especially those that circulate through the whole organism via the blood, can simultaneously act on multiple pathways, leading to unspecific and unexpected cellular outputs [10][11][12] .To avoid such off-target effects, orthogonal signaling modalities that are independent of endogenous pathways may be preferable due to their highly selective and predictable modes of action [13][14][15] . In recent years, different engineering approaches have been employed to design orthogonal receptors for efficient cell signaling control 8,[16][17][18] .One of the main strategies is repurposing molecular components from different parts of known proteins and/or from different species.However, a major challenge is that some of these components do not function as expected when ectopically expressed in mammalian cells.The majority of proteins required proper folding to form the specific three-dimensional shapes necessary for their activity, and this may require the assistance of species-and/or organelle-specific enzymes and chaperones [19][20][21][22][23] .Thus, the same primary polypeptide sequence may be folded in different ways when expressed in different species or even in different cellular organelles of a single species. The endoplasmic reticulum (ER) is the first destination of proteins following their translocation into the secretory pathway 24,25 .Many such proteins undergo several types of post-translational modifications (PTMs) mediated by ER-resident enzymes.For example, covalent disulfide bond formation between two distantly located cysteine residues, N-linked glycosylation, and O-linked glycosylation are among the major PTMs that occur in the ER [26][27][28] .Following their transport from the ER, these PTMs may be further modified, and/or new ones may be introduced in the Golgi apparatus.After these structural processings, the mature proteins may be secreted into the extracellular space (soluble proteins), or sorted into other cellular organelles, such as the plasma membrane 29,30 .Thus, it should be taken into account that a polypeptide that enters the secretory pathway might encounter a variety of structural changes as a result of misfolding and/or decoration with PTMs.These structural modifications may dramatically alter the ligand recognition and signal transduction capabilities of the protein. To address this problem during the construction of artificial receptors, we employed a systematic approach to restore synthetic receptors' activity in the cytoplasm, ER, and on the plasma membrane, for orthogonal signaling control.Here, we started with topology adjustment of the synthetic receptors in the cytoplasm in order to exclude the involvement of PTMs in the secretory pathway.This spatial engineering focuses on the functional evaluation of different topologies of the receptor parts in a combinatory manner.Evaluating receptors' activities in the cytoplasm is a critical step before starting with compartment-oriented engineering, as a nonfunctional receptor in the cytoplasm is most likely to be inactive also inside the secretory organelles.Following their successful construction in the cytoplasm, active receptors were then targeted into the secretory pathway.We found that following their translocation into the secretory organelles, most receptors lost their inducibility, but we could rescue the expected activity of all these receptors by blocking functionally harmful PTMs through systematic site-directed mutagenesis of PTMsusceptible residues.Here, we chose three different small molecules as soluble ligands; rapamycin (RAPA), abscisic acid (ABA), and gibberellin (GA), collectively referred to as RAG.The ligand detection part of all these synthetic receptors consists of one of the protein domain pairs FRKB/FPB, ABI/PYL1, and GAI/GID1 that dimerize upon the addition of RAPA, ABA, or GA, respectively [31][32][33][34][35] .The site-specific split form of tobacco etch virus protease (TEVp) was used as a signal transducer domain in cytoplasmic and the ER-localized receptors 36,37 .In the cell membrane, we used the Notch1 receptor core.We think that these signaling moieties will be valuable tools for fine-tuning orthogonal cell signaling in mammalian cells, and they can be re-purposed during the engineering of protein architectures within the secretory pathway for cell-based medicines, diagnostics, and basic science applications. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Engineering a membrane-docked, dormant signaling effector activated by topologically adjusted cytoplasmic receptors For reliable activity assessment of synthetic receptors in the cytoplasm, and to engineer an efficient protease-activated expression system, we first built an orthogonal signaling effector.This modular mediator is composed of a transcription factor (TF) flanked with two successive protease-cleavable pleckstrin homology (PH) domains.PH domains are evolutionarily conserved β-sandwich protein constructs found in various proteins in mammalians.They possess a strong and highly specific binding affinity for cell membrane-anchored phosphoinositides, and play a key role in targeting different proteins to the inner part of the cell membrane [38][39][40][41] .We hypothesized that decorating a TF with PH domains would cause it to be docked in the plasma membrane and would thus diminish its availability in the nucleus.In addition to their membrane-directing features, multiple PH structures generate a remarkable steric hindrance, which cooperatively reduces the basal TF activity.Thus, proteolytic cleavage of the PH domains should liberate an active form of TF that can initiate gene regulation of protein of interest (POI) (Fig. 1a).For proof of concept, we initially used tTA (tetracycline-controlled transactivator) as a transcription factor, and SEAP (human placental secreted alkaline phosphatase) as a sensitive and quantitative reporter gene. Since the basal activity of most inducible gene switches dramatically influences their performance and robustness, we first focused on the engineering of PH-tTA chimeras with low basal activity.To do this, we systematically investigated the basal performance of a series of PH-tTA fusions constructed in various fashions (Fig. 1b).These constructs were co-transfected with a fixed amount of tTA-regulated reporter plasmid, tetO 7 -SEAP, in HEK-293 cells.As can be seen from the SEAP levels in the supernatants, the addition of one PH domain to tTA diminished the basal activity as expected; however, the decrease was not sufficient.Therefore, we systematically incorporated multiple PH domains in different orientations.We found that the PH x2 -tTA-PH x2 hybrid exhibited the lowest basal leakiness, with a SEAP output comparable to that of reporter gene-expressing cells.Since the basal activity was negligible, we focused on the PH x2 -tTA-PH x2 scaffold for further engineering.To explore whether the removal of PH domains enables tTA-mediated transcription, two TEVp cleavage sites (TCS) ENLYFQ ▼ S were introduced between tTA and the PH x2 adducts to yield a membrane-docked, dormant tTA (MDD-tTA), PH x2 -TCS-tTA-TCS-PH x2 .The cotransfection of MDD-tTA along with an intact cytoplasmlocated TEVp expression vector led to activation, and a significant increase (33.6-fold change) in SEAP levels was observed (Fig. 1c).The performance of MDD-tTA was also compared to other strategies frequently employed in protease-activated gene circuits.The performance of tTA fusions with a TEVp-cleavable membrane-directing Src myristylation signal (MS), Escherichia coli dihydrofolate reductase destabilizing domain ecDHFR(DD), and dopamine receptor D 1 (DRD1) transmembrane domain were evaluated 42,43 .As can be inferred from the SEAP levels in Fig. 1c, introducing Src MS and ecDHFR(DD) resulted in leaky systems and the fold changes are around 3-4 fold upon TEVp expression.Fusing of DRD1, however, could significantly reduce the basal expression, but the fold change was not high as that of MDDA-tTA (14.1 vs 33.6), which exhibits activation output comparable to that of unmodified tTA. In order to engineer chemically inducible systems, we utilized FKBP/FRB, PYL1/ABI, and GAI/GID1 protein-dimerizing domains that become juxtaposed upon the addition of RAPA, ABA, or GA, respectively.Each of the dimerization domains was fused to one part of the split cytoplasmic TEVp (scTEVp), affording scTEVp (1-118) and scTEVp (119-245) .The performance of these constructs was evaluated in HEK-293 cells in the absence or presence of the relevant DA (dimerization agent).From the SEAP levels in the supernatants (Fig. 1d), we can conclude that FKBP/FRB, PYL1/ABI fusions liberate tTA when stimulated by RAPA or ABA, though the GAI/GID1 split system proved unresponsive to GA treatment.While the dimerization ability of GAI/ GID1 in the presence of GA is well validated by many studies, they have rarely been used in the reconstitution of split protease systems 33,44,45 .We considered that in addition to GAI/GID1 dimerization, a specific orientation of scTEVp parts may also be required for functional reconstitution of this split system.To test this idea, we constructed different structural topologies of GAI/GID1 that are N' or C' terminally fused to scTEVp (1-118) /scTEVp (119-245) , annotated A-D, to cover all folding possibilities (Fig. 1e).The performance of these constructs was monitored in HEK-293 cells in a combinatory manner.We found that only one mode of assembly, B+C, could successfully restore scTEVp proteolytic activity in response to GA treatment (Fig. 1f).employed similar strategies for engineering gene circuits 47,48 .In this system, the protein of interest (POI) is C' terminally tagged with KDEL, a retention signal that prevents protein emigration from the ER due to its constitutive interaction with ER-dwelling KDEL receptors (Fig. 2a).Here, the KDEL signal is designed to be proteolytically cleaved from the POI by secTEVp (by introducing TCS between the POI and KDEL), allowing secretion of the POI into the extracellular space within a few minutes following RAPA addition.The cotransfection of SEAP-TCS-KDEL with intact secTEVp significantly increased the SEAP level in the supernatant, compared to cells that express SEAP-TCS-KDEL alone (Fig. 2b).To engineer chemically inducible systems, protein-dimerizing domains FKBP/FRB, PYL1/ABI, and GAI were fused N' terminally with a secretion signal in order to target the receptors into the secretory pathway.Exceptionally, GID1 was inverted relative to other constructs, as we noted before (Fig. 1e,f).Split secTEVp domains, ssTEVp 1-118 and ssTEVp 119-245 , were introduced with the same topologies that were functional in the cytoplasm (Fig. 1d,f).In addition, these split constructs were engineered with secTEVp non-cleavable KDEL tags in order to prevent their auto-proteolysis and consequent secretion.The inducibility of these constructs was evaluated in HEK-293 cells, using SEAP-TCS-KDEL as a reporter gene.The RAPA-regulated system was inducible as we previously reported, and only the topologically engineered version of GID1/GAI (the inverted form) responded to GA as shown in Fig. 1f.However, the ABA-regulated system did not respond to inducer addition (Fig. 2c).Interestingly, when ABA-regulated receptors were re-targeted from the ER to the cytoplasm (by removing the secretion signal and retaining TCS-KDEL), they regained their proteolytic activity when the cytoplasmic MDD-tTA (see Fig. 1) was used instead (Fig. 2d).This result implies that ABI/PYL1-ssTEVp receptors undergo ER-mediated PTMs that alter their physicochemical features and impair their dimerization ability in the presence of ABA.The formation of disulfide bonds between distantly located cysteine residues plays a pivotal role in shaping protein spatial structures in the secretory pathway, and the ER is the major cellular compartment where S-S bridges are stably formed by ER-resident enzymes, the protein disulfide isomerases (PDIs) 49 .In order to rescue these ER-retained receptors from functionally detrimental PTMs, we decided to block the formation of specific S-S bridges in the ABI /PYL1 dimerization domains by site-directed mutagenesis of cysteine residues.For this purpose, we performed a systematic mutagenesis screen, replacing cysteine with serine residues (Fig. 2e).The reason why we selected serine is its structural similarity to cysteine (the only difference is the oxygen atom instead of sulfur in cysteine), and its inability to form inter/intramolecular bridges.As can be seen from the SEAP levels in the supernatants of the cells transfected with the different mutants, singly mutated analogs could not rescue these constructs, so we next examined systematic double and triple mutagenesis.Fortunately, we found that ABI double-mutated at C29 and C102 could partially rescue the dimerization capability of PYL1/ABI in the presence of ABA when expressed in the ER. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To enhance the activity of ABI/PYL1-based receptors inside the ER, we also investigated the involvement of asparagine (Asn) N-linked glycosylation, since the addition of bulky sugars to synthetic moieties may modify their structures and affect the expected activity.Biochemically, N-linked glycosylation consists of the selective attachment of glycans to the nitrogen atom of asparagine within the canonical Asn-X-Ser/ Thr consensus sequence, and this common PTM occurs mainly in the ER 50 .To explore whether ABI/PYL1 undergo such PTMs, we initially constructed both ER-and cytoplasm-localized ABI/PYL1 receptors with ssTEVp 1-118 and ssTEVp 119-245 , as depicted in Fig. 3a (annotated as I C , I ER , II C , II ER ).The only difference between the cytoplasmic versions and the ER-dwelling analogs is the presence of the secretion signal (SS).All of these constructs were FLAG-tagged just before the KDEL retention signal, to enable their detection by western blotting.To monitor whether these constructs undergo N-linked glycosylation, we transfected these chimeras into HEK-293 cells and prepared the total lysate from each sample.The cleared lysates were then treated with endo-H, an endoglycosidase that cleaves asparagine-linked oligosaccharides 51 .The resulting lysates were separated by SDS-PAGE and immunoblotted against FLAG tag (Fig. 3b).Receptors inserted into the ER were endo-H sensitive, as they exhibited lower molecular weight due to the removal of covalently attached sugar (lane #3 vs #4 and lane #7 vs #8).In contrast, the cytoplasmic versions were not affected by endo-H treatment (lane #1 vs #2 and lane #5 vs #6).These results indicate that ABI/PYL1-ssTEVp constructs do undergo N-linked glycosylation. To investigate whether these receptors could be rescued from potentially detrimental N-linked glycosylation, we conducted asparagine (N) to glutamine (Q) mutagenesis analysis of PYL1 and ABI for asparagine residues located within the Asn-X-Ser/Thr consensus motif (X is any amino acid except proline) (Fig. 3c).Indeed, asparagine 308 in ABI appears to be involved in N-linked glycosylation, because converting this amino acid to glutamine significantly improved its dimerization ability with PYL1 WT in the presence of ABA.To further improve the ABAinducibility of ABI/PYL1-ssTEVp, we performed combinatory mutagenesis analysis using ABI N308Q with the C29S/C102S mutations that we had already characterized (Fig. 2e).It is clear from the SEAP levels in the supernatants that triple-mutated ABI N308Q, C29S, C102S is effectively induced inside the ER, showing more than 10-fold change versus nonfunctional ABI WT in response to ABA (Fig. 3d).Since changing these residues may also affect the binding affinity, we next examined the binding efficiency of ABI WT , ABI N308Q and ABI N308Q/ C29S/C102S with PYL1 wt .A dose-response analysis showed that the introduced mutations did not affect the binding affinity, and the EC 50 value was maintained at around ~4 µM (Fig. 3e) 31 .To demonstrate a potential application of the engineered ABI N308Q/ C29S/C102S , a fast-release experiment was conducted in HEK-293 cells.The cells were transfected with SEAP-TCS-KDEL, ABI N308Q/ C29S/C102S -ssTEVp 1-118 , and PYL1 WT -ssTEVp 119-245 .Indeed, the addition of ABA triggered the secretion of SEAP into the supernatant in less than 40 min (Fig. 3f). ",
"section_name": "Engineering an efficient ABA-inducible fast-release system through combinatory mutagenesis of N-linked glycosylation sites and disulfide bond-forming residues",
"section_num": null
},
{
"section_content": "The main disadvantage of integrating a synthetic gene switch into a cell's endogenous pathways is the poor predictability of the activation pattern, because many endogenous signaling cascades are highly interconnected, and are stimulated by multiple signal inputs.Thus, orthogonal pathways are needed to ensure high selectivity and operational reliability, especially for in vivo applications.We therefore engineered an orthogonal ABA-activated receptor in the plasma membrane.For this system, which we call orthogonal chemically activated receptor (OCAR), we utilized the Notch1 receptor core transmembrane domain (Notch1 core ) 7 .This core architecture was N' terminally connected with ABA-sensitive protein-dimerizing domains Fig. 1 | Engineering RAG-inducible intracellular receptors using a membranedocked, dormant transcription factor for orthogonal cell signaling control.a Schematic illustration showing the molecular mechanism of chemically regulated intracellular receptors using membrane-docked, dormant transcription factor (MDD-TF).b Basal activity analysis of different tTA-PH hybrids.SEAP levels in culture supernatants of HEK-293 cells transfected with 5 ng of pMMH171 (P hPGK -tTA-pA), pMMH172 (P hPGK -PH-tTA-pA), pMMH173 (P hPGK -PH-tTA-PH-pA), pMMH174 (P hPGK -PH x2 -tTA-pA), or pMMH125a (P hPGK -PH x2 -tTA-PH x2 -pA) plasmids.All samples were transfected with 20 ng of reporter plasmid pTS1017, which encodes tetO 7driven SEAP (P hCMV*-1 -SEAP-pA bGH ; P hCMV*-1 , O tetO7 -P hCMVmin ).Data are presented as mean ± SD of n = 8 biologically independent samples.c SEAP levels after 24 h in culture supernatants of HEK-293 cells transfected with 5 ng of pMMH201 (P hPGK -Src MS -TCS-tTA-pA bGH ), pMMH202 (P hPGK -ecDHFR(DD)-TCS-tTA-pA bGH ), pMMH203 (P hPGK -DRD1-TCS-tTA-pA bGH ), or pMMH125b which encode MDD-tTA (P hPGK -PH x2 -TCS-tTA-TCS-PH x2 -pA) in combination with pcDNA3.1(+)or cytoplasmic intact TEVp pTS2405 (P hCMV -TEVp-pA).Data are shown as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.d SEAP levels in culture supernatants of HEK-293 cells transfected with 5 ng of pMMH125b in addition to 1 ng of pMMH139 (P hCMV -FKBP-(GGGGS) 2 -scTEVp 1-118 -pA bGH ) with pMMH140 (P hCMV -FRB-scTEVp 119-245 -pA bGH ), pPW20 (P hCMV -ABI-(GGGGS) 2 -scTEVp 1-118 -pA bGH ) with pPW21 (P hCMV -PYL1-scTEVp 119-245 -pA bGH ), or pPW22 (P hCMV -GID1-(GGGGS) 2 -scTEVp 1-118 -pA bGH ) with pPW23 (P hCMV -GAI-scTEVp 119-245 -pA bGH ).Cells were treated with DMSO, RAPA (100 nM), ABA (100 µM), or GA (100 µM) for 24 h.Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.e Illustrative depiction of different spatial topologies of GID1/GAI chimeras that are linked to csTEVp parts, annotated A-D.f HEK-293 cells were transfected with A-D constructs in different transfection combinations using 5 ng of pMMH125b and 1 ng of pPW22 (P hCMV -GID1-(GGGGS) 2 -scTEVp 1-118 -pA bGH ), pPW23 (P hCMV -GAI-scTEVp 119-245 -pA bGH ), pPW24 (P hCMV -scTEVp 1-118 -(GGGGS) 2 -GID1-pA bGH ), pPW25 (P hCMV -scTEVp 119-245 -GAI-pA bGH ).SEAP levels were measured 24 f following GA (100 µM) treatment.Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.Source data are provided as a Source Data file. ABI and PYL1.At the C' terminus, however, these receptors were fused to a cleavable tTA for gene regulation.Mechanistically, upon the addition of the dimerization-inducing agent (DA), the OCAR parts bind to each other and undergo conformational change.These changes in the receptor parts stimulate their cleavage by endogenous membraneanchored proteases (such as ADAM and γ-secretase) (Fig. 4a).Once liberated, the tTA translocates to the cell nucleus, and controls gene expression. In this study, we used TetR as a DBD (DNA binding domain) and VP64 as a transcriptional transactivator.SEAP under tetO 7 regulation was used as the reporter.Cotransfection of HEK-293 cells with pPW86 WT (P PGK -SS-PYL1 WT -Notch1 core -TetR-VP64-pA) together with different amounts of pPW116 WT (P PGK -SS-ABI WT -Notch1 core -TetR-VP64-pA) did not result in any marked change in SEAP levels when ABA was added (Fig. 4b, left half).Analysis of the primary structure of Notch1 core revealed that this scaffold contains an exceptionally large number of cysteine residues (21 residues), which comprise more than 15% of the total amino acids.Thus, functionally detrimental disulfide bond formation between Notch1 core and the dimerization domains was considered likely to be responsible for the non-inducibility of OCAR parts.To validate this hypothesis, we systematically replaced cysteine residues with serine residues.First, we showed that C29S and C102S mutations of ABI rescued ABI/PYL1 dimerization of the ER receptor (Fig. 2e).Then, to validate these mutations in the OCAR system, we designed ABI C29S,C102S -Notch1 core -TetR-VP64 (pPW116 C29S,C102S ).We found that this synthetic OCAR receptor was indeed functional in HEK-293 cells after cotransfection with its heterodimerizing companion receptor (pPW86 WT , SS-PYL1-Notch1 core -TetR-VP64), in contrast to its wild-type version (pPW116 WT ) (Fig. 4b).To elucidate its activation mode, we performed co-culturing experiments to examine whether ABA-regulated OCAR works in cis (on the same cell) or in trans upon inducer addition. To explore this, two cell populations were separately transfected with one of the receptor parts, and subsequently, they were co-cultured in absence/presence of ABA.We also co-transfect both receptor parts in the same population.As a positive control, we use trans-acting anti-CD19 SynNotch 7 .In ABA-induced OCAR, co-culturing of the two cell populations that express one of the receptor parts did not lead to any marked increase in SEAP levels when ABA was added (Fig. 4c, top left and right), in contrast to cells that simultaneously transfected with both parts of the receptor (Fig. 4c, bottom left).Using the same experimental settings, we found that anti-CD19 synNotch-expressing cells are stimulated when co-cultured with CD19-overexpressing cells (Fig. 4c, bottom right), suggesting that ABA-inducible OCAR functions in a cis fashion.To investigate which intracellular part of ABA-regulated OCAR is cleaved following the inducer addition, we tested the activity of pPW86 WT and pPW116 C29S,C102S with their TetR-VP64-lacking counterparts pPW97 (P hPGK -SS-PYL1-Notch1 core -pA bGH ) and pMMH241 C29S,C102S (P hPGK -SS-ABI C29S,C102S -Notch1 core -pA bGH ), respectively (Fig. 4d).From the SEAP levels in the supernatants, we can conclude that both intracellular parts of the receptor (TetR-VP64) are cleaved in the presence of ABA and both contribute to the overall SEAP signal. Engineering RAPA-and GA-inducible OCARs through systematic mutagenesis of cysteine residues in the receptor-dimerizing domains To expand the OCAR platform for other dimerization agents (DA), and to show that cysteine mutagenesis can rescue the receptors' activity, we engineered RAPA-and GA-inducible OCAR systems. Here, we fused FKBP/FRP dimerization domains to Notch1 core -TetR-VP64, as depicted in Fig. 5a (left).To avoid increasing the rigidity of the protein, which may hamper the ability of FKBP/FRP to dimerize upon RAPA addition, we introduced a flexible linker (GGGGS) x2 between these domains and Notch1 core .Activity screening of a series of constructs was performed to evaluate the effect of linker presence and C to S mutagenesis of FKBP/FRP.Transfection of HEK-293 cells with these receptors in different combinatory patterns demonstrated that C22S mutation in FKBP restored OCAR functionality (lane #10), resulting in 9.4-fold induction (Fig. 5a, right).The presence of a flexible linker in the FKBP C22S mutant seems to have a negative impact on its activity, and significantly increases the basal expression (lane #12).Increased leakiness was also observed when a (GGGGS) x2 linker was introduced into FRB, even though inducibility was preserved (lane #10 vs #11).RAPA treatment decreased the basal expression of nonfunctional receptors, and this can be also seen in cells that express reporter gene alone.This decrease in signal is due to RAPA inhibition of the mTOR pathway, which diminishes global protein synthesis (see Fig. 5a, lanes #1-5) [52][53][54] .In active receptors, however, the signaling output of OCAR exceeds the RAPA-mediator protein attenuation, thereby overriding the unspecific rapamycinmediated decrease.This was confirmed by employing the rapalog FK506, which activated OCAR without any unspecific decrease (Supplementary Fig. S2). To evaluate whether RAPA-regulated OCAR also functions in cis, as does the ABA-induced variant (Fig. 4c), we performed the same type of co-culture experiments combining sender cells encoding SS-FRB-Notch1 core -TetR-VP64 (pPW113) and receiver cells encoding SS-FKBPC22S-Notch1 core -TetR-VP64 (pPW112C22S).Surprisingly, SS-FKBPC22S-Notch1 core -TetR-VP64 alone was sufficient for RAPA-triggered activation (Fig. 5b, middle).To engineer GA-inducible OCAR, we first cloned pPW124 WT and pPW125 WT as depicted in Supplementary Fig. S1a (left), and then conducted systematic mono and di mutagenesis screening of GID1 (GAI does not contain any cysteine residue).Unfortunately, neither mono nor di-mutated GID1 could rescue GID1/GAI inducibility (Supplementary Fig. S1a, right).However, unexpected behavior was observed in this C to S mutagenesis screen in GID1.All the mutants (and the WT) showed remarkably high basal expression levels as inferred from the SEAP levels of reporter-expressing cells, denoted as (-), compared to the other samples (see red arrow).To determine which construct is responsible for this leakiness, we separately transfected pPW124 WT and pPW125 WT into HEK-293 cells (Supplementary Fig. S1b).It turned out that the transfection of pPW125 WT alone leads to high leakiness, which could have masked the activities of some mutants that we previously generated.To solve this problem, we removed the intracellular part of pPW125 WT (TetR-VP64 transactivator) to produce pMHH130 as a \"silent\" dimerization anchor (Fig. 5c, left), and repeated the same screen using pMHH130 and pPW124 mutants instead (Fig. 5c,right).Here, the signal intensity was significantly reduced and it can be seen that the double mutations C155S, C157S and C174S, C362S in GID1 partially restore the dimerization ability of OCAR receptors in the presence of GA (1.8-and 2.6-fold change, respectively).To further increase the fold change and to enhance GA-induced OCAR, we performed combinatory triple mutagenesis of cysteine hotspots C155S, C157S, C174S, and C362S (Fig. 5d).Finally, the triplemutated GID1 C155S, C157S, C362S showed significant induction by GA, with a more than 5-fold change.Co-culture experiments combining cells expressing the SS-GID1-Notch1 core -TetR-VP64 variant Fig. 2 | Design of RAG-inducible modules for fast secretion control of ERaccumulated glycoproteins with avoidance of disulfide bridge formation in CID domains.a Schematic illustration of the molecular components that comprise the endoplasmic reticulum RAG-inducible system.b SEAP levels after 24 h in culture supernatants of HEK-293 cells transfected with 5 ng of pMMH51 and pMMH10 plasmids, which encode intact secTEVp-KDEL (P hCMV -ssTEVp-KDEL-pA) and SEAP-TCS-KDEL (P hCMV -SEAP-TCS-KDEL-pA) separately or in combination.Data are presented as mean ± SD of n = 5 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.c SEAP levels in culture supernatants of HEK-293 cells transfected with pMMH10 (5 ng) with pMMH26 (P hCMV -SS-FKBP-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA) and pMMH27 (P hCMV -SS-FRB-ssTEVp 119-245 -KDEL-pA), pMMH44 (P hCMV -SS-PYL1-ssTEVp 119-245 -KDEL-pA bGH ) and pMMH45 (P hCMV -SS-ABI-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA bGH ), pMMH43 (P hCMV -SS-GAI-ssTEVp 119-245 -KDEL -pA bGH ) and pPW26 (P hCMV -SS-ssTEVp 1-118 -(GGGGS) 2 -GID1-KDEL-pA bGH ) (5 ng each).Cells were treated with DMSO, RAPA (100 nM), ABA (100 µM), or GA (100 µM).Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.d Left: SEAP levels in culture supernatants of HEK-293 cells transfected with pMMH125b, which encodes MDD-tTA, along with 1 ng of the cytoplasmic pPW20 (P hCMV -ABI-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA bGH ) and pPW21 (P hCMV -PYL1-ssTEVp 119-245 -KDEL-pA bGH ).Right: SEAP levels in the supernatants of cells transfected with the ER-resident counterparts, e.g., pMMH44 (P hCMV -SS-PYL1-ssTEVp 119-245 -KDEL-pA bGH ) and pMMH45 (P hCMV -SS-ABI-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA bGH ), in combination with 5 ng of the reporter gene pMMH10, which encodes SEAP-TCS-KDEL (P hCMV -SEAP-TCS-KDEL-pA).Transfected cells were treated either with DMSO or ABA (100 µM).Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.e Site-directed mutagenesis of ER-localized ABI and PYL1 dimerization domains fused with ssTEVp 1-118 and ssTEVp 119-245 , respectively.SEAP levels in culture supernatants of cells transfected with 5 ng pMMH10 in addition to 5 ng of different S to C mutants of pMMH44 (P hCMV -SS-PYL1-ssTEVp 119-245 -KDEL-pA bGH ) and pMMH45 (P hCMV -SS-ABI-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA bGH ).Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.Source data are provided as a Source Data file.*** Fig. 3 | Engineering an efficient ER-localized ABA-inducible fast-release system through combinatory mutagenesis of C and N residues in CIDs to circumvent functionally detrimental PTMs. a Structure depiction of the cytoplasmic and the ER-localized versions of ABI-ssTEVp 1-118 and PYL1-ssTEVp 119-245 , annotated as I c (pPW32), I ER (pPW34), II c (pPW33), and II ER (pPW35).A secretion signal (SS) was introduced into I ER and II ER fusions.All constructs were tagged with FLAG for further analysis.b Immunoblotting against FLAG tag of HEK-293 cells transfected separately with the I c , I ER , II c , or II ER following endo-H treatment of the total lysates.Actin was used as a loading control.c Site-directed mutagenesis of ER-localized ABI-ssTEVp 1-118 and PYL1-ssTEVp 119-245 fusion proteins.SEAP levels in culture supernatants of HEK-293 cells transfected with 5 ng pMMH10, which encodes SEAP-TCS-KDEL (P hCMV -SEAP-TCS-KDEL-pA), in addition to 5 ng of different N to Q mutants of pMMH44 (P hCMV -SS-PYL1-ssTEVp 119-245 -KDEL-pA bGH ) and pMMH45 (P hCMV -SS-ABI-(GGGGS) 2 -ssTEVp 1-118 -KDEL-pA bGH ).Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.d Combinatory site-directed mutagenesis of ER-localized ABI-ssTEVp 1-118 , pMMH45.SEAP levels in culture supernatants of cells transfected with 5 ng pMMH10 in addition to 5 ng of different N to Q and/or C to S mutants of pMMH45 with non-mutagenized pMMH44 WT .Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using twotailed, unpaired Student's t-test.e Dose-response analysis of pMMH45 WT pMMH45 N308Q , and pMMH45 C29S, C102, N308Q mutants.Data are presented as mean ± SD of n = 4 biologically independent samples.f SEAP levels in culture supernatants of HEK-293 cells transfected with pMMH10, pMMH44 WT , and pMMH45 C29S, C102, N308Q (5 ng each).At 24 h after transfection, the culture medium was replaced with 30 µl of fresh medium containing either DMSO or ABA (100 µM) and 20 µl aliquots were collected for analysis at different time points.Data are presented as mean ± SD of n = 5 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.Source data are provided as a Source Data file.and with different amounts of either pPW116 WT (P PGK -SS-ABI WT -Notch1 core -TetR-VP64-pA) or pPW116 C29S,C102S (P PGK -SS-ABI C29S,C102S -Notch1 core -TetR-VP64-pA).All samples were transfected with 20 ng of reporter plasmid pTS1017, which encodes to tetO 7 -driven SEAP (O tetO7 -P hCMVmin TetO 7 -SEAP).Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.c SEAP levels in the supernatants of cocultured HEK-293 cells.Receiver cells were transfected with different amounts of pPW116 C29S,C102S and pPW86 WT either separately or in combination.Similar transfection procedure was performed using anti-CD19-synNotch-TetR-VP64.All receiver cells were co-transfected with the reporter plasmid pTS1017 (see methods). Sender cells were separately transfected either with pPW116 C29S,C102S , pPW86 WT , or with CD19 expression vector INS-2A-luciferase-2A-CD19-GFP.pcDNA3.1 (+) transfected HEK-293 sender cells are indicated as (-).ABA and DMSO were immediately added to the mixed cells, and SEAP measurements were performed 24 h following the co-culturing.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.d SEAP levels in the supernatants of cells transfected with 40 ng pPW86 WT , pPW116 C29S,C102S , pPW97 (P hPGK -SS-PYL1-Notch1 core -pA bGH ), and pMMH241 C29S,C102S (P hPGK -SS-ABI C29S,C102S -Notch1 core -pA bGH ).All samples were transfected with 20 ng of reporter plasmid pTS1017.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.Source data are provided as a Source Data file. (pPW124 C155S,C157S,C362S ) with cells encoding SS-GAI-Notch1 core (pMHH130) revealed that, as in the case of the RAPA-triggered OCAR, the SS-GID1-Notch1 core -TetR-VP64 OCAR variant (pPW124 C155S,C157S,C362S ) was also sufficient for GA-triggered activation (Fig. 5e). OCAR platform possesses intrinsic off-switch control, and ABAregulated OCAR can be used to increase synNotch activity during cell-cell communication Notch receptors rely on membrane-localized proteases, ADAM and γsecretase, for their cleavage upon binding to suitable ligands presented on the sender cells 55 .Potent and selective γ-secretase inhibitors have recently been developed and some are found in various clinical trials [56][57][58] .To examine whether OCAR receptors can also be regulated by γ-secretase inhibitor, we treated the cells with different concentrations of LY411575 (Fig. 6a, left).Indeed, the addition of LY411575 abolished OCAR activity despite the presence of ABA.To exclude the possibility that the effect of LY411575 is due to cell toxicity, we also evaluated cell viability using alamarBlue assay (Fig. 6a, right).Similar characteristics were also observed for RAPA and GA-stimulated OCARs (Fig. 6b,c).This built-in characteristic could be utilized as safety switch to stop gene expression during cell-based therapy.Next, we investigated whether we could influence the behavior of trans-acting Syn-Notch using our engineered cis-acting ABA-regulated OCAR during cell-cell communication.Here, we first sequestered one part of ABAregulated OCAR to anti-CD19 SynNotch receptor through coiled-coil interactions in order to prevent ABA-induced activation in the absence of CD19-presenting sender cells.To do this, we cloned several constructs using high-affinity coiled-coil interacting peptides, Ex3 and Kx3, as depicted in Fig. 6d 59,60 .The effect of cotransfection of different combinations of these receptors was analyzed in the presence/absence of ABA (Fig. 6d).Interestingly, the cotransfection of pMMH209, pMMH212, and pPW116 C29S, C102S almost abrogated the response to ABA, suggesting an efficient intermolecular interaction between pMMH209 and pMMH212.To explore whether the co-culture of CD19overexpressing cells (sender cells) can restore ABA-responsiveness and thus enhance SynNotch signaling by liberating pMMH212 from the receiver cells, we performed co-culturing experiments as shown in Fig. 6e.Indeed, the transfection of pMMH209, pMMH212, and pPW116 C29S, C102S significantly enhanced the performance of anti-CD19 SynNotch signaling when CD19-positive sender cells were co-cultured in addition to ABA.Comparative SDS-PAGE-based Western blot analysis of wild-type and mutant OCARs showed no significant changes in band size or intensity, suggesting that OCARs show similar expression levels and stability and do not form intermolecular disulfide bonds with other proteins (Fig. 6f,g).However, treatment of the OCARs with DTT and/or AMS resulted in band shifts, indicating that these receptors form intramolecular disulfide bonds (Fig. 6h). ",
"section_name": "Engineering of cis-functional transmembrane ABA-regulated orthogonal chemically activated receptors (OCARs)",
"section_num": null
},
{
"section_content": "In this work, we have engineered several synthetic receptors that reside in different organelles of mammalian cells by focusing on orientation adjustment of the receptor parts, and by employing rational mutagenesis to block functionally destructive PTMs in the secretory pathway.This approach enabled us to engineer various artificial receptors located in the cytoplasm, the ER, and on the cell surface, orthogonally controlled by small soluble ligands (RAPA, ABA, and GA).Here, we first engineered MDD-tTA as a robust and highly efficient signaling effector in the cytoplasm.Structurally, this signaling mediator consists of tTA, flanked with multiple TEVp-cleavable PH domains, which serve as a bulky steric shield and promote transfer to the membrane, synergistically suppressing the basal activity.To design an inducible system, several cytoplasmic receptors were engineered by fusing chemical dimerizing domains (CID) with scTEVp parts.Upon the addition of DA, the proteolytic activity is restored, and the PH adducts are cleaved to afford de-shielded tTA.The addition of RAPA and ABA reconstituted the receptor's proteolytic activity when the CID domains were integrated N' terminally.The GID/GAI-based receptor was nonresponsive to GA, but this was overcome by ensuring the proper orientation of the receptor parts. For ER-resident receptors, however, a different engineering approach was employed.In this compartment, the POI was retained and accumulated inside the ER by tagging its C'-terminus with a TEVp-cleavable KDEL sequence.The GA-regulated receptor again required the appropriate topological orientation, as in the cytoplasm, while the ABA-controlled receptor required specific mutagenesis to abolish detrimental PTMs: disulfide bridge formation and N-linked glycosylation.The same strategy, namely site-directed mutagenesis of disulfide-bond-forming residues, was employed to maintain the activity of cell-membrane-anchored OCAR receptors for regulated cell signaling.Since these OCAR receptors are among the few known Notch-based receptors regulated by soluble small ligands, we examined their mode of action.For this end, we co-cultured two cell populations, each expressing only one part of the receptor, in the absence/presence of the relevant inducer.As can be seen in Fig. 4c, ABA-regulated OCAR works in a cis-manner and both receptor parts are required for proper activation.However, RAPA-and GA-regulated OCARs required only the engineered part of the receptor to respond to their inducers (Fig. 5b,e).Although the precise molecular mechanism of RAPA-and GA-mediated OCAR activation remains elusive, we established that these synthetic receptors are functional and reliably trigger the orthogonal signaling cascade in the presence of functional γ-secretase (Fig. 6b,c). The N308Q mutation in the ABI domain significantly improved the dimerization ability with PYL1 inside the ER when ABA was added (Fig. 3c).In addition, the two cysteine mutations C29S and C102S further enhanced its performance in this compartment (Fig. 3d).To better understand how these mutations rescued the receptor activity, we looked at the x-ray structure of PYL1/ABA/ABI complex 61 .Yin et al. have demonstrated that following ABA binding, PYL1 protein undergo a conformational change in the CL2 loop which enables efficient dimerization with the ABI domain.Furthermore, it has been shown that the interacting part of ABI contains a Mn +2 -binding domain that participates in the interface interactions with ABA-bound PYL1 complex.Interestingly, we noticed that the D307 residue of ABI (referred as D413) participates in the formation of the Mn +2 -containing domain, which is required for proper dimerization with PYL1-ABA.Therefore, it seems that glycosylation of adjacent N308 may disturb the formation of this domain and abolish its dimerization capability.The precise molecular mechanism underlying the effects of the C29S and C102S mutations is unclear.However, it seems likely that these mutations may play a role in preventing the formation of functionally harmful intra-/ intermolecular disulfide bonds.To validate our hypothesis regarding N308Q mutation, and to establish how cysteine mutagenesis rescues ABI dimerization ability in the secretory pathway, further structural studies will be needed. Structurally, Notch receptors are composed of a ligand-binding domain, negative regulatory region (NRR), and intracellular domain 55,62 .Following ligand binding, Notch receptor undergo a conformational change that triggers cleavage by membrane-located proteases (such as ADAM and γ-secretase).This process leads to liberation Fig. 5 | Rational engineering of RAPA-and GA-regulated OCARs by C to S sitedirected mutagenesis in CID domains.a Left: structural architectures of the initial, non-mutagenized constructs pPW108 (P PGK -SS-FKBP-(GGGS) x2 -Notch1 core -TetR-VP64-pA), pPW109 (P PGK -SS-FRB-(GGGS) x2 -Notch1 core -TetR-VP64-pA), pPW112, and pPW113.\"L\" symbolizes a flexible linker.Right: SEAP levels in supernatants of HEK-293 cells transfected with pPW108, pPW109, pPW112, and pPW113 using either WT and/or different C to S mutants (40 ng each).Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.b SEAP levels in the supernatants of cocultured cells.Receiver cells were transfected with different amounts of pPW113 and pPW112 C22S either separately or in combination (see methods).Sender cells were separately transfected either with pPW112 C22S or pPW113.RAPA and DMSO were immediately added to the mixed cells, and SEAP measurements were performed 24 h following the co-culturing.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.c Systematic C to S mutagenesis of pPW124 (P PGK -SS-GID1-Notch1 core -TetR-VP64-pA).Cells were transfected with pMMH130 (P PGK -SS-GAI WT -Notch1 core -pA) and different C to S mutants of pPW124 (40 ng each).SEAP levels were measured at 24 h after GA (100 µM) treatment.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.d Combinatory C to S mutagenesis analysis of pPW124 and SEAP was measured as described in (c).Data are presented as mean ± SD of n = 6 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.e SEAP levels in the supernatants of co-cultured cells.Sender cells were transfected with pMMH130 and receiver cells were transfected with different amounts of pPW124 C155S, C157S, C362S .GA was used as the inducer.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.Source data are provided as a Source Data file. of the intracellular part of the receptor, which can be translocated to the nucleus to regulate gene expression.The NRR part is located between the ligand-binding domain and transmembrane part of Notch receptors and serves to prevent ligand-independent cleavage.This part of the receptor is composed of three cysteine-rich LIN-12-Notch repeats (LNRs) and a hetero-dimerization domain.In the OCAR design, we used the minimal Notch1 receptor core scaffold, which includes the NRR part.However, following their construction, initial evaluation showed that none of these receptors responds to its chemical activator.This led us to hypothesize that a functionally destructive intramolecular disulfide bond(s) between two distantly located cysteine residues in the dimerization domain and Notch1 core may be responsible for the lack of activity of the unmodified OCARs.Indeed, we saw that cysteine mutagenesis could restore the activity of the OCAR receptors activities, presumably by abolishing these functional harmful PTMs in the secretory pathway.reducing conditions In summary, our engineered receptors are expected to be useful tools for fine-tuning orthogonal signaling in mammalian cells.We have provided a number of proof-of-concept applications and also characterized these synthetic constructs.The ER-retained receptors enable rapid secretion of POI within a few minutes using small molecules as a signal input.Such fast-responding systems could be utilized in cellbased therapy for diseases, such as diabetes, pain, and epilepsy, where the rapid release of therapeutics is required.The cell-membraneanchored OCARs also enable different levels of signaling control.Due to their membrane location and their mechanism of action, OCAR systems possess an intrinsic off-switch, which could be used as a safety switch to shut down therapeutic cells' gene circuits in the event of toxicity or malfunction.This would be especially important for inducers with a long half-life, which may remain in the blood circulation for several days after their discontinuation.In addition, we showed that ABA-regulated OCAR could be adjusted, through coiled-coil interactions, to enhanced SynNotch signaling during cell-cell communication.We think that the receptor architectures introduced here will find multiple applications in synthetic biology, basic science, and cellbased therapy. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Rapamycin (cat.no.553210), gibberellic acid (cat.no.48880), γ-secretase inhibitor LY411575 (cat.no.SML0506), FK506 (cat.no.342500), and cycloheximide (cat.no.01810) were purchased from Sigma-Aldrich, Buchs, Switzerland.(+)-Abscisic acid (Cat.No. 6554) was purchased from Tocris Bioscience, Bio-Techne AG, Switzerland.4-Acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt (AMS) (cat.no.A485) was purchased from Thermo Fisher Scientific, Reinach, Switzerland. ",
"section_name": "Chemical reagents",
"section_num": null
},
{
"section_content": "Gene expression vectors were constructed either by using restriction enzymes (New England BioLabs, Ipswich, MA, USA) followed by ligation with T4 DNA ligase (New England BioLabs, cat.no.M0202L) or by Gibson assembly (New England BioLabs, cat.no.E2611L).For restriction enzyme-based cloning, digested plasmid backbones were dephosphorylated with Antarctic phosphatase before ligation (New England BioLabs, cat.no.M0289L).PCR reactions were performed using Q5 High-Fidelity DNA polymerase (New England BioLabs, cat.no.M0491L).For Gibson assembly, the PCR products were amplified using primers having 15-20 bp complementary sequences to each end of the linearized vector.Site-directed mutagenesis was performed using a Q5® Site-Directed Mutagenesis Kit (New England BioLabs, cat.no.E0552S).Detailed information about plasmid cloning is presented in Supplementary Tables S1 andS2.Plasmids were transformed and propagated in XL10-Gold® ultra-competent Escherichia coli (New England BioLabs, cat.no.C2992) and extracted using a plasmid miniprep kit (Zymo Research, Irvine, CA, USA, cat.no.D4054) or a Zymo-PURE II Plasmid Midiprep Kit (Zymo Research, cat.no.D4200). ",
"section_name": "Plasmid construction",
"section_num": null
},
{
"section_content": "Human embryonic kidney cells (HEK-293T, ATCC: CRL-3216) were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, cat.no.10566016) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, cat.no.F7524, lot no.022M3395) and penicillin (100 U)-streptomycin (100 µg) solution (Sigma-Aldrich, cat.no.P433) under a humidified atmosphere of 5% CO 2 in air at 37 °C.Passaging of pre-confluent HEK-293T cultures was performed by trypsinization with 0.05% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA; cat.no.25300-054) for 5 min at 37 °C.Cells were transferred to 10 ml cell culture medium, and centrifuged for 1 min at 200 × g.The supernatant was discarded and the cells were resuspended in a fresh medium.Cell number and viability were quantified using an electric field multichannel cell-counting device (Casy® Cell Counter and Analyzer Model TT, Roche Diagnostics GmbH, Rotkreuz, Switzerland). ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "For plasmid transfection in a 96-well format, HEK-293T cells were seeded at a density of 50,000 cells per 1 cm 2 in 100 μl medium for 24 h. 100 μl of serum and antibiotics-free minimum essential medium MEM (Thermo Fisher Scientific, cat.no.11095080) containing a 1:5 DNA:PEI mixture (polyethylenimine, MW 40,000; Polysciences Inc., Warrington, FL, USA, cat.no.24765) with a total DNA amount of 350 ng/cm 2 was added dropwise to the cells, and the plate was incubated for 16 h. ",
"section_name": "Transient transfection",
"section_num": null
},
{
"section_content": "Unless stated otherwise, the culture medium of the transfected cells was replaced with 100 µl of fresh culture medium containing the indicated drugs and incubated for 24 h. ",
"section_name": "Pharmacological treatment",
"section_num": null
},
{
"section_content": "SEAP levels in the cell culture medium were determined as follows: 20 µl of the culture supernatant was mixed with 80 µl ddH 2 O and heat-inactivated for 30 min at 65 °C.Then, 80 µl of 2x SEAP buffer (20 mM homoarginine, 1 mM MgCl 2 , 21% (v/v) diethanolamine, pH 9.8) and 20 µl of 120 mM para-nitrophenyl phosphate (Acros Organics, Geel, Belgium, cat.no.128860100) solution in 2× SEAP buffer were added to each well, and the absorbance at 405 nm was measured at 37 °C using a Tecan M1000 plate reader (Tecan Group Ltd., Maennedorf, Switzerland).SEAP concentrations were calculated from a standard curve. Fig. 6 | OCAR platform possesses an intrinsic off-switch control, and ABAregulated OCAR can be used to increase synNotch activity during cell-cell communication.a Left: SEAP levels in the supernatants HEK-293 cells transfected pPW116 C29S,C102S , pPW86 WT , and reporter plasmid pTS1017.Following the transfection, cells were treated with different concentrations of γ-secretase inhibitor LY411575 along with ABA (100 µM) or DMSO for 24 h.Right: alamarBlue cell viability assay of the previous samples.Data are presented as mean ± SD of n = 4 biologically independent samples.b, c The same experiment as shown in (a) for OCARs regulated by RAPA (b) and GA (c).For analysis of the RAPA-regulated OCAR, the cells were transfected with pPW112 C22S .For analysis of the GA-regulated OCAR pPW124 C155S, C157S, C362S was transfected.Data are presented as mean ± SD of n = 4 biologically independent samples.d SEAP levels in the supernatants of cells transfected with the indicated plasmids.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.e SEAP levels in the supernatants of co-cultured cells.Receiver cells were transfected either with pMMH209, pMMH212, and pPW116 C29S,C102S , combination or with anti-CD19-synNotch-TetR-VP64.The transfected receiver cells were co-cultured with sender cells that overexpress CD19 or sender cells that were transfected with pcDNA3.1(+), in presence/absence ABA.Data are presented as mean ± SD of n = 4 biologically independent samples.***P < 0.0001 was calculated using two-tailed, unpaired Student's t-test.f Immunoblotting against FLAG and β-actin of cells separately transfected with pMMH234 (P hPGK -SS-ABI-Notch1 core -TetR-VP64-2xFLAG-pA bGH ), pMMH233 (P hPGK -SS-ABI C29S , C102S -Notch1 core -TetR-VP64-2xFLAG-pA bGH ), pMMH230 (P hPGK -SS-FKBP-Notch1 core -TetR-VP64-2xFLAG-pA bGH ), pMMH231(P hPGK -SS-FKBP C22S -Notch1 core -TetR-VP64-2xFLAG-pA bGH ), pMMH232(P hPGK -SS-GID1-Notch1 core -TetR-VP64-2xFLAG-pA bGH ), and pMMH235 (P hPGK -SS-GID1 C155S , C157S , C362S -Notch1 core -TetR-VP64-2xFLAG-pA bGH ) followed by cycloheximide chase (see Methods).g Immunoblotting against FLAG and β-actin of cells separately transfected with the same plasmids as above.For each sample, the cleared lysate was divided equally into two parts and sample buffer with or without DTT was added.h Immunoblotting against FLAG following AMS shift assay of cells transfected with the same plasmids as above (see Methods).Source data are provided as a Source Data file. ",
"section_name": "SEAP quantification",
"section_num": null
},
{
"section_content": "Cell viability was evaluated using alamarBlue™ Cell Viability Reagent (Thermo Fisher, cat.no.DAL1025) according to the manufacturer's instructions. ",
"section_name": "Cell viability assay",
"section_num": null
},
{
"section_content": "Receiver cells: HEK-293 cells were seeded in 6-well plates at a density of 0.4 × 10 6 cells per well.The following day, cells were transfected with 2.5 µg total DNA.0.4 µg reporter plasmid was used for all samples.Sender cells: HEK-293 cells were seeded in 10-cm plates at a density of 2 × 10 6 cells per plate.On the following day, cells were transfected with 10 µg plasmid that encodes the relevant receptor/protein or with pcDNA3.1(+)as a negative control.24 h following the transfection, sender and receiver cells were harvested and co-cultured in a 96-well plate by mixing 2 × 10 4 receiver cells with 8 × 10 4 sender cells (1:4 ratio).Drugs were added immediately, and samples were analyzed after 24 h. ",
"section_name": "Co-culture experiments",
"section_num": null
},
{
"section_content": "Cells were harvested, centrifuged at 1000 × g for 5 min, and washed twice in cold PBS.For cell lysis, RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher, cat.no.A32963) was added to the cell pellet and the mixture was vortexed for 20 min at 4 °C.Lysates were cleared by centrifugation at 12,000 × g for 30 min at 4 °C.5× reduced Laemmli sample buffer was added, boiled for 5 min at 95 °C, and loaded on SDS-PAGE.Protein quantification was performed using a Pierce™ BCA Protein Assay Kit (Thermo Fisher, cat.no.23227).Following SDS-PAGE, gels were blotted onto PVDF membranes using a Biorad PowerPac TM .Blots were blocked with 10% skim milk in TBST buffer at room temperature for 1 h.The following primary antibodies were used (in 1:1000 dilution): monoclonal anti-FLAG® M2 antibody produced in mouse (Sigma, cat.no.F1804, clone M2), rabbit (Cell Signaling, cat.no.4970, clone 13E5).Secondary HRPconjugated goat anti-rabbit (cat.no.111-035-144, polyclonal) and antimouse (cat.no.115-035-003, polyclonal) antibodies were purchased from Jackson Immunoresearch, West Grove, PA and used at a dilution of 1:10,000 dilution.PageRuler™ Plus Prestained Protein Ladder, 10-250 kDa (Thermo Fisher, cat.no.26619) was used as protein molecular weight marker.Blots were developed using FUSION Pulse TS (cat.no.37480003, Vilber, France), and images were analyzed using Adobe Illustrator software.All uncropped and unprocessed western blot images are provided in the Source Data file. ",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Thiol alkylation of proteins using the sulfhydryl-specific reagent AMS was performed as described 63 .Briefly, the cleared lysates were treated with dithiothreitol (DTT) at 200 mM final concentration.Water was added to untreated samples.All samples were incubated for 5 min at 100 °C.Next, all samples were precipitated with 10% (w/v) trichloroacetic acid (TCA) for 30 min at 4 °C, and the precipitated proteins were recovered by centrifugation for 30 min at 14,000 g.Protein pellets were washed three times with 1 ml of ice-cold acetone followed by centrifugation for 15 min at 14,000 g.Protein pellets were air-dried and resuspended in 40 µl of an alkylating buffer (150 mM Tris-HCl (pH 7.5) and 2% SDS) containing/not containing 20 mM AMS and incubated for 2 h at 22 °C with moderate shaking.10 µl of 5× sample buffer (containing DTT) was added to all samples, followed by boiling for 5 min.A 10 µl aliquot of each sample was subjected to SDS-PAGE. ",
"section_name": "AMS shift assay",
"section_num": null
},
{
"section_content": "To evaluate protein stability, cells were first transfected with the indicated expression vectors in a well of a 6-well plate.24 h after transfection, cells were harvested, washed twice with PBS, and resuspended in cycloheximide-containing PBS (50 µg/ml).Cells were incubated at 37 °C and samples were collected at different time points.Lysates were prepared from the samples and subjected to SDS-PAGE. ",
"section_name": "Cycloheximide chase experiment",
"section_num": null
},
{
"section_content": "Cell lysates were isolated and quantified as described before.Endoglycosidase treatment was performed using Endo-H (New England BioLabs, cat.no.P0702L) according to the manufacturer's instructions. ",
"section_name": "N-linked glycosylation analysis",
"section_num": null
},
{
"section_content": "Figures were designed and assembled using Adobe Illustrator 2021 software. ",
"section_name": "Figures",
"section_num": null
},
{
"section_content": "The statistical significance of differences among groups was evaluated with a two-tailed, unpaired Student's t-test using GraphPad Prism.Differences are considered statistically significant at P < 0.05.The statistical test used and the significance are reported in the figure legends.All the presented data has been independently repeated three times. ",
"section_name": "Statistics and reproducibility",
"section_num": null
}
] |
[
{
"section_content": "The work in the laboratory of M.F. is financially supported through a European Research Council advanced grant (ElectroGene, no.785800) and in part by the National Centre of Competence in Research (NCCR) for Molecular Systems Engineering, the EC Horizon 2020 Framework Programme ENLIGHT, Innosuisse (no.59511.1IP-LS)as well as an EMBO fellowship granted to M.M. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors declare that all data generated in this study are provided within the paper and in the Supplementary Information/Source Data file.All vector information is provided in Supplementary Tables S1 andS2.Requests for materials should be made to the corresponding author.All plasmids generated in this study are available upon request.Source Data are provided with this paper. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. M.M. and M.F.designed the project.M.M., P. W. and S.X.conducted the experiments.M.M. and M.F.analyzed the data and wrote the manuscript. The authors declare no competing interests. ",
"section_name": "Reporting summary",
"section_num": null
},
{
"section_content": "Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. ",
"section_name": "Reporting summary",
"section_num": null
},
{
"section_content": "M.M. and M.F.designed the project.M.M., P. W. and S.X.conducted the experiments.M.M. and M.F.analyzed the data and wrote the manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1101/2022.02.28.482396
|
A BALB/c IGHV Reference Set, Defined by Haplotype Analysis of Long-Read VDJ-C Sequences From F1 (BALB/c x C57BL/6) Mice
|
<jats:p>The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized. This compromises data analysis. To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals. Strain variations were identified in the<jats:italic>Ighm</jats:italic>and<jats:italic>Ighg2b</jats:italic>genes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles. 169 alleles are not present in the C57BL/6 genome reference sequence. To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset. Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences. A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain. Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype. The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted at<jats:uri /><jats:uri>https://ogrdb.airr-community.org</jats:uri>. We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci. Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly. This suggests that current MGP assemblies are unsuitable for the comprehensive documentation of germline IGHVs and more efforts will be needed to establish strain-specific reference sets.</jats:p>
|
[
{
"section_content": "The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized.This compromises data analysis.To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals.Previously unreported strain variations were identified in the Ighm and Ighg2b genes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles.169 alleles are not present in the C57BL/6 genome reference sequence.To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset.Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences.A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain.Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype.The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted at https://ogrdb.airrcommunity.org.We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci.Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly.This suggests that Adaptive Immune ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Our understanding of B cells and the antibody response have long been informed by studies of immunoglobulin genes.For example, early genetic studies revealed processes such as somatic point mutation and affinity maturation (1,2).Other studies focused upon the expression of particular immunoglobulin genes, in an effort to better understand the aberrant immune responses that are seen in allergic (3) and autoimmune diseases (4).Today, new insights are coming from the study of immunoglobulin gene repertoires using high-throughput sequencing. Immunoglobulin genes are found within the genome as multiple sets of highly similar genes.The immunoglobulin heavy chain (IGH) that is the focus of this study is encoded by Variable (IGHV), Diversity (IGHD) and Joining (IGHJ) genes.Multiple genes of each type are found together within the IGH gene locus.During early B cell development, genetic recombination joins one of each of these gene types to form a functional VDJ gene (5).The VDJ gene is expressed in association with a single Constant (C) region gene to produce the immunoglobulin heavy chain, while the light chain is produced by similar processes acting on separate sets of light chain genes. The study of human VDJ genes was facilitated first by the documentation of the complete set of germline immunoglobulin genes that are available for recombination (6,7), and later by the documentation of allelic variation and structural variation within the human population (8)(9)(10).With this knowledge to hand, analysis of the expressed antibody repertoire became possible.It is now known that the diversity of the antibody repertoire is an outcome of processes that are stochastic, but that are nevertheless influenced by germline variants (11)(12)(13). Adaptive immune receptor repertoire sequencing (AIRR-seq) studies now often report the sequencing of thousands and even millions of different rearranged immunoglobulin genes from a single individual.Over the last decade, such sequencing studies have transformed our understanding of the nature of the human antibody repertoire, and of fundamental aspects of the antibody response in health and disease (14)(15)(16)(17)(18).But despite the importance of animal models for our understanding of antibody-mediated immunity, autoimmunity and allergic disease, there have been relatively few reports of the antibody repertoires of laboratory mice.In part, this is a consequence of our lack of understanding of the germline immunoglobulin genes of the mouse, for only the genes of the C57BL/6 mouse have been comprehensively documented.Without an understanding of their germline genes, reliable analysis of the repertoires of other mouse strains is impossible. The germline genes of the BALB/c mouse have previously been explored using AIRR-seq data and a process of gene inference (19).In such analyses, the presence of multiple examples of identical sequences within the set of VDJ gene rearrangements is used to identify each germline gene.The reliable inference of germline IGHV genes within datasets of VDJ gene rearrangements, particularly those of naïve B cells, is a trivial exercise for highly expressed genes, but it is challenging for rarely expressed genes.Many BALB/c genes appear to consistently rearrange at frequencies as low as 0.01%.Given the sequencing depth of the study by Collins and colleagues (19), many of their BALB/c germline gene inferences were only supported by a handful of rearranged VDJ gene sequences.It is therefore important that these inferences be further tested using validated inference tools (20,21). High-throughput AIRR-Seq using long read single-molecule real-time (SRMT; Pacific Biosciences) sequencing can cover the entire V(D)J region and extend into the CH3 exon of the constant region.It was recently applied to the study of the mouse VDJ repertoire (22).Here, we used this approach to amplify VDJ rearrangements in association with constant region genes (VDJ-C).The F1 IGHV genotype was defined from rearranged VDJ-C amplicons and IGHJ1based haplotyping was used to explore genetic variation within the Ighm and Ighg-encoding constant region genes.Strain-defining single nucleotide polymorphisms (SNPs), including variants that are not present in the IMGT database, were identified, allowing the RAbHIT haplotyping tool (23) to be used to assign each IGHV to one or other of the parental strains.This analysis largely confirms the reported germline IGHV sets for the C57BL/6 and BALB/c strains.Only four IGHV sequences are shared by the two strains. It is now known from studies of wild-derived and classical inbred mice that the IG gene loci are highly divergent between strains (19,22).The divergence is so great that it is impossible to assign new mouse sequences as allelic variants of genes that have been defined by the C57BL/6 genome.Strain-specific IGHV Reference Sets should allow accurate analysis of the immunoglobulin repertoire, without reference to C57BL/6 genes, but there is presently a lack of strain-specific IGHV Reference Sets for non-C57BL/6 strains.To begin to address the need for such sets, the BALB/c sequences identified in this study were used to establish a curated BALB/c IGHV Reference Set at the Open Germline Receptor Database (OGRDB) website (https://ogrdb.airr-community.org/ ) (24).The errors that can arise from the use of incomplete Reference Sets in AIRR-Seq analysis are highlighted here, and we suggest that it will be critical for the IGHV repertoires of additional inbred strains to be properly documented if we are to better understand the roles of antibodies in mouse models of human disease. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "RNA was extracted from BALB/cByJ x C57BL/6J F1 hybrid dissected spleens (n = 4) preserved in RNA later (Thermofisher, Cat.No. AM7020; Waltham, MA, USA).Briefly, total RNA was extracted from 30 mg of preserved spleen using the RNeasy Mini kit (Qiagen, Cat.No. 74104; Germantown, MD, USA).First-strand cDNA was generated from 1 µg of RNA for each sample using the SMARTer RACE 5'/3' Kit (Takara Bio, Cat.No. 634858; Mountain View, CA, USA).For each sample, long-read single molecule real-time (SMRT) sequencing (Pacific Biosciences) libraries were prepared using IgM and IgG constant region primers that amplified off the firststrand cDNA synthesis product.Rearranged VDJ IGM and IGG amplicons were generated using a universal forward primer (Takara Bio;10 µM), and either an IgM-CH3 (5'-CAGATCCCTGTGAGTCACAGTACAC-3'; 10 µM) or IgG-CH3 (5'-ATGAAGTAAGAACCATCAGAGTCCAGGAC-3; 10 µM') reverse primer.First-strand cDNA was amplified using Thermo Fisher Phusion HF Buffer (Thermo Fisher, Cat.No. F530S; Waltham, MA, USA) for 30 PCR cycles.Final amplicons were used to construct SMRTbell libraries and sequenced on either the RSII or Sequel system (Pacific Biosciences; Menlo Park, CA, USA). ",
"section_name": "Mice and Library Construction",
"section_num": null
},
{
"section_content": "Genotype inference tools such as TIgGER (21,25) are able to identify previously unreported germline IGHV in AIRR-seq datasets, but they work best with reference datasets that correspond fairly closely to the genotypes under investigation.If an unreported sequence is substantially different to any sequence in the reference dataset, it may not be detected.We therefore sought to confirm the results of an earlier study (19), rather than determining the F1 genotype de novo.We compiled a reference set of all BALB/c and C57BL/6 genes identified in the study of Collins and colleagues, as well as all other reportedly functional C57BL/6 or BALB/c IGHV genes in the Reference Directory of the ImMunoGeneTics (IMGT) group.Some IGHV gene sequences and names in the IMGT Reference Directory have changed since the publication of the Collins study.Sequence changes mostly involve the 3' terminal nucleotides, and are largely undocumented at the IMGT website, but were identified through archived webpages.Name changes and sequence changes are documented in Table I. The performance of annotation tools as well as germline gene inference tools can be compromised by the presence of truncated sequences in a reference set.A number of truncated genes from IMGT and other sources are annotated as being present in the BALB/c strain.To maximize the likelihood of their detection by genotyping tools, reference set sequences that appeared to have short truncations were manually extended by reference to their most similar full-length IGHV sequences (Supplementary Table I).Substantial extensions were required for other IGHV sequences that are referred to, by IMGT, as being 'unmapped'.These sequences can be identified by IMGT's 'S' nomenclature: for example, IGHV1S22*01.These truncations involve both 3' and 5' nucleotides, with as many as 57 nucleotides being missing from individual sequences (Supplementary Table I). Extensions of eight of the ten 'unmapped' sequences were made with reference to other Genbank sequences that were exact matches to the truncated sequences (Supplementary Table I).In these eight cases, the Genbank sequences were recorded as being derived from BALB/c animals, or from animals of the BALB/c-derived D-limited strain (26).A ninth sequence, IGHV9S8*01, was originally reported in BALB.K mice.It was extended using the closely matching genomic sequence (AY169679) reported from the DBA strain (27).Both the BALB.K and DBA stains are closely related to the BALB/c strain.An exact match to the final 'unmapped' sequence, IGHV1S75*01, was found in the BALB/c genome sequence of the Sanger Mouse Genome Project (MGP) at Ensembl 104 (28).All the extended sequences were used in the knowledge that errors in the extensions could be readily identified in this study as part of genotyping and the search for novel alleles using inference processes. The incorporation of all these changes led to the definition of a reference set made up of modified and unmodified IMGT and other sequences called the Combined and Extended (ComEx) Reference Directory.It was used as the starting point for this study.Over the course of the study, the ComEx Reference Directory was modified by the inclusion of additional inferred novel alleles, and final analysis was based upon this modified version of the Reference Directory. ",
"section_name": "Definition of Germline Gene Reference Sets",
"section_num": null
},
{
"section_content": "UMIs were extracted from PacBio SMRT-seq Q30 fastq files and primers were trimmed using MaskPrimer from Immcantation's presto package (v0.5.4) (29).Reads were aligned using IgBLAST (v1.14) (Ye et al., 2004) to the ComEx IGHV and IMGT's mouse IGHD and IGHJ reference directory (reference obtained from IMGT 16-01-2020) to generate AIRR-formatted output (-outfmt 19).Change-O databases were generated using the MakeDB command from the Change-O tool (v 1.1.0)with --failed option to retain both the 'pass' and 'fail' subsets (30). The Ighc portion of each read was extracted based on the position of the final IGHJ nucleotide in the IgBLAST output, with the Ighc part running from 1 nucleotide downstream from the IGHJ through to the end of the primer trimmed read.Upon definition of the strain-specific spliced constant region exons for the strains, the Ighc portions were insertion/deletion (indel) corrected via blastn alignment (31) and processing with a custom script.This permitted positional extraction of the Ighm and Ighg2b SNPs. ",
"section_name": "Processing of long read VDJ-C datasets",
"section_num": null
},
{
"section_content": "Change-O databases for the IgM datasets, both pass and fail, were merged with the extracted Ighc sequences and combined to generate a single F1 dataset.VDJ-C reads that were 5' truncated or which lacked a CDR3 or an IGHJ gene call were removed, and the dataset was collapsed to unique VDJ nucleotide sequences.TIgGER's FindNovelAlleles was called on this F1 IgM dataset with germline_min = 50 and min_seqs = 10. Before proceeding to genotyping, novel alleles were added to ComEx.IgBLAST was re-run and new Change-O databases were created.Genotyping of the IgM F1 datasets was performed using TIgGER (v1.0.0) (21) with the inferGenotypeBayesian function.Following genotyping, alleles were reassigned using the reassignAlleles function as input for haplotyping.The F1 genotype and associated fasta set were exported from TIgGER and processed with OGRDBstats (https://github.com/airr-community/ogrdbstats),providing data for the clarification of the 3' ends of the genes. ",
"section_name": "Defining the F1 Genotype",
"section_num": null
},
{
"section_content": "The BALB/c strain carries the IGHJ1*01 allele, which differs from the C57BL/6 IGHJ1*03 allele at a single SNP (32,33).IGHJ1-based haplotyping was performed to explore variability between strains in the constant region exons.Reads were filtered for IGHJ1 usage and then subsets were created based on either IGHJ1*01 or IGHJ1*03 usage.Unique Ighc sequences associated with the *01 and *03 VDJs were sorted according to their frequency.The top Ighm sequence associated with each IGHJ1 allele was selected and aligned to the C57BL/6 and BALB/c genomes at ensembl (ensembl.org).The IGHJ1-based haplotyping was repeated on the IgG F1 dataset, but with the top 2 sequences for each IGHJ1 allele extracted to account for IgG2a and IgG2b in BALB/c and IgG2b and IgG2c in C57BL/6.Identification of strain-defining SNPs in the Ighm gene allowed haplotyping of the IGHV loci of the two strains to be determined using unmutated IgM-associated VDJ sequences.Haplotyping was performed using RAbHIT (v0.1.8)(23,34).Input to haplotyping was the TIgGER reassigned F1 IgM dataset joined to the IgM SNP-typed data in the form of a c_call with format IgM*B6 or IgM*BALB.Sequences lacking an Ighc call were excluded, as were those with ambiguous calls (for example, the same VDJ nucleotide sequence that was associated with both IgM*B6 and IgM*BALB).Haplotyping was also limited to unmutated, unique sequences with read counts greater than 1.The RAbHIT function createFullHaplotype was called with the following parameters: toHap_col = v_call, hapBy_col = c_call, hapBy = IgM, kThreDel = 0.1, relative_freq_priors = FALSE and single_gene = TRUE.IGHV8 haplotypes were plotted with the plotHaplotype function using a custom genes_order and removeIGH = FALSE.C57BL/6 and BALB/c IGHV sets were output from RAbHIT haplotypes using the TIgGER germline inferred database.Prior to incorporation into the OGRDB BALB/c Reference Set, gene ends were manually reviewed using analysis of terminal nucleotide triplets. To assess the consequences of the use of an incomplete Reference Set for the analysis of BALB/c data, we analyzed publicly available datasets from the study of Corcoran and colleagues (20).The data was generated from peripheral blood lymphocytes using 5' RACE and Illumina MiSeq sequencing.The animals were of the BALB/cJ substrain, giving an opportunity to also explore IGHV gene and repertoire differences between the BALB/cByJ and BALB/cJ substrains.IgBLAST was first run using the IMGT Reference Set and then using the OGRDB BALB/c Reference Set, noting the number of mismatches in each IGHV alignment. ",
"section_name": "Defining strain specific genes via haplotyping",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "IgM CH3 primed libraries were sequenced from four F1 mice (J1, J2, J3, J4), with replicate sequencing from mouse J2 (three replicates) for a total of 6 IgM datasets.IgG CH3 primed libraries were sequenced from the same four animals.IgG from J1 generated a low read count and was excluded from analysis. A combined total of 383,550 IgM reads and 67,237 IgG reads passed primer trimming.Complete VDJ-C amplicons were found in 117,262 IgM reads and 59,217 IgG reads.Filtering of the IgM dataset retained 51,988 unique, full length IgM sequences that were used for novel allele predictions and a subset of 36,294 unmutated sequences were used for genotyping of the F1.Haplotyping utilized 7,896 sequences with highly confident Ighm constant region SNP calls. ",
"section_name": "Long read VDJ-C sequencing of F1 mice",
"section_num": null
},
{
"section_content": "The analysis of 51,988 reads aligned against the ComEx IGHV dataset for novel alleles with TIgGER detected 271 alleles found in the ComEx dataset as well as four putative additional alleles.IGHV alleles are described here using four naming schemes.Alleles that are present in the IMGT Reference Directory are referred to using IMGT names of the type 'IGHV1-2*01'.Other non-IMGT alleles that were first reported by Haines and colleagues have been named using a schema producing labels such as 'J558.1.85'(35).Non-IMGT sequences that were not named by Haines and colleagues but that appear in the VBASE2 database are referred to by their VBASE2 labels, such as 'musIGHV021'.Finally, alleles that were first described by Collins and colleagues (19), or were first described in this study have been assigned names such as 'balbIGHV034' or 'b6IGHV040', according to strain.All confirmed BALB/c IGHV sequences were also ultimately assigned names using the OGRDB Naming Schema (https://github.com/williamdlees/IgLabel,manuscript in preparation). A novel allele named here as balbIGHV036 was previously reported as a BALB/c IGHV by Corcoran and colleagues (20).A second novel allele appeared to be a variant of balbIGHV027 that differed at the 3' end of the sequence.No alignments were found to balbIGHV027 itself.This variant allele was also previously reported by Corcoran et al (20).It was assigned the name balbIGHV037.A third novel allele had a single difference to balbIGHV009 (balbIGHV009_G32A) and was named balbIGHV038.A truncated version of balbIGHV038 appears as a BALB/c IGHV sequence in the VBASE2 database.The fourth inferred allele was present in the IMGT database but had not been included in ComEx.It was found as IGHV1-12*01_C5T_T6G_T7C_T9A_A12G_T19C_G41A_A139G_G216A_C232T_T239C_A291G_T3 08A and corresponds to IGHV1S121*01. When the absence of musIGHV398-utilizing sequences was investigated, a variant with a 7nucleotide deletion was suspected.It was named balbIGHV039.Investigation of musIGHV269utilizing sequences highlighted the likely association of this reported IGHV gene with additional 5' and 3' nucleotides.A possible allelic variant was also seen.These two sequences were assigned the names b6IGHV040 and balbIGHV041.Re-analysis of the VDJ-C dataset with TIgGER, using an expanded ComEx dataset, identified the presence of balbIGHV039, b6IGHV040 and balbIGHV041. The six non-IMGT sequences are reported in Table I.The seven inferred sequences were added to ComEx, and balbIGHV027 was removed, before a final genotyping and haplotyping analysis was performed. ",
"section_name": "Novel alleles identified in F1 dataset",
"section_num": null
},
{
"section_content": "IgM haplotyping began with manual exploration of the IgM constant region exons based on IGHJ1 allele usage.There were 29,593 reads using IGHJ1; 14,071 *01 and 15,522 *03.Alignment of the top IGHJ1*01-and IGHJ1*03-associated Ighm sequences to the BALB/c and C57BL/6 genomes confirmed 100% identical matches to the BALBc Ighm exons for the *01associated sequence and 100% identity to the C57BL/6 Ighm exons for the *03-associated sequence.A single SNP, rs29176517 (http://www.informatics.jax.org/snp/rs29176517), was identified in the CH1 exon that differentiates IgM from the two strains (see Table II).Given the propensity of homopolymer tract errors in PacBio sequencing data from RSII and Sequel I platforms, constant region sequences were indel corrected and the rs29176517 SNP extracted.IGHC calls were assigned to each VDJ-C read using the SNP calls: IgM_B6 (rs29176517a), IgM_BALB (rs29176517g) or 'uncalled'. Among the IGHJ1 subset, 85.77% of VDJ-Cs carrying rs29176517g were associated with the BALB/c IGHJ1*01 gene, while 85.98% of VDJ-Cs with rs29176517a were associated with the C57BL/6 IGHJ1*03 gene.This suggests some PCR-based chimerism within the datasets.Overall, there were 58,762 VDJ-Cs matched to the IgM_BALB and 55,445 VDJ-Cs with the IgM_B6. The C57BL/6 CH1 sequence is identical to an allele that was recently added to the IMGT Reference Directory as IGHM*04.This sequence maps to chr12: 113386037 -113386351 of the mouse GRCm39/mm39 genome reference sequence.In contrast, the IGHM*01 allele, which is annotated in the IMGT Reference Directory as a C57BL/6 allele, was not observed.The BALB/c CH1 sequence aligns perfectly to chr12: 111920727 -111921041 of the 'house mouse 09 May 2016/BALB_cJ_v1' sequence of the Mouse Genome Project (MGP).This sequence is not in the IMGT Reference Directory and is shown in Table II.All other amplified IGHM exons were shared between the two strains, and match sequences in the IMGT Reference Directory. ",
"section_name": "Haplotyping identifies a SNP that distinguishes the C57BL/6 and BALB/c Ighm CH1 exons",
"section_num": null
},
{
"section_content": "The IGHJ1 manual haplotyping approach was applied to the IgG data.The strain specific IgG2a and IgG2c regions were identified along with two IgG2b sequences.Four SNPs were found to distinguish the Ighg2b genes between the strains; rs45969375 in the CH1 and rs49934817, rs45822066 and rs46899601 in the CH2.SNP genotyping therefore allowed IgG2b alleles to be assigned to either BALB/c or C57BL/6.The C57BL/6 IGHG2B*rs45969375t CH1 exon sequence has 100% identity to a new IMGT allele, IGHG2B*03, and to chr12: 113271264 -113271554 of the mouse GRCm39/mm39 genome reference sequence.The C57BL/6 hinge, CH2 and partial CH3 sequences that were identified here also match IGHG2B*03.The BALB/c IGHG2B* rs45969375c sequence has 100% identity to chr12: 111806470-111806760 of the 'house mouse 09 May 2016/BALB_cJ_v1' reference sequence and is shown in Table I.It did not match any of the IMGT alleles.The BALB/c hinge, CH2 and partial CH3 sequences are also matches to the IMGT IGHG2B*02 rather than IGHG2B*01 which is annotated as being BALB/c-derived.BALB/c Ighg2a sequences matched perfectly to the IMGT sequence IGHG2A*01 (data not shown).C57BL/6 Ighg2c when compared to the IMGT reference directory matched the IGHG2C*01 sequence.The full length of the CH3-CHS exon was not captured by the amplicons and a search of GRCm39 suggests that C57BL/6 CH3-CHS exon matches the IGHG2C*03 sequence (data not shown).The IgG primer was not optimized for Ighg1 and Ighg3 associated sequences.Consequently, there was insufficient capture to allow investigation of those genes. ",
"section_name": "Haplotyping identifies SNPs that distinguish C57BL/6 and BALB/c Ighg2b genes",
"section_num": null
},
{
"section_content": "TIgGER identified 278 IGHV germline alleles in the F1 genotype.109 known C57BL/6 sequences (IMGT Functionality: 94F, 10P and 5 ORF) were present in the F1 genotype.The pseudogenes were seen as low frequency non-functional rearrangements.We initially confirmed the presence of the musIGHV269 sequence in the C57BL/6 haplotype, and then determined that this sequence is identical to the pseudogene IGHV1-2*01 as it appears in the IMGT Gene Table.This relationship was not identified earlier as IGHV1-2*01 does not appear in the IMGT Reference Set.Closer inspection of the musIGHV269 alignments revealed that both the VBASE2 and IMGT sequences are incomplete.The complete sequence was designated balbIGHV040 (see Table I).The full-length sequence is readily identified in the C57BL/6 genome reference sequence.It can also be seen in the IMGT annotations of the genome reference sequence AC073561.Rare reads that represent rearranged sequences of this IGHV allele are also seen in another dataset (ENA: ERR1759753) generated from transcripts derived from C57BL/6 mice (data not shown). Only two sequences that were previously identified in the C57BL/6 strain were not seen here (IGHV1-74*04 and IGHV8-6*01).These sequences have previously been reported in VDJ repertoires at frequencies of less than 0.1% (19). Twenty-seven sequences that were identified here, including 14 pseudogenes, were not seen in the previous analysis of the parental strains (19).All but six of them were seen at very low frequencies (<0.01%-0.03%).This low level of expression could explain their absence from the previous analysis that was undertaken at lower sequencing depth (19).Three of the more abundant sequences were newly inferred alleles that are not present in the IMGT database (see Table I). Nine sequences that IMGT reports as functional C57BL/6 sequences were not seen in either this or previous studies (19) Of the 278 identified IGHV, 169 identified sequences are not present in the C57BL/6-derived GRCm39/mm39 genome reference sequence and are of likely BALB/c origin.This included 17 sequences that were not previously seen (19), and were mostly identified here at very low frequencies (<0.03%).Six sequences that we formerly associated with the strain were not identified.Five of them (balbIGHV016, balbIGHV031, balbIGHV033, IGHV1S75*01 and IGHV1S136*01) were previously seen at frequencies between 0.01% and 0.1% (19).The sixth sequence, musIGHV398, was previously seen at a frequency of 1.56%.Although the musIGHV398 sequence was not seen in the present study, a single full-length, perfect alignment was seen to a sequence that appears to be a pseudogene variant of musIGHV398, with a sevennucleotide deletion in the FR1 region.This sequence was designated balbIGHV039 (see Table I). The terminal nucleotides of each identified IGHV sequence were subjected to further analysis, as exonuclease processing of the gene ends in VDJ rearrangements can make it difficult to infer the final nucleotides of an IGHV sequence with certainty.Analysis of the frequency distribution of terminal nucleotides at the 3' ends of each identified BALB/c sequence supported previously reported gene ends in all but a handful of cases.The penultimate nucleotide of the extension of IGHV1S82*01 was shown to be in error, being determined to be G, rather than T. The final nucleotides of IGHV10-1*02 were also shown to be in error, with analysis confirming that the sequence ending is GAGACA, rather than GAGCGA, and is identical to the ending of the IGHV10-1*01 allele.balbIGHV015, musIGHV672, J558-27 and J558-44 may be truncated by 1 or 2 nucleotides, however these sequences are sufficiently distinct from other BALB/c IGHV genes that there was no trouble identifying them in VDJ rearrangements.Finally, the 3' terminal nucleotides of IGHV13-2*02 (beyond base 320) could not be confirmed, and it is likely that the sequence as described by IMGT is two nucleotides too long. Haplotype analysis was then conducted using RAbHIT (23), to provide further evidence in support of the strain-specific origins of each gene.To limit the possibility of errors, haplotype analysis was limited to unmutated IgM data.A total of 7,876 VDJ-C sequences were suitable for haplotyping: 3,616 sequences associated with the C57BL/6 IGHM and 4,260 sequences associated with the BALB/c IGHM.Two-hundred and sixty IGHV were successfully assigned to one or both strains, and a representative haplotype plot for genes of the IGHV8 subgroup is shown as Figure 1.Eighteen IGHV that were present in the genotype could not be haplotyped.These sequences are listed in Supplementary Table IV.In most cases the sequences could not be haplotyped because they were of low frequency and too few reads met the additional haplotype filtering criteria.Seven of these IGHV sequences can be found in the C57BL/6derived GRCm39/mm39 genome reference sequence, and their identities as rearrangeable C57BL/6 IGHV cannot be doubted.The 11 non-C57BL/6 IGHV are likely BALB/c sequences, but this cannot be confirmed.Seven of the 11 sequences are present in the IMGT reference directory where they are assigned to either the BALB/c strain or the 129 group, and one is the pseudogene variant of musIGHV398 that appears in the BALB/cByJ strain. Only four sequences (IGHV2-3*01, IGHV2-5*01, IGHV5-2*01 and IGHV5-6*01) were seen in both strains.IGHV1-69*01 was previously reported in both strains (19), but was only confirmed here in C57BL/6 mice.Instead, IGHV1-69*02 was inferred to be present in the BALB/c-derived haplotype. The strength of the haplotype confirmation process is illustrated by the very low percentage of VDJ-C sequences that apparently included IGHV genes from one strain with IGHM genes from the other, after appropriate filtering to remove sequences for which the IgM allele identification was ambiguous or uncertain.Only 66 (0.84%) of the 7,876 IgM VDJ sequences in the haplotype analysis appear to include chimerism or other problems leading to such haplotyping errors. ",
"section_name": "Genotype and Haplotype inferences using long-read sequencing largely confirm the reported C57BL/6 and BALB/c IGHV genotypes",
"section_num": null
},
{
"section_content": "The utilization frequencies of the four gene sequences that were confirmed in both strains varied substantially between the parental strains (19), and were different again in the F1 animals.For example, it was previously reported that the IGHV5-6*01 gene was utilized by 0.38% of all C57BL/6 VDJ sequences and the identical IGHV5-6-1*01 gene by 0.27% of BALB/c VDJ sequences.In this study, the sequence was seen in 1.08% of all VDJ rearrangements, including 1.24% of the C57BL/6 chromosomal rearrangements and 0.94% of VDJ of the BALB/c chromosomal rearrangements.Similar increases as well as decreases in utilization were seen for many other sequences.For example, IGHV1S113*01 was previously seen in only 0.01% of BALB/c rearrangements (19), but in F1 animals it was seen in 0.99% of rearrangements of the BALB/c chromosome.In the parental C57BL/6 strain, IGHV1-18*01 was seen in just 0.01% of rearrangements but it was seen in 1.34% of rearrangements of the C57BL/6 chromosome in F1 mice.In contrast, IGHV1-59*01 was seen in 3.40% of all C57BL/6 rearrangements (19), but in the F1 animals it was seen in just 0.41% of rearrangements of the C57BL/6 chromosome.Expression frequencies for each gene in the parental strains and in F1 animals are shown as Figure 2. ",
"section_name": "Utilization of IGHV genes varies between F1 and parental strains",
"section_num": null
},
{
"section_content": "To provide a measure of the errors that can arise from the use of an IGHV Reference Set that poorly represents the IGHV genotype of a mouse strain, we analyzed a dataset of 772,837 BALB/cJ VDJ reads derived from the IgM-encoding transcriptome, documenting the apparent levels of somatic point mutation in each sequence, by counting nucleotide mismatches in the sequence alignments (see Figure 3).Although 65.9% of sequences were shown to be unmutated, in analysis using the OGRDB BALB/c Reference Set, only 37.8% of sequences appeared to be unmutated in analysis using the IMGT Reference Set.Differences were conspicuous for sequences with 2, 6, 8, 9, 18 and 19 mismatches.A conspicuous group of sequences was also seen in analysis using the OGRDB Reference Set, with 15 mismatches to the germline alignments.Investigation of these sequences showed them to be almost entirely alignments to the balbIGHV005 sequence.Further investigation showed these sequences to align perfectly to musIGHV398, and the inclusion of this sequence in the Reference Set resulted in 72.1% of sequences being perfect alignments.musIGHV398 was subsequently identified in the MGP BALB/cJ_V1 genome assembly. The musIGHV398 sequence and its variant (balbIGHV039), as well as the other 162 IGHV sequences that were confirmed as BALB/c genes by haplotype analysis, were used to establish a BALB/c IGHV sequence Reference Set at the OGRDB website (https://ogrdb.airrcommunity.org/) (24).This set can also be found in the Supplementary Material as the file BALB.xlsx.In addition to their previously-assigned names, the sequences in the BALB/c Reference Set have been given new labels, using the OGRDB Temporary Nomenclature (manuscript in preparation).This Schema recognizes the impossibility of presently determining 'relationships by descent' between the genes of the C57BL/6 strain which were used to develop the IMGT nomenclature, and the genes of the BALB/c strain.It is intended that these labels will facilitate the reporting of BALB/c IG sequences until a new community-supported nomenclature can be developed for use with inbred mouse strains. ",
"section_name": "Importance of valid databases for annotation and analysis of hypermutation status",
"section_num": null
},
{
"section_content": "To investigate the potential to define the IGH loci of inbred mouse strains by annotation of shortread genomic assemblies, we conducted BLAST searches (31) of the Mouse Genome Project (MGP) BALB/cJ_V1 genome assembly (36) for each of the IGHV genes in the OGRDB BALB/c Reference Set.Of the 162 BALB/c sequences, only 70 are present in the MGP assembly.Missing sequences were by no means confined to those sequences that were newly identified in our earlier study.85 of the sequences are present in the IMGT Reference Directory, but only 37 (43.5%) of these sequences can be found in the MGP assembly.Of the 77 sequences that are not present in the IMGT Reference Directory, only 33 (42.9%) of the sequences are present in the MGP assembly, including 5 of 12 sequences originally reported by Haines and colleagues (35) and 16 of 35 sequences that can be found in the VBASE2 database (37). ",
"section_name": "Current genomic sequencing does not reliably define IGHV loci content",
"section_num": null
},
{
"section_content": "Despite the central role of inbred mice in biomedical research, and their historical importance in immunoglobulin gene studies, the germline IGHV genes of inbred mouse strains remain poorly understood.For the IGHV locus, only the genes of the C57BL/6 strain have been fully documented, and this presently limits the use of antibody repertoire analysis in mouse studies. The Mouse Genome Project has produced genome assemblies of many mouse strains, and analysis of these assemblies might seem like a sensible way to extend the documentation of mouse IGHV genes.Genomic analyses of this kind recently led the IMGT Group to report and name sequences identified in the IGH loci of species including the horse, rat and Rhesus macaque (http://www.imgt.org/IMGTinformation/creations/).There are strong grounds for believing that NGS assemblies of complex genomic regions like the immunoglobulin loci are unlikely to be sufficiently accurate for reliable and comprehensive gene discovery (38,39).The results of the analysis presented here supports this view.The majority of BALB/c IGHV genes that were identified in this study are not present in the MGP BALB/cJ genome assembly.This is true even for those IGHV sequences that are present within the IMGT Reference Directory, and that are annotated by IMGT as being either BALB/c genes or genes of the highly similar 129S1 strain.The accuracy of NGS assemblies is improving over time, and may well reach the point at which they can be used for receptor gene discovery, however, these results emphasize that their accuracy cannot be assumed without validation against established results and methods. Germline immunoglobulin gene discovery by inference from AIRR-seq data is an alternative pathway that is well established for the human (23,40,41), and confidence in these inferences is often strengthened by haplotype analysis (41,42).As VDJ rearrangement is a chromosomal event, the association of different IGHV alleles with different human IGHJ6 alleles in IGHJ6heterozygous individuals allows gene data to be phased.This can lead to the identification of sequences that may appear to be novel alleles, but which arise because of sequence chimerism.Such sequences can easily arise during the PCR amplification of any genes belonging to large sets of highly similar sequences, and it is a problem that has been recognized in studies of antibody genes for decades (27). It is possible to monitor the extent of the problem within AIRR-seq datasets by careful analysis of gene rearrangements at heterozygous loci.Unfortunately, such loci are not present in inbred mice.An alternative approach identifies chimeric sequences by abnormal clustering of mutations.This approach can also be problematic in the mouse, for it depends upon a thorough knowledge of the germline IGHV gene repertoire of the species.Such analysis is also compromised where multiple genes and their allelic variants are identical or highly similar to one another.This appears to often be the case in inbred mice (22). We have previously identified IGHV genes in AIRR-seq data generated from inbred BALB/c mice (19,22).Many of the IGHV genes appeared to rearrange at very low frequencies (0.01% -0.05%), making their unequivocal identification by inference difficult.It is possible that these inferred IGHV are not real, but rather are chimeric gene products generated by PCR amplifications.In this study, we therefore analyzed VDJ rearrangements from F1 hybrid animals, so that the generation of strain-specific haplotypes could give sufficient confidence in the inferences for the compilation of a BALB/c IGHV Reference Set. Previous exploration of the C57BL/6 and BALB/c germline repertoire was performed using manual analysis (19), but in recent years, suitable genotyping and haplotyping inference tools have been developed for this purpose (21,23,25).Allelic variation is recognized at the mouse IGHJ1 gene.Variation at this locus might be used for IGHV gene haplotype analysis, but the relatively low utilization frequency of the IGHJ1 gene makes it poorly suited to direct IGHJ vs IGHV haplotyping.Genetic variation is also seen within the constant region genes of inbred mice (43).When expressed as VDJ-C genes, however, these constant region (C) genes are too remote from the VDJ sequences to allow haplotype analysis using standard AIRR-Seq sequencing methods (typically based on Illumina's MiSeq technology).In this study, we therefore used high-throughput single molecule, real time (SMRT) sequencing.Long-read sequencing allowed us to explore allelic variation in genes spanning the entire length of the heavy chain locus, and to perform IGHV gene haplotyping using constant region genes.Against expectations, we identified allelic variation in the CH1 region of the IGHM gene.We also determined that the BALB/c strain carries a previously unreported variant of the Ighg2b gene. The documentation that the BALB/c strain carries an Ighg2b constant region gene that differs from that of the C57BL/6 strain will provide researchers with new opportunities for analysis of long-read AIRR-Seq data.In this study, however, we chose to use the heterozygous IGHM locus as the anchor point for haplotype analysis.The general lack of somatic point mutations in the IgM-associated VDJ sequences made the dataset ideal for the documentation of a BALB/c Reference Set by inference. The results of the haplotype analysis largely confirmed the results of the study of Collins and colleagues (19), though some rarely utilised genes were seen in one study but not the other.This is to be expected, given the sequencing depth of the two studies.Five new BALB/c IGHV genes were identified, and are reported here as balbIGHV036, balbIGHV037, balbIGHV038, balbIGHV039 and balbIGHV041.The agreement between the studies, despite the substantial differences in methodology, demonstrates the reliability of the inference process. The strain-specific IGHV gene sets defined in this study are comprehensive, but they are still likely to lack some germline genes that are poorly expressed or unexpressed.Some evidence was seen in support of additional sequences -e.g.Genbank IDs: KY199076, KY199111, KY199136 and KY199153 reported from BALB/c studies by Corcoran et al (20) -but the evidence was judged to be insufficient to confirm the existence of the genes in this study.Other genes that have apparently been utilized at low frequencies in BALB/c repertoires (19) were not seen here, and have therefore not been included in the OGRDB Reference Sets.Many of these genes may be of questionable functionality, mitigating the impact of their absence from the Reference Set.The IMGT definition of functionality is based upon the presence or absence of key regulatory elements.It therefore may fail to identify the lack of function of genes that encode aberrant nonfunctional polypeptides.Many poorly expressed or unexpressed genes such as IGHV6-7*01, IGHV1-74*04 and IGHV8-6*01 carry unusual codons at critical positions within the sequences.In the human, such unusual codons have been associated with a lack of expression of reportedly functional IGHV genes (44).Some genes may not have been detected because of differences between the utilization frequencies in the F1 animals, and frequencies previously reported in the parental strains.The expression of antibody genes is relatively predictable.This was first demonstrated in human twin studies (45,46), and it has also been noted in inbred mice (11).Differences could arise, however, as a result of the genomic context in which the F1 repertoire forms.The naïve antibody repertoire is affected by the processes of negative selection that act to restrict the survival or functionality of self-reactive B cells (47).In F1 animals, the C57BL/6 IGHV gene set and the BALB/c IGHV gene set are each subject to negative selection resulting from both C57BL/6 and BALB/c derived self-antigens.The larger light chain repertoire of F1 animals could also allow those IGHV genes with a tendency to self-reactivity to form more self-tolerant heavy and light chain pairings, thereby increasing the expression of those genes.Some changes in expression frequencies that we observed could also be a consequence of difference in the methodologies of the two studies. Substrain differences could also explain differences between expression levels reported here for genes such IGHV1S113*01 in the BALB/c mouse and IGHV1-18*01 in the C57BL/6 mouse.The most striking example of a previously-reported IGHV gene that was not seen in this study is the gene musIGHV398.Although it was not called in the F1 genotype, investigation of the absence of musIGHV398-utilizing VDJ in the F1 repertoire led to the identification of a small number of sequences that appeared to utilize a variant of the gene.This variant (balbIGHV039) was subsequently confirmed by genotype analysis.It was also identified in a BAC clone prepared from the BALB/cByJ genome (Kos and Watson: unpublished data).musIGHV398 is present in the MGP BALB/cJ genome assembly and was also seen here in our analysis of BALB/cJ VDJ datasets.It therefore seems that this highly utilized gene defines a critical genetic difference between the BALB/cJ and BALB/cByJ substrains.Both musIGHV398 and the pseudogene allelic variant balbIGHV039 have been included in the BALB/c Reference Set that is available at the OGRDB website (https://ogrdb.airrcommunity.org/)(24).Reference Sets for wild-derived strains are also now available at the OGRDB website.These, and the corresponding C57BL/6 Reference Set, will enable better annotation of AIRR-seq studies in comparison to reference germline gene sets that do not take strain differences into account and in some cases even fail to incorporate some highly expressed genes and allelic variants that contribute substantially to the IGHV repertoire. The BALB/c set that is defined here includes many IGHV genes that are present as truncated sequences in the IMGT reference directory and a large number of genes that have not been named by IMGT or the responsible IUIS Nomenclature Committee.All the BALB/c sequences have been assigned temporary OGRDB labels that will serve adequately until issues relating to the official nomenclature of the mouse can be resolved (48).This resolution will not be possible until careful genomic sequencing allows a proper comparison of the BALB/c and C57BL/6 IGH loci.It will then be possible to determine whether or not there is sufficient correspondence between the two loci for the IMGT C57BL/6-based nomenclature to be applied to the BALB/c strain.The resolution of the assignment of alleles to particular genes is important, for it impacts how some tools handle annotation and analysis.Hopefully, genomic validation of the inference process will also end any uncertainties that might still surround 'discovery by inference', and will lead to the exploration of the immunoglobulin genotypes of the many important mouse strains for which high quality genome assemblies are currently unavailable. MO was supported by a grant from the Swedish Research Council (grant number 2019-01042).GY and AP were supported by a grant from the Israel Science Foundation (grant number 2940/21).MC was funded by the Swedish Research Council, grant No. 532 2017-00968.WL and GY were also supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 825821.The contents of this document are the responsibility of the authors and can under no circumstances be regarded as reflecting the position of the European Union. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"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. The study was designed by KJ, CW and AC; library construction and sequencing was performed by CW, JK, WG and ML; data analysis was performed by KJ, MO, WL, CW and AC; manuscript was prepared and edited by KJ, MO, CW, CB, MC, AP, GY and AC. ",
"section_name": "Conflict of Interest",
"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": "The study was designed by KJ, CW and AC; library construction and sequencing was performed by CW, JK, WG and ML; data analysis was performed by KJ, MO, WL, CW and AC; manuscript was prepared and edited by KJ, MO, CW, CB, MC, AP, GY and AC. ",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.1186/1742-6405-11-38
|
Expression of activating receptors on natural killer cells from AIDS-related lymphoma patients
|
Abnormal NK phenotype and cytotoxic functions have been described in acute myeloid leukemia, chronic lymphocytic leukemia, myeloma and myelodysplastic syndromes. Defective NK cytotoxicity is due to decreased expression of the Natural Cytotoxicity Receptors (NCRs), 2B4/CD244/p38, or NKG2D. This prompted us to test the expression of these molecules on circulating NK cells from patients with AIDS-related lymphomas (RL) in comparison with HIV + patients without lymphoma, healthy subjects and HIV-negative patients with lymphoma.Blood samples were analyzed by flow cytometry for NCRs, 2B4/CD244/p38 and NKG2D expression on NK cells defined as CD3-/CD56+ lymphocytes. We also analyzed by quantitative PCR specific RNA for NKp30/NCR3 and NKp46/NCR1.We could not detect any defect in NKp46/NCR1 expression between all groups. NKp44/NCR2, NKp30/NCR3 and NKG2D had lower expression in AIDS-RL in comparison with HIV + patients without lymphoma when compared to patients with similar (>0.3 G/L) CD4+ lymphocyte levels. Expression of 2B4/CD244/p38 was lower in AIDS-RL than in HIV-negative lymphoma. Comparison of specific NKp30/NCR3 and NKp46/NCR1 RNA showed increased steady state levels, despite decreased surface expression for NKp30/NCR3, suggesting abnormal post-transcriptional regulatory mechanisms.We show a more pronounced defect in NK activating molecule when HIV infection is associated with lymphoma than when only one condition (HIV positivity or lymphoma) is present. Defective NK phenotype, in addition to CD4+ depletion and dysfunction, may participate to the increased incidence of lymphoma in HIV patients.
|
[
{
"section_content": "Advances in lymphoma treatment prolong progressionfree survival.Nonetheless, many patients relapse.Deficient cytotoxic functions of natural killer (NK) cells [1], which can be infected by HIV [2], may participate in the failure to cure AIDS-related lymphomas (AIDS-RL).Engagement of inhibitory receptors by human leukocyte antigen (HLA)-class-I molecules inhibits NK cytotoxicity.Thus, according to the \"missing self hypothesis\", absent or deficient expression of HLA-class-I molecule activates NK if an additional activating signal is delivered by the natural cytotoxicity receptors (NCR) NKp30/ NCR3, NKp44/NCR2 or NKp46/NCR1, 2B4/CD244/p38 and NKG2D.Deficient NK functions interfere with the anti-tumor response: 1) during treatment, via decreased efficiency of anti-CD20 antibody-driven cell cytotoxicity (ADCC) [3][4][5] 2) during the complete remission phase by favoring residual HLA-class-I negative lymphoma cells to escape from NK-mediated immunity [6].Abnormal NK functions have been described in hematological malignancies such as acute myeloid leukemia, chronic lymphocytic leukemia, myeloma and myelodysplastic syndromes [7][8][9][10].Of note, down-regulation of NCRs is associated with HIV infection [11].We compared the NK cell surface activating molecules expression between patients with AIDS-RL, HIV-positive patients without lymphoma, lymphoma patients not infected by the HIV, and healthy subjects. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Among the 31 AIDS-RL (mean age: 43 ± 8 years) of the study, 20 had CD4+ lymphocytes <300/mm 3 (mean 133 ± 70/mm 3 ), 11 had CD4+ lymphocytes >300/mm 3 (mean 630 ± 260/mm 3 ).Only 2 patients (6.4%) were not treated by highly active antiretroviral therapy (HAART) at study inclusion.The control cohort included 56 HIV-positive patients without lymphoma (mean age: 44 ± 9 years) selected to be matched for the CD4+ lymphocyte count with the AIDS-RL: among this population, 9 patients [16%] were not treated by HAART.Two groups were designed: HIV patients with <300 CD4+ lymphocytes/mm 3 (n = 12; mean age 44 ± 8.4 years, 1 patient without HAART) and HIV patients with >300 CD4+ lymphocytes/mm 3 (n = 44; mean age 44 ± 9.8 years patients without HAART).Two other control cohorts of 33 HIV-negative lymphoma patients (mean age: 62 ± 14 years) and 19 healthy subjects (HS, mean age: 41 ± 16 years) were included. ",
"section_name": "Population characteristics",
"section_num": null
},
{
"section_content": "There was no significant difference in total lymphocytes and T-lymphocytes count among the different groups (p > 0.05).Lymphoma groups had more B lymphocytes (1700 ± 7000/mm 3 ) than HIV (200 ± 200/mm 3 ) and HS (300 ± 100/mm 3 ) because of lymphoma circulating cells.Total NK cells counts in AIDS-RL with <300 CD4+ lymphocytes/mm 3 and HIV + patients without lymphoma but with <300 CD4+ lymphocytes/mm 3 were lower than in the other groups (mean 41 ± 35/mm 3 for AIDS-RL <300 CD4+ lymphocytes/mm 3 and 51 ± 52/mm 3 for HIV + without lymphoma with <300 CD4+ lymphocytes/mm 3 vs 137 ± 257/mm 3 for AIDS-RL with >300 CD4+ lymphocytes/mm 3 , 131 ± 148/mm 3 for HIV + with >300 CD4+ lymphocytes/mm 3 , 276 ± 510/mm 3 for HIV-negative lymphoma patients and 150 ± 70/mm 3 for HS (p = 0.04 and 0.02).No difference in NKp46/NCR1 expression was observed (Figure 1, panel B, p > 0.05, NS).The analysis of NKp44/ NCR2 expression, an activating receptor only expressed on activated NKs (Figure 1, panel C) showed no difference between AIDS-RL and HIV-negative lymphoma patients (p > 0.05, NS) and between AIDS-RL and HIV patients without lymphoma but with <300 CD4/mm 3 patients (p > 0.05, NS).However, AIDS-RL with >300 CD4+ lymphocytes/mm 3 expressed lower NKp44/NCR2 than HIV + patients without lymphoma with >300 CD4+ lymphocytes/mm 3 (MFIr median (M) = 1.52; interquartile range (IQR)[1.24-1.850]vs M = 1.80 IQR[1.540-2.110],p = 0.011) and than HS (M = 1.75 IQR[1.650-2.000],p = 0.006).In spite of a very high NKp44/NCR2 expression by one HIV + lymphoma with <300 CD4+ lymphocytes/mm 3 (MFIr =6.21), this group tended to express lower NKp44/NCR2 than HIV + patients with >300 CD4+ lymphocytes/mm 3 (M = 1.42 IQR[1.320-1.765]vs 1.80[1.543-2.115],p = 0.06).HIV-negative lymphoma patients expressed more NKp44/NCR2 than HIV + patients without lymphoma and >300 CD4+ lymphocytes/ mm 3 (M = 1.48 IQR[1.330-1.690]vs 1.80[1.543-2.115],p = 0.02) or HS (M = 1.75[1.650-2.000],p = 0.002).There was no difference in NKp30/NCR3 expression (Figure 1, panel D) between AIDS-RL and HIV-negative lymphoma patients (p > 0.05, NS) or between AIDS-RL and HIV + patients without lymphoma and <300 CD4+ lymphocytes/mm 3 (p > 0.05, NS).NKp30/NCR3 expression was lower in AIDS-RL with >300 CD4+ lymphocytes/mm 3 than in HIV + patients without lymphoma and >300 CD4+ lymphocytes/mm 3 Regarding NKG2D (Figure 1, panel E), AIDS-RL patients with <300 CD4+ lymphocytes/mm 3 had a lower expression of NKG2D than AIDS-RL patients with >300 CD4+ lymphocytes/mm 3 We failed to detect 2B4/CD244/P38 expression difference (Figure 1, panel F) between AIDS-RL and HIV + patients without lymphoma (NS, p > 0.05).2B4/CD244/P38 expression was lower in AIDS-RL with <300 CD4+ lymphocytes/ mm 3 Quantitative RT-PCR of NKp30/NCR3 and NKp46/NCR1 (Figure 2) ",
"section_name": "Lymphoid cells repartition (Figure 1-Panel A)",
"section_num": null
},
{
"section_content": "In line with cell surface expression, AIDS-RL had no modification of NKp46/NCR1 specific RNA level (0.5 < normalized ratio <2 for the 6 of the 7 patients analyzed) compared with HIV + population without lymphoma (Panel A).We did not find any correlation between NKp46/NCR1 expression and CD4+ lymphocyte count.On the contrary, NKp30/NCR3 RNA (Panel B) was overexpressed in AIDS-RL patients (normalized ratio >2 for the 7 patients analyzed).No significant RNA level was detected for NKp44/NCR2, a results in line with normal physiology since this molecule is only expressed by stimulated NKs (data not shown). ",
"section_name": "NK activating receptors expression (",
"section_num": null
},
{
"section_content": "The total NK cells count in patients with <300 CD4+ lymphocytes/mm 3 was lower in AIDS-RL in comparison with the other groups, suggesting a poor prognosis as shown in low or high grade HIV-negative lymphomas [12,13].Low circulating counts concerned the NK CD56 bright and CD56 low subsets, while the ineffective CD56 negative subpopulation was elevated in AIDS-RL (data not shown).Regarding the NCR we found no difference in NKp46/NCR1 expression in the different groups but, in contrast, a significant decrease in both NKp44/NCR2 and NKp30/NCR3 was observed in AIDS-RL with >300 CD4/mm 3 in comparison with HIV + patients with comparable CD4+ lymphocytes.Thus in moderately immune-suppressed patients the development of lymphoma is associated with low expression of two activating molecules.Regarding NKp44/NCR2, low levels should be of good prognosis since NK cells in HIV-controller patients do not up-regulate NKp44/ NCR2 thus protecting uninfected CD4+ lymphocytes from inadequate NK killing [14].Regarding the mechanism of NCR regulation, quantitative RT-PCR measured comparable NKp46/NCR1 levels in AIDS-RL and controls, suggesting an identical regulation at both transcriptional and post-transcriptional levels.However elevated level of NKp30/NCR3 specific RNA was detected in AIDS-RL in comparison with HIV patients without lymphoma, despite identical surface expression of NKp30/ NCR3.This suggests that a post-transcriptional mechanism negatively interferes with NKp30/NCR3 RNA traduction or protein stability, leading to identical surface expression despite higher specific RNA levels.Regarding NKG2D, we observed a gradient of expression from lower level (AIDS-RL <300 CD4/mm 3 ) to HS/HIV-negative lymphoma patients, with intermediary levels for AIDS-RL with >0.3 G/L CD4+ lymphocytes followed by HIV + patients without lymphoma.The NKG2D ligands MICA/ MICB/ULBP are stress molecules expressed on tumor cells, and secreted at high levels in HIV patients, leading to down-regulated NKG2D expression on NK and impaired anti-lymphoma cytotoxicity [15].Expression of 2B4/CD244/p38 was lower in AIDS-RL than in HIVnegative lymphoma patients.The 2B4/CD244/p38 ligand is the CD48 molecule [16,17] which is expressed on B normal and neoplastic lymphocyte and is drastically up-regulated by Ebstein-Barr virus (EBV) infection.The (See figure on previous page.)Figure 1 Flow cytometry analysis of whole PBMC population (panel A) and of NK cells (panels B to F). Results are expressed as absolute numbers of cells per volume unit, i.e.Giga/Liter in panel A. Results are expressed as mean fluorescence intensity ratio (in comparison with isotype controls, cf.Material and Methods).When significant, statistical results are indicated with the corresponding p-value.The number of analyzed patients was: AIDS-RL/CD4 < 300/mm3 = 20, AIDS-RL/CD4 > 300/mm3 = 11, HIV + <300 CD4/mm3 = 12, HIV+ > 400 CD4/mm3 = 44, non AIDS-RL lymphoma = 33, control HS = 19. down-regulation of 2B4/CD244/p38 could thus impair the cytotoxicity against EBV-positive B-cell lymphomas. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The AIDS-RL patients had decreased levels of 2 out of 3 NCRs, of NKG2D and of 2B4/CD244/p38.The most significant difference concerned NKG2D, which expression was significantly decreased regarding both HIV patients without lymphoma, non-HIV lymphoma patients and HS.This specific abnormality is of great interest since lymphoma cells express the stress ligands MICA/B and ULBP, but may escape to NK cytotoxicity due to impaired NKG2D expression.Of note, HAART was not sufficient to restore a normal phenotype since most of our patients were already treated at the time of NK phenotype analysis, with NK abnormalities also detected in patient with CD4+ lymphocytes >300/mm 3 .Since defects in NK immune surveillance may also impair the anti-infectious immunity, they could also partly explain the susceptibility to infection of HIV patients during chemotherapy, even in patients with high CD4+ T-lymphocytes levels.Altogether our data suggest than immune intervention aiming at NK cell function restoration could be of interest in AIDS-RL patients. ",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Material and methods",
"section_num": null
},
{
"section_content": "According to previous data [8], we hypothesized that 75% ±10% of AIDS-RL patients and 15% ±10% of HIV patients without lymphoma had low NCR expression (NCR dull ).In order to show a statistically significant difference between the 2 groups with a risk α = 5% and β = 75%, we included in our study 31 AIDS-RL patients and 56 HIV positive patients without lymphoma. ",
"section_name": "Study design",
"section_num": null
},
{
"section_content": "From July 2006 to June 2011 patients from Marseille, Nice and Paris were included in first line of therapy.Inclusion criteria were the co-existence of HIV infection with biopsy-proven lymphoma.The 56 HIV positive patients without lymphoma were recruited from Service des Maladies Infectieuses (Hôpital Nord, Marseille).According to Helsinki declaration, patients were informed and signed a consent form.Biological samples were collected at diagnosis time, before lymphoma treatment.Additional comparison of our data was also performed with 33 non-HIV patients with lymphoma and 19 healthy subjects (HS). This study was approved by the Comité de Protection des Personnes (CPP) Aix-Marseille II. ",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "Blood samples were collected on EDTA and analyzed by flow cytometry.Dry pellets of PBMC were frozen at -80°C for subsequent quantitative RT-PCR analysis. qRT-PCR analysis qRT-PCR analysis concerned 7 AIDS-RL patients and 32 HIV-positive patients without lymphoma.qRT-PCR analysis was performed with the Applied Biosystems 7900HT Fast Real-Time PCR system using Taqman detection.Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies).Capture of fluorescence was recorded on the ABI Prism 7900HT scanner and the Ct (threshold cycle) was calculated for each assay (Sequence Detection System Software 2.3, Applied Biosystems).We used GAPDH as endogenous control (ΔCt = Ct target gene -Ct GAPDH).GAPDH TaqMan Gene Expression assays were from Applied Biosystems.Since the NCR expression is almost exclusively restricted to NK, the PCR was performed on the whole PBMC population, but the values were adjusted to the percentage of NK present in each sample.We compared ΔCt with the mean of VIH ΔCt using a ratio (ΔCt HIV + Lymphoma/ΔCt HIV), considering that a ratio >2 corresponded to RNA overexpression. ",
"section_name": "Blood samples and cell separation",
"section_num": null
},
{
"section_content": "Flow cytometry was performed on an Epics XL R flow cytometer (Beckman Coulter).The NK cells were defined as CD3-/CD56+/CD16+.The following mAbs were used (Beckman-Coulter, Marseille, France): anti-CD3 FITC (UCHT1), anti-CD56 PC5 (N901-NKH1), anti-NCR1/ NKp46 PE (BAB281), anti-NCR2/ NKp44PE (Z231), anti-NCR3/NKp30 PE (Z25), anti-NKG2D-PE (ON72), anti-P38 (C1.7), anti-IgG1 -FITC , anti-CD19 -PC5 (J4.119),CD4 -PE / CD8 -ECD /CD3 -PC5 , anti-CD3 -FITC /CD16 -PE (UCHT1/3G8).All our results were expressed as the mean fluorescent intensity ratio molecule of interest/isotypic control (MFIr). ",
"section_name": "Phenotypic analysis",
"section_num": null
},
{
"section_content": "Data were compared between the 4 groups using a nonparametric Kruskal-Wallis test; post hoc tests for multiple comparisons were performed when the test was significant (macro Marta Garcia-Granero [07/2008] for SPSS).The statistical analyses were performed using the SPSS software package, version 17.0 (SPSS Inc., Chicago, IL, USA).All tests were two-sided.Statistical significance was defined as p <0.05. ",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "Special thanks to Bernadette Barbarat for kind help for qRT-PCR, and special thanks to Marta Garcia-Granero for additional help in statistical analysis.Special thanks to Jean Gabarre for helpful discussion. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Abbreviations NK: Natural killer; NCR: Natural cytotoxicity receptors; AIDS-RL: AIDS-related lymphoma; HS: Healthy sublects; MFI: Mean fluorescent intensity; PBMC: Peripheral blood mononucleated cells; PCR: Polymerase chain reaction. The authors declare that they have no competing interests. Authors' contributions DMB, CS, TLT, CB, RC performed or contributed to the flow cytometry experiments, DMB performed the qPCR experiments, RC, DMB, CS, TLT, CB, DO, GS, CB, NM, SM, KB, GS participated to the interpretation of data and to the revising of the manuscript, RC, SM, NM contributed to subject recruitment, KB, RC contributed to the conception of the study and performed statistical analysis.All authors have given final approval of the version to be published, and are accountable for all aspects of the work. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations NK: Natural killer; NCR: Natural cytotoxicity receptors; AIDS-RL: AIDS-related lymphoma; HS: Healthy sublects; MFI: Mean fluorescent intensity; PBMC: Peripheral blood mononucleated cells; PCR: Polymerase chain reaction. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests. Authors' contributions DMB, CS, TLT, CB, RC performed or contributed to the flow cytometry experiments, DMB performed the qPCR experiments, RC, DMB, CS, TLT, CB, DO, GS, CB, NM, SM, KB, GS participated to the interpretation of data and to the revising of the manuscript, RC, SM, NM contributed to subject recruitment, KB, RC contributed to the conception of the study and performed statistical analysis.All authors have given final approval of the version to be published, and are accountable for all aspects of the work. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.12669/pjms.36.3.541
|
Prognostic markers in Chronic Lymphocytic Leukaemia - A flow cytometric analysis
|
<jats:p>Objective: To find out the frequency of ZAP-70, CD38 and CD49d in patients diagnosed with CLL in our population.
 Methods: This is a cross sectional study conducted in Army Medical College in collaboration with Armed Forces Institute of Pathology and Military Hospital Rawalpindi from 1st January 2018 to 30th November 2018. Permission from Institutional Ethical Committee was obtained. Blood samples were collected by non-probability consecutive sampling technique and analyzed for blood counts and flow cytometry was done for ZAP-70, CD38 and CD49d. Manufacturer’s instructions for the kits were strictly followed.
 Results: Fifty-one newly diagnosed patients with CLL were studied for the prognostic markers in CLL. CD 38 was expressed in 25(49%) and CD49d in 21(41.2%). ZAP-70 expression was not detected in our series of patients.
 Conclusion: We conclude that CD38 and CD49d expression was detected in almost half of the patients of CLL in our series. CD49d showed statistically positive correlation with CD38, showing that it is a more pragmatic choice for reliable prognostication of CLL along with CD38.
 doi: https://doi.org/10.12669/pjms.36.3.541
 How to cite this:Haq H, Uddin N, Khan SA, Sunia Ghaffar4. Prognostic markers in Chronic Lymphocytic Leukaemia - A flow cytometric analysis. Pak J Med Sci. 2020;36(3):338-343. doi: https://doi.org/10.12669/pjms.36.3.541
 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</jats:p>
|
[
{
"section_content": "Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of small mature looking, ineffectual, CD5+ B-cells in the peripheral blood, bone marrow and secondary lymphoid tissues. 1 These cells have a characteristic immunophenotype i.e.CD19+, CD20+ and CD23+ with relatively low expression of CD22 and CD79b. 2 The peak incidence of CLL is between 60 to 80 years, and only 10% of patients are younger than 55 years of age.In Pakistan it accounts for 20.1% of all leukemias. 3It is more common in the West with an incidence of 4.2:100,000 per year.Most cases are diagnosed during routine complete blood counts done for other reasons. 4iginal Article ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Leukaemia -A flow cytometric analysis Its diagnosis is based upon clinical presentation and laboratory features. 5Peripheral blood counts with absolute lymphocyte count more than 5 x 10 9 /L, which may reach to 300 x 10 9 /L.The peripheral smear shows small mature looking lymphocytes with numerous smudge cells.A bone marrow biopsy, if done, shows predominance of mature lymphocytes, replacing 95% of normal hemopoietic tissue.Immunophenotyping is done to confirm the diagnosis. 6he Rai and Binet staging systems are well recognized systems which serve as standard for assessing treatment requirements and overall survival in the patients diagnosed with CLL.However, both these systems are unable to categorize the patients into groups requiring treatment from those in whom the disease remains indolent. 7The outcome of patients can be predicted by a number of immunophenotypic markers (ZAP-70, CD38 and CD49d), Immunoglobin heavy variable (IGHV) gene mutation and chemical analysis. 8AP-70 and CD38 are well-established immunophenotypic markers indicating poor prognosis in CLL.ZAP-70 is a protein rarely present on normal B-lymphocytes whereas CD38 is expressed on them.Patients who are positive for ZAP-70 and CD38 have a poor prognosis with aggressive disease course and a shorter overall survival.On the other hand those who are negative for ZAP-70 and CD38 have much better results. 7,9D49d is a recently added prognostic marker.It belongs to the integrin family with an important function in leukocyte activation, trafficking and survival. 10Its expression promotes unfavorable progressive disease course and can identify patients with poor disease outcome, independent of CD38 and ZAP-70. 8Studies show that CD49d positive patients have increased risk of death and lower overall survival, independent of ZAP-70 and CD38.Comparing models of these three prognostic markers, with and without CD49d by several prediction performance measures indicated that excluding CD49d significantly reduced the prognostic power of the model. 11nother study compared the combined expression of CD38 and CD49d.They showed that all those patients who require treatment had strong double expression of both.On the contrary, those with double negative expression had good prognosis and long treatment free survival. 12Patients with > 30% of CLL B-cell expressing CD49d are labeled positive. 11Till to date no study has been done in Pakistan to study presence and frequency of CD49d in Pakistani patients with CLL. This study helps us to evaluate the prognosis of CLL at the time of diagnosis in our population.ZAP-70, CD38 and CD49d are prognostic markers of CLL.They help in segregating those patients of CLL which will need treatment from those that can be placed in the \"Wait and watch\" group.Globally, studies show that CD49d is gaining acceptance as an independent prognostic marker and may replace the combination of ZAP-70 and CD38.Foregoing in view, we planned a study to find out the frequency of ZAP-70, CD38 and CD49d in CLL patients our setup. ",
"section_name": "Prognostic markers in Chronic Lymphocytic",
"section_num": null
},
{
"section_content": "This cross sectional study was conducted in Department of Hematology, Army Medical College in collaboration with Armed Forces Institute of Pathology Rawalpindi from 1 st January, 2018 to 31 st October, 2018 after the approval of Institutional Review Board dated on January 24, 2019. Total 51 newly diagnosed cases of CLL were included in our study.Sample size (n=44) was calculated by WHO calculator (Confidence interval at 95% and Anticipated population as 13% with absolute precision required as 5%).Sample collection was done by non-probability purposive sampling. Three (3) ml of venous blood was drawn under aseptic conditions.It was transferred to Ethylenediaminetetraacetic acid (EDTA) tube.Complete blood counts were generated through Sysmex KX-21TM automated hematology analyzer after adequate quality control.Immunophenotyping was performed by flow cytometry to analyze for ZAP-70, CD38 and CD49d by BD FACS caliber and BD FACS CANTO.Known negative samples were used as normal controls.Manufacturer's instructions for the kits used were strictly followed.Statistical analysis: Data was analyzed by statistical package for social sciences (SPSS 23).For qualitative variables frequency and percentages were calculated and quantitative variables Mean and Standard Deviation (SD) were calculated.Correlation between CD38 and CD49d was calculated by applying the Pearson Chi-Square.P-value <0.05 was considered statistically significant. ",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "Our study included a total of 51 newly diagnosed cases of CLL.Out of these, 40 (78%) were male and 11 (21.5%) were females.The mean age of the patients was 65 years± 10.64 (mean ± SD) years with a range of 39 -86 years.The mean hemoglobin, white cell and platelets counts were 11.1 ± 2.7 g/dl (range 3.6 -16.1); 80.8 ± 74.2 x 10 9 /L (range 11.7 -538x10 9 /L) and 169 ± 67 x 10 9 /L(range 08 -383x10 9 /L) respectively.The mean absolute lymphocyte count was 68.2 ± 71.6 x 10 9 / L (range 7.9 -505.7 x 10 9 /L) (Table-I). CD38 expression was detected in 25 (49%) cases with the mean value of 34.4% (range 0-96) while CD49d was expressed in 21 (41.2%)cases with a mean of 35.4 (range 0-99).ZAP-70 expression was not detected in any case (Fig. 1).The mean of positive expression of CD38 was 70.9 ± 18.2 with range of 42%-96% and CD49d was 76.2 ± 16.2 with a range of 42%-99% (Table-II). We calculated the correlation between CD38 and CD49d by applying the Pearson Chi-Square.The p-value was <0.05 showing that the two variables are statistically significant (Fig. 2).We divided our data into two groups i.e.Group-1 (below 60 years of age) and Group-2 (61 and above).In Group-1, 3/13 cases were positive for CD49d and CD38.In Group-2, 18/38 cases were positive for both CD38 and CD49d.No positive association was found between CD markers and age group (p-value >0.05). ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia is the most frequently occurring chronic leukemia in the Western world. 7The progression and response to treatment is variable.Previously, prognosis of patients with CLL was based on the clinical features alone.However with the advent of new techniques, significant progress has been seen like identification of immunophenotypic markers and molecular genetics which help us to predict the progression of the disease. 13,14e studied 51 newly diagnosed patients of CLL.Among these, 25 (49%) were positive for CD38.Our study is consistent with other studies conducted in China, India and Iraq showing almost similar results i.e.6][17] However, our values are slightly higher than those reported by Wiestner A et al. in UK and D'Arena G et al. in Italy which were 30% and 29% respectively. 18,19D49d was positive in 21 (41.2%)cases which is comparable to results by Bulian P et al. in Italy, Uzay A et al in Turkey and Gattei V et al. in Italy who reported CD49d positivity in 52%, 47% and 39% of cases respectively. 11,20,21Our results were slightly lower than study done by Al-Rubaie HA et al. 15 in Iraq who showed positivity for CD49d (60%).This might be explained by the smaller sample size they studied (n=30).We could not find any similar studies conducted in our region with which our statistics could be compared.So we believe that this is the first study of this kind in Pakistan. The expression of ZAP-70 was not recorded in our cases.A study conducted by Zeeshan R et al. showed ZAP-70 expression in only 13% cases. 7n the contrary, an Indian study revealed that expression of ZAP-70 by flow cytometry was weak in a vast majority of cases (n=60) with small shifts above the baseline threshold thus, was not a robust assay.This may explain why ZAP-70 could not be detected in any of our studied Prognostic markers in Chronic Lymphocytic Leukaemia cases.And hence low frequency in South Asian population. 22here was a significant correlation between CD38 and CD49d (p-value <0.05).No correlation was found between ZAP-70 and either of the other two markers (CD38 & CD49d).We divided our data into two groups i.e.Group-1 (below 60 years of age) and Group-2 (60 and above).No association was found between the immunophenotypic markers and age. In an analysis of 3000 patients done in Italy for flow cytometric based prediction of overall survival (OS) in CLL, CD49d+ had a significantly high risk of death and lower poor survival rate as compared to CD49d-patients.By a Cox analysis for OS, they showed that CD49d+ patients have a two-fold increase risk of death and it was the only flow cytometry based marker with independent prognostic relevance for OS.They also compared models for prognostic markers with and without CD49d by several prediction performance measures indicating that excluding CD49d significantly reduced the prognostic power of the model. 11n 2017, a study conducted by Ahmed S et al. 12 compared the expression of CD38 and CD49d in patients with CLL.They concluded that patients with strong double expression of CD38 and CD49d required treatment.Whereas, patients with negative expression of CD38/CD49d had a good prognosis and long treatment free survival. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "The limitation in our study was that only a small number of patients were studied.It is strongly recommended that all the three prognostic markers i.e.CD38, ZAP-70 and CD49d are studied in all the newly diagnosed patients of CLL.As the study was single centered, that's why it's difficult to extrapolate it to whole Pakistani population.Therefore, it is highly recommended that these immunophenotypic markers should be studied on a large cohort of patients in centers which extend diagnostic and therapeutic facilities to patients of Chronic Lymphocytic Leukemia in Pakistan.Thus, this pilot study can form a foundation for larger studies on the subject. ",
"section_name": "Limitation of the study:",
"section_num": null
},
{
"section_content": "We conclude that assessing prognosis of CLL at the time of diagnosis is essential for all patients to segregate patients into groups who need urgent treatment because of presence of adverse prognostic markers from those who can be placed in watch and wait group.CD38 and CD49d expression was detected in almost half of the patients and were significantly correlated.CD49 is gaining acceptance internationally as an independent prognostic marker and is more reliable for prognostication of CLL along with CD38. ",
"section_name": "CONCLUSION",
"section_num": null
},
{
"section_content": "We recommend that along with the conventionally used biomarkers i.e.ZAP-70 and CD38, CD49d should also be added to the immunophenotyping panel for stratification of prognostic groups in CLL.Further studies should also be done on larger groups of patients to evaluate the frequency of these biomarkers in our population and re-evaluation should be done for the inclusion of ZAP-70 in the immunophenotyping panel. ",
"section_name": "Recommendation:",
"section_num": null
}
] |
[
{
"section_content": "There is no conflict of interest among the authors. HH conceived, designed and did statistical analysis & manuscript writing.NUD did overall supervision, proof reading and final drafting.SAK did the conceptualization of the study and critical revision of article.HH, SG did data collection and statistical analysis.HH takes the responsibility and is 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. ",
"section_name": "Conflict of interest:",
"section_num": null
},
{
"section_content": "There is no conflict of interest among the authors. ",
"section_name": "Conflict of interest:",
"section_num": null
},
{
"section_content": "",
"section_name": "Source of funding: Higher Education Committee.",
"section_num": null
},
{
"section_content": "HH conceived, designed and did statistical analysis & manuscript writing.NUD did overall supervision, proof reading and final drafting.SAK did the conceptualization of the study and critical revision of article.HH, SG did data collection and statistical analysis.HH takes the responsibility and is 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. ",
"section_name": "Authors' Contribution:",
"section_num": null
}
] |
10.1186/s13045-014-0104-2
|
Notch1 phenotype and clinical stage progression in non-small cell lung cancer
|
Notch1 transmembrane receptor is activated through ligand-binding- triggered proteolytic cleavages and, upon release, the intracellular domain (N1-ICD) translocates into the nucleus and modulates target gene transcriptions. Notch activation has been implicated in tumorigenesis in an increasing number of human malignancies including non-small cell lung cancer (NSCLC). However, Notch1 in distinct expression patterns and activation status with tumor progression remains to be defined in NSCLC.Notch1 and activated Notch1, N1-ICD, were examined by immunohistochemistry in 58 cases of stage I to IV NSCLC tumors. Association between Notch1 or N1-ICD expression and clinicopathological factors was assessed via correlation coefficient r statistics. P-values are two-sided.Detectable tumor Notch1, predominantly localized to the membrane and cytoplasm, was observed in 29 cases (50%, 95% Blyth-Still-Casella confidence interval 37 - 63%). It was negatively associated with stage (r=- 0.43, P<0.001) and nodal status (r=- 0.33, P = 0.01), but not tumor size. In contrast, nuclear N1-ICD expression level was low and found in 12% of NSCLC patients, neither significantly associated with stage nor nodal status. Upon Notch1 activation in vitro, a mostly extra-nuclear staining was substantially turned into the nuclear signal in cancer cells.Notch1 in the largely inactivated phenotype is inversely associated with clinical stage progression in NSCLC. Notch1, rather than activated N1-ICD, may be a context-dependent restrictive factor to nodal metastasis.
|
[
{
"section_content": "Lung cancer is the leading cause of cancer-related mortality in men and women in the United States and Europe [1].Approximately 85% of all lung cancers are non-small cell lung cancer (NSCLC), which includes squamous cell carcinoma, adenocarcinoma and large cell carcinoma (LCLC).The Notch signaling pathway is a highly evolutionally conserved signal transduction network that is critical for cell fate specification in a context-dependent manner during and after development in various organ tissues [2,3].Notch signaling is frequently deregulated in human hematological malignancies [4,5] and solid tumors including NSCLC, through gene mutations and aberrant expression of Notch receptors [6,7].Specifically, Notch signaling maintains a balance between cell proliferation and apoptosis and has been shown to be oncogenic or tumor-suppressive depending on the cancer types; it can be both oncogenic and tumor-suppressive within one cancer type such as in B-cell malignancies [8].Activated Notch1, in co-operation with Myc or through regulation of expression of epidermal growth factor receptor (EGFR), was implicated in the tumorigenesis, proliferation and survival of NSCLC models in preclinical studies [9,10].Moreover, hypoxia via HIF1α stabilizes and activates Notch1 in lung adenocarcinomas.In turn, Notch1 activates the IGF-1R pathway, promoting cancer cell survival under hypoxia [11,12].Inhibition of ADAM-17a critical step of ligand-dependent activation of Notch signaling led to substantial cell death and reduced tumorigenesis in NSCLC cell lines and xenograft tumor models.Activated Notch1 was associated with poor survival in NSCLC patients without p53 mutations [13].Despite the substantial data of Notch1 in tumorigenesis and tumor cell survival, association of Notch1 expression levels and patterns with tumor progression in terms of tumor size and metastasis remains to be delineated. The Notch receptors are non-covalently bound heterodimeric proteins, consisting of a large N-terminal extracellular portion (N EC ) featuring multiple EGF-like repeats and the Notch transmembrane domain (N TM ), which includes an extracellular stub, transmembrane segment and intracellular domain (NICD) [14,15].The NICD, upon sequential cleavages of N TM by ADAM10 or 17 and gamma-secretase, is released from the plasma membrane and translocates into the nucleus, where it activates target gene transcriptions.In addition, the EGF repeats in the N EC is calcium-dependent.Calcium depletion by EDTA can destabilize N EC and lead to its dissociation from N TM , activating the signaling of some of the Notch receptors such as Notch1 and 2 [16]. Agents targeting the Notch pathway including γsecretase inhibitors (GSIs) and monoclonal antibodies (mAbs) to Notch ligands and Notch receptors are currently in early clinical development across a range of advanced human malignancies.GSIs have numerous possible targets, but their anti-neoplastic effects are thought to be due mostly to Notch inhibition, primarily Notch1, observed by several studies [15,17,18].However, single-agent antitumor activity of Notch inhibitors, as determined by radiologic responses, was observed in only about 3% of patients treated with a Notch pathway inhibitor according to available clinical trial data [15].Unexpectedly, clinical activity has not been observed in patients whose tumors harbor Notch1 mutations thus far [19,20].Moreover, systemic Notch inhibitor administration causes significant toxicities with dose limiting gastrointestinal adverse events (goblet cell metaplasia and secretory diarrhea) [15]. Given the current clinical challenges and specific features of the Notch signaling, it is important to characterize expression levels and patterns in association with activated or inactivated Notch1, and in turn elucidate the target status quo for therapeutic intervention.In this study, we examined Notch1expression in NSCLC, and identified those tumors with abundant Notch1 as well as activated Notch1, N1-ICD, at γsecretase cleavage site (N1-ICD-V1754).Notch1 and N1-ICD-V1754 expression patterns were characterized in cancer cell lines via modulation using EDTA and in human NSCLC tumors.Particularly, this investigation tested the hypothesis that inactivated Notch1 may play a suppressive role in tumor progression through defining Notch1 or N1-ICD-V1754 status in relation to clinicopathological factors such as tumor differentiation, nodal status and clinical stage. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To characterize Notch1 expression patterns, NSCLC and breast cancer cells were treated with EDTA to activate Notch1.With and without EDTA exposure, EP1238Y antibody identified Notch1 protein of ~125 kDa, corresponding to the approximate size of N TM of Notch1 (Figure 1A); D3B8 antibody detected N1-ICD-V1754 of ~110 kDa in NSCLC NCI-H23, NCI-H522 and breast cancer MCF-7 cells after EDTA treatment.Immunocytochemistry analysis revealed that Notch1 was mainly bound to the membrane and cytoplasm, and upon EDTA treatment, N1-ICD-V1754 was rapidly induced and translocated to the nucleus in NCI-H23 cells (Figure 1B).These results indicate specific modulation of Notch1 expression patterns upon Notch1 activation by EDTA, switching a mostly membranous/cytoplasmic staining to the darkbrown signal in the nucleus in NSCLC cells expressing Notch1.In addition, specificity and utility of the two antibodies were validated for use in paraffin-embedded sections.Further, the cell adhesion molecule E-cadherin was decreased in MCF-7 and NCI-H23 cells (Figure 1A). To ascertain and extend the findings, NCI-H358 and NCI-H322M NSCLC cells were also subjected to EDTA treatment.As expected, N1-ICD-V1754 was markedly induced in these cells that express Notch1 (Figure 2A).The reduction of E-cadherin by EDTA treatment was also studied further with these two cell lines.It was again noted that appearance of N1-ICD-V1754 was coupled with a significant reduction in E-cadherin expression by both Western blot and immunocytochemistry (Figure 2A andB).Treatment with EDTA in the presence of RO4929097 prevented Notch1 activation and partially rescued E-cadherin from the decrease (Figure 2A). ",
"section_name": "Modulation of Notch1 expression patterns following activation by EDTA in cancer cells, and validation of antibodies",
"section_num": null
},
{
"section_content": "All levels of tumor Notch1expression were found in 29 of the 58 NSCLC cases (50%, 95% CI 37% -63%), with 8 cases (14%) exhibiting strong staining.These included 5 of the 35 cases of squamous cell carcinomas, 2/19 adenocarcinomas, and 1/4 undifferentiated or large cell undifferentiated carcinomas (Table 1; Figure 3).Notch1expression was not statistically different between adenocarcinomas and squamous cell carcinomas (P = 0.57; Table 1).Similar to the expression pattern observed in cancer cells, membranous/ cytoplasmic/nuclear Notch1, with an extra-nuclear staining as the major signal, was observed in NSCLC tumors.In addition, it was high levels of Notch1 expression, 6 out of 8, which were coupled to heterogeneous N1-ICD-V1754 expression (Figure 3). Expression of N1-ICD-V1754 was exclusively nuclear and detected in 7 of the 58 cases (12%, 95% CI 6-22%). The seven were all from 29 Notch1-positive cases, and none from Notch1-negative ones.Among those, the expression levels were low in 6 cases and intermediate in one sample, relative to NCI-H23 cells treated with EDTA.In the context of heterogeneous Notch1 activation, we observed a heterogeneous Ki67 labeling/tumor cell proliferation (Figure 3). ",
"section_name": "Expression of Notch1 and N1-ICD-V1754 in human NSCLC",
"section_num": null
},
{
"section_content": "Analysis of disease-related clinicopathological parameters showed neither a significant association between Notch1 expression and demographics including age and sex, nor a correlation between Notch1 and tumor grade or tumor size (Table 2).However, there was a significant negative association between Notch1 and clinical stage (r = -0.43;P < 0.001) or between Notch1 and nodal status (r = -0.33;P = 0.01).In contrast, N1-ICD-V1754 was not significantly associated with stage (r = -0.06;P = 0.65) and nodal status (r = -0.09;P = 0.50). ",
"section_name": "Association between Notch1 or N1-ICD-V1754 and clinicopathological covariates in NSCLC",
"section_num": null
},
{
"section_content": "Our data demonstrate that all levels of Notch1are detected in 50% of stage I to IV NSCLC tumors, and it is predominantly localized in the cell membrane and cytoplasm.Notch1 is largely inactivated in NSCLC as only a small fraction of NSCLC samples heterogeneously express low levels of N1-ICD-V1754 [21].Immunocytochemistry results clearly show that Notch1 translocation from the cell membrane to the nucleus upon activation by EDTA treatment, indicative of the function of the canonical Notch signaling in NSCLC and breast cancer cells [15,16,22].EDTA treatment could elicit a degree of Notch1 activation comparable to that resulted from Notch ligand Delta-1 exposure [16].Pre-treatment with and in the presence of RO4929097 during EDTA treatment abolishes Notch1 activation, suggesting that Notch1 activation mediated by EDTA is gamma-secretase-dependent in NSCLC cells. Importantly, the inactivated Notch1 configuration in NSCLC is inversely associated with locoregional node metastasis, whereas it is not significantly associated with tumor size.Thus, the negative association between Notch1phenotype and clinical stage progression is likely driven by nodal status.The findings support our hypothesis that inactivated Notch1 may serve as a context-dependent restrictive factor of tumor cells to local-regional spread.The scenarios that inactivated Notch1 is protective against nodal spread could be explained as follows.First, as the single-pass heterodimeric transmembrane receptor and/ or in couple with Notch ligands on the cell surface, Notch1 mediates cell-to-cell interactions in adjacent cells and may therefore physically limits the migration of tumors cells [14].Secondly, other lines of experimental evidence showed that without Notch1 activation, the adherens junctions complex containing E-cadherin is intact, which suppresses tumor cell migration and metastasis [22,23].In NSCLC A549, NCI-H1650 and NCI-H596 cells, E-cadherin expression was decreased after transfection with a N1-ICD vector [24].Notch1 down-regulated E-cadherin through upregulation of the snail family of transcriptional factors in these cell lines [24].The reduction in E-cadherin by EDTA treatment can be somewhat rescued by RO4929097 in NCI-H358 and NCI-H322M cells.The data suggest that Notch1 activation is in part responsible for reducing E-cadherin.By contrast, activated Notch1 is not negatively associated with nodal metastasis.Rather, heterogeneous N1-ICD-V1754 expression is associated with heterogeneous tumor cell proliferation in NSCLC.In agreement with our results, a study found that Notch1 expression was inversely correlated with stage, despite lack of correlative data on nodal metastasis, in 395 NSCLC tumor samples by immunohistochemistry using a semi-quantitative scoring method [25].Moreover, Notch1 expression predicted not only less progressive disease but also better overall survival in lung adenocarcinoma patients [26] Taken together, Notch1 plays distinct roles depending on its activation status in patients with all stages of NSCLC from I to IV. Interestingly in this study, we found that Notch1and N1-ICD-V1754 are expressed in undifferentiated carcinomas of the lung, which has not been described previously [27].Notch1and N1-ICD-V1754 were also found highly expressed in a case of undifferentiated carcinoma of the ovary [28].Undifferentiated carcinoma is an epithelial malignancy that lacks morphologic or functional indicators of its embryonic origin, capable of deriving from many organ sites including bladder, cervix, colon, esophagus, larynx, pancreas, salivary glands, thyroid and uterine, besides lung and ovary [29,30].Given the implication of the Notch pathway signaling in early development, Notch signaling may be of significance in the pathogenesis of undifferentiated carcinoma.Additional studies should investigate the role of Notch1 signaling in the pathogenesis of undifferentiated carcinomas. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Expression of membranous and cytoplasmic Notch1expression is common, in contrast to the activated Notch1 in the nucleus, in human NSCLC [9,10].Such pattern of expression or inactivated Notch1 may serve as a marker of low-metastatic propensity while high levels of or activated Notch1 a more pertinent therapeutic target in NSCLC or advanced NSCLC.We anticipate clinical validation of Notch1 and N1-ICD-V1754 expression levels and patterns for the purpose of evaluation of efficacy, ultimately patient stratification, and as a pharmacodynamics biomarker to document inhibition of Notch cleavage after treatment with the Notch pathway inhibitors in clinical trials. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The NSCLC cell lines NCI-H23 (adenocarcinoma), NCI-H522 (adenocarcinoma), NCI-H322M (adenocarcinoma) and NCI-H358 (adenocarcinoma) were obtained from the Tumor/Cell Line Repository, Division of Cancer Treatment and Diagnosis, National Cancer Institute.MCF-7 breast cancer cells were obtained from ATCC (Rockville, MD).Exponentially growing cells in 10% fetal bovine serum RPMI media at ~80% confluence were washed with serum-free RPMI.After washing, cells were treated with EDTA at a concentration of 0.53 mM or serum-free RPMI for 15 minutes and subsequently subjected to either formalin fixation and paraffin-embedding or cell lysis for lysates collection.Additionally, NCI-H358 and NCI-H322M cells were treated with 5 μM of a GSI RO4929097 (Selleck Chemicals, Boston, MA), for 10 min before adding EDTA solution containing 5 μM of RO4929097.After 15 min, lysates were collected or cells were fixed with 10% neutral buffered formalin before paraffin embedding. ",
"section_name": "Cells, cell culture, and EDTA and GSI treatment",
"section_num": null
},
{
"section_content": "Equal numbers of cells were lysed in Laemmli sample buffer and proteins in the lysates were separated by electrophoresis [31].After protein separation and transferring, nitrocellulose filters were probed with monoclonal antibodies to Notch1 (clone EP1238Y, Abcam Inc., Cambridge, MA) and cleaved Notch1, N1-ICD-V1754 (clone D3B8, Cell Signaling Technology Inc., Danvers, MA) in dilutions of 1 to 5000 and 1 to 500, respectively.The Notch1 antibody recognizes the cytoplasmic portion of human Notch1 receptor.The cleaved Notch1 antibody specifically recognizes an epitope of Notch1 intracellular domain when cleaved at the protein sequence between G1753 and V1754 for human Notch1 (N1-ICD-V1754) or G1743 and V1744 for mouse and rat [16,32].E-cadherin was detected by a monoclonal antibody against E-cadherin (clone HECD-1, Invitrogen Corp., Camarillo, CA) in 1 to 500 dilution and incubated for 1 hour at 37°C.ß-actin was probed with mAb at a dilution of 1: 10,000 as loading control (Sigma, St. Louis, MI).Reactive proteins were revealed using an enhanced chemiluminescence (Pierce Chemical Co., Rockford, Illinois, USA). ",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "Lung cancer tissue microarrays and clinicopathological data were obtained from Cybrdi, Incorporation (Rockville, MD), and FOLIO Biosciences (Powell, OH).Approval of the biomarker study on de-identified human tissues was obtained from the Office of Human Research Protections, National Institutes of Health, Bethesda, Maryland.Tumor presence and histology were confirmed on HE stained sections. ",
"section_name": "Human lung cancer tissue microarray specimens",
"section_num": null
},
{
"section_content": "Immunohistochemistry method on formalin-fixed and paraffin-embedded sections has been described previously [33,34].In brief, after incubation of the primary antibodies to Notch1 and cleaved Notch1 in dilutions of 1: 200 and 1: 20 or antibody to E-cadherin in 1:100 dilution or antibody to Ki67 in 1: 200 dilution (clone MIB-1, DAKO) for 1 hour, binding of the antibodies to their antigen binding sites in sections was amplified using Vectastain Elite avidin-biotin-peroxidase complex kits (Vector Laboratories, Burlingame, CA).The antigen-antibody reaction sites were visualized using 3,3-diaminobenzidine for 7 min and, subsequently, sections were counterstained with Mayer's hematoxylin.Paraffin-embedded NCI-H23 cells or MCF-7 cells treated with and without EDTA described above were used as controls; negative controls were performed using isotype immunoglobulins appropriate to the primary antibodies used.Areas of tumor staining on each tissue core were analyzed with assistance of a digital imaging system (DAKO, Carpinteria, CA) by reporting intensity and percentage of staining.Staining Index (SI) for Notch1 or N1-ICD-V1754 was calculated as the percentage multiplied by the intensity of staining (after subtracting the tissue readout of the corresponding negative control) divided by 100 (SI = intensity x percentage/100) [34].It was defined as negative if SIs were < 2, and positive if the SIs were ≥ 2. As for defining the levels of expression, it was considered as the low level if SIs were ≥ 2 and <15 (1+ by manual scoring of intensity), as the moderate level if SIs were ≥ 15 and < 30 (2+), and as the high level if SIs were ≥ 30 (3+). ",
"section_name": "Immunohistochemistry or immunocytochemistry and quantitative analysis",
"section_num": null
},
{
"section_content": "The correlation between Notch1 or N1-ICD-1754 and age was analyzed using the Pearson's correlation coefficient r statistic.All other correlations between Notch1 or N1-ICD-1754 and sex, grade, tumor size, nodal status or clinical stage were assessed by the Spearman's rank order correlation coefficient.The two-sided P-values were calculated by means of Fisher transformation, and P values < 0.05 were considered statistically significant.Comparison of Notch1 percent positivity between squamous and adenocarcinoma was assessed by Fisher's Exact test, two-sided P-value. ",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "The Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States in part supported this work.We are grateful to Dr. Mark Sherman for his review of H&E stain of undifferentiated carcinoma of the ovary. 1 National Clinical Target Validation Laboratory, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 3 Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 4 Stanley Scott Cancer Center, Louisiana State University Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, LA, USA. 5 Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States in part supported this work.We are grateful to Dr. Mark Sherman for his review of H&E stain of undifferentiated carcinoma of the ovary. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "1 National Clinical Target Validation Laboratory, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 3 Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 4 Stanley Scott Cancer Center, Louisiana State University Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, LA, USA. 5 Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. ",
"section_name": "Author details",
"section_num": null
},
{
"section_content": "Abbreviations CI: Blyth-Still-Casella confidence interval; EDTA: Ethylenediaminetetraacetic acid; EGF-R: Epidermal growth factor receptor (EGF-R); GSI: Gamma-secretase inhibitor; mAb: Monoclonal antibody; N1-ICD: Notch1 intracellular domain; NICD: Notch intracellular domain; NSCLC: Non-small cell lung cancer. The authors declare that they have no competing interests. Conceptual design: SY, JT, JD, LR, NT and PI.Provision, collection and assembly of the data: DN and SY.Data analysis and interpretation: LR, SY, NT.Manuscript writing: SY, LR, LM.All authors read and approved the final version of the manuscript. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations CI: Blyth-Still-Casella confidence interval; EDTA: Ethylenediaminetetraacetic acid; EGF-R: Epidermal growth factor receptor (EGF-R); GSI: Gamma-secretase inhibitor; mAb: Monoclonal antibody; N1-ICD: Notch1 intracellular domain; NICD: Notch intracellular domain; NSCLC: Non-small cell lung cancer. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Conceptual design: SY, JT, JD, LR, NT and PI.Provision, collection and assembly of the data: DN and SY.Data analysis and interpretation: LR, SY, NT.Manuscript writing: SY, LR, LM.All authors read and approved the final version of the manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.1038/s41598-020-76069-3
|
A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats
|
<jats:title>Abstract</jats:title><jats:p>Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.</jats:p>
|
[
{
"section_content": "Single-chain variable fragment (scFv) immune phage libraries are convenient tools for antibody discovery.Immune libraries are constructed by incorporating scFv derived from an immunized animal into a phage display system.Panning of an immune scFv phage library combines advantages of both hybridoma and phage display methodologies and can quickly identify multiple diverse binders 1,2 . Immune libraries are typically derived from immunized mice; but several species have served as the source of variable region diversity for antibody phage libraries including mouse, rabbit, and human [3][4][5] .However, the generation of rat immune libraries is still relatively unstudied 6,7 , despite rats having several advantages for this application 8,9 .For instance, rats typically undergo rapid seroconversion, have a large set of variable chains and can generate unique binders.Here, we describe a primer set for rat immune library generation which covers all rat germline heavy and kappa light chain variable genes.We exclude bias by comparing deep sequencing of pooled amplified variable regions with rat variable sequence usage. To determine how well our primers perform, we generated binders against CD160 as a test antigen.CD160 is a GPI-anchored protein with potential to be a useful cancer target 10,11 ; it has been detected on endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues 12,13 CD160 has been also shown to be aberrantly expressed on B cell malignancies such as B cell chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia (HCL) 14,15 .Following successful identification of several lists 232 Rattus norvegicus germline heavy variable (VH) sequences with 13 families, and 164 kappa light variable (VLK) sequences in 21 families.We designed 39 forward primers which anneal to the VH genes and 29 to the variable kappa light chains.Predicted pseudogenes and not in-frame Open Reading Frames (ORFs) were excluded.In rodents, the kappa light chain is predominantly used, with only 5% of mouse immunoglobulin expressing the lambda light chain, hence primers for the lambda light chain were not designed 17 . Three strategies allowed the size of the primer set to be kept to a minimum: (1) The similarity shared within each family at the start of the VH genes allowed the design of several primers which cover many functional ORFs. (2) In addition, judicious incorporation of up to 3 ambiguous DNA nucleotides per primer increased the numbers of genes primed per primer; (3) Locked Nucleic Acids (LNA) 18 were incorporated to anneal to conserved residues of the variable chains' genes.This avoided the need to design long primers that further extend into the target gene; ensuring specific amplification while covering a large number of genes with the same primer (Tables 1,2). For VH regions, on average each primer amplified 23 variable genes, although smaller families required 1 or 2 primers (Supplementary Table S1).The kappa light chain genes contain a higher number of families, but a smaller number of different subgroups (Supplementary Table S2).This allowed the coverage of each family with fewer primers per family.In this instance, an average of 19 subgroups were covered per primer.We designed 4 reverse primers for VH [annealing to the heavy chain joining regions (HJ)] and 5 reverse primers for the VLK [annealing to the kappa chain joining regions (KJ)].Together these primers cover all the possible joining regions in rat VH and VLK. The primer tail at the 5′ end of the VH gene and the 3′ end of the VLK gene contained the annealing sites for the nested PCR outer primers and SfiI/NotI restriction sites for cloning into the phagemid vector (pHEN1 19 ).The tails at the 3′ end of the VH and the 5′ end of the VLK included the serine-glycine linker sequence (3xGGGGS) as overlapping regions.The primary PCR amplification of VH and VLK chains incorporated the outer tail regions.Overlap extension PCR created a single amplicon encoding an scFv in the VH-VLK orientation separated by a serine-glycine linker sequence. Primer set effectively amplifies VH and VL genes without PCR-biases.We first sought to better understand the VH/VLK usage in rats, since this has not been well studied.We then could determine if our novel primer set result in biased amplification of certain VH/VLK families.Deep-sequencing of VH/VLK genes from 5′RACE and primer set amplified cDNA were studied and compared; cDNA was isolated from three wild type naïve, antigen unchallenged, Wistar rats.The material was used as template for both an Ig specific 5′RACE PCR and for primer set amplification and sequenced using the Illumina MiSeq Next-generation sequencing (NGS) platform. Analysis of the VH and VLK usage in naïve rat following 5′ RACE showed that VH family 5 was the most represented (39%) followed by family 2 and 1 with 28 and 16% respectively.The other families were all present at a lower frequency (1-4%) while family 12 and 15 were the least represented with a frequency of less than 0.1%.Analysis of VLK usage showed that family 22 and 12 and 1 were the most represented (28%, 20%, 12% of the total VLK usage respectively) while the majority of VLK families were present at similar frequencies between 3 to 5%.Families, 5, 9, 17, 19 and 21 were present at less than 1% and family 7, 13, 18 and 20 were less than 0.1% (Fig. 1). In order to determine whether our primer set was able to amplify the rat VH/VLK repertoire in an unbiased fashion, we compared the repertoire from 5′RACE described above with that amplified by our primer set, again in the context of unchallenged naïve rats.The relative frequency of variable heavy (VH) and joining (HJ) genes was similar in both primer and 5′RACE amplified sets (Fig. 1a).In the 5′ RACE amplified dataset 55% of the VH genes were paired with J2 the next most frequently used J region was J3 while a similar proportion of J1 and J4 genes were used.The V-J pairing distribution was maintained after primer set amplification in which J2 remained predominantly used (49%), followed by J3 (33%).Thus, the frequency of the VH family usage was preserved before and after V region specific PCR. The kappa light chain (VLK) samples were similarly analysed.Here we observed that the V-J pairing pattern was comparable in both 5′RACE and primer set amplified products (Fig. 1b).For example, the VLK22-KJ2 pairing frequency was observed at 55% and 50% in the 5′RACE and primer amplified samples respectively.This again suggested that the PCR amplification using gene specific primers did not bias the repertoire towards specific clones.The general frequency of the usage for each kappa V chain family and J genes did not show differences in the two samples. The PCR products obtained after V region specific amplifications demonstrated a similar representation of VLK families as the 5′RACE sample.IgVLK7, IgVLK13, IgVLK18, IgVLK20, IgVLK21 were present at low frequency in the pre-PCR sample and were observed at a similar proportion post-amplification with the primer set.A notable omission in the primer set sample was the absence of IgVLK13 which had a low representation in the 5′RACE dataset and a reduction in the frequency of the J4 region in the kappa light chain pairing.The larger proportion of J5-assigned sequences suggest that the J5 reverse primer, which shares homology with J4, may have preferentially amplified this region over the J4.Overall, both the frequency of the gene-usage and the V-J pairing suggest that PCR amplification using this primer set did not skew the rat's immune repertoire, but enabled generation of V-J products as close as possible to the originally repertoire present in these rats. As additional confirmation, we have looked at the frequency distribution of unique CDR3 sequences within both the VH and VLK genes in the naïve rat data sets.The percentage unique clones containing CDR3 sequences of different length was almost identical in 5′RACE and primer set amplified genes for both heavy and light chain (Fig. 1c).Gaussian analysis of the data indicated a mean CDR3 length of 14.09 and 13.78 for the heavy chain and 10.87 and 10.94 for the light chain, in 5′RACE and primer amplified data sets respectively (Supplementary Fig. S1).This data supports the lack of bias introduced by the PCR amplification of the variable genes and demonstrates that the overall diversity is faithfully represented with our primer-set.Table 1.Sequence of the primers used to amplify and fuse the heavy variable chain genes into an scFv phage display library.The table includes forward and reverse primer specific for different genes of the heavy variable chain families and joining regions of Rattus norvegicus germline sequences.An outer forward primer was used for the overlap extension PCR with the kappa variable chains products to generate the scFv phagemid library.The primer may include an ambiguous DNA nucleotide (Y, R, W, S, K and M) to increase the coverage for families containing large number of subgroups while only 1 or 2 primers were required for smaller families.Four reverse primer were designed to cover all the joining regions of the VH.Three Wistar rats were genetically vaccinated using the plasmid pVAC2 encoding CD160.Twenty-one days post vaccination, serum-conversion was observed in all three rats (Supplementary Fig. S2).CD160 was also cloned into the retroviral vector SFG and SupT1 cells expressing high levels of CD160 were generated (Supplementary Fig. S3).Pooled cDNA from the three vaccinated rats was used as template with 68 individual PCR reactions (29 for the VH and 39 for VLK primers).Reverse primers for the joining region were used in an equimolar mixture for each individual forward primer PCR reaction.(Supplementary Fig. S4).A notable feature was the presence of the VH family 3 which was sequenced at low frequency (0.4%) from 5′RACE amplicon in naïve rats.In the case of CD160-immunised animals this region was successfully amplified with only one of the two primers designed to anneal to this region (MP20891).After overlap extension PCR, the approximately 800 bp scFv insert was subsequently cloned in the phagemid vector and displayed on the phage surface for biopanning. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "We displayed the human GPI-anchored protein CD160 on Strep-Tactin magnetic beads.This facilitated direct capture on to beads from the supernatant of transfected HEK293T cells and convenient elution with biotin (Fig. 2a).To confirm quality of the CD160 protein from the transfected HEK293T, we prepared it to 95% purity, analysed it by size exclusion chromatography (SEC) and differential scanning fluorimetry (DSF), and compared it with different sources of CD160 protein (Supplementary Fig. S5).The success of the biopanning process was assessed by flow cytometry staining of the beads at the various stages using an anti-CD160 antibody (Fig. 2b).Three sequential biopanning rounds of the phage-scFv library were conducted using the CD160-Strep-Tactin magnetic beads; enrichment was tested after each round of selection.To increase the likelihood of finding functional binders against the native conformation of the target antigen, we screened individual clones using flow cytometry.Successful enrichment was assessed using IPTG-induced bacterial supernatant to stain CD160 positive SupT1 cells (Fig. 2c).A clear binding of the supernatant was observable in selection rounds 2 and 3, but a small enrichment is also evident in round one, highlighting the presence of specific binders after a single round of phage selection.Screening clones derived from individual bacterial colonies identified 15 unique scFvs that specifically bind CD160 positive cells (Fig. 2d). ",
"section_name": "VH family",
"section_num": null
},
{
"section_content": "VH genes to the IMGT database of rat germline variable genes revealed that the 15 unique scFv clones which contained 5 different HCDR3 originating from rearranged germline family V2 and V5.Two of these CDR3s were found to account for 46.67% and 26.67% of the total diversity (Fig. 3a). Five binders (SC14, C10, C25, C83, C123) carrying unique CDR3s were studied further as recombinant chimeric rat scFv/mouse IgG2aFc antibodies.Flow cytometric analysis determined that the recombinant antibodies selectively bound cell lines engineered to express CD160 (Fig. 3b). The kinetic profile of the scFvs was studied by SPR using purified CD160 as the analyte.We found a range of high affinity scFvs (Fig. 3c).SC14, C25 and C123 were the highest affinity with KD values of 0.399, 0.869 and 0.206 nM respectively.These clones all exhibited rapid association and slow dissociation kinetics as expected for high affinity binding antibodies, (k a = 1.8-5.2. Sequence of the primers used to amplify and fuse the kappa light variable chain genes into an scFv phage display library.The table includes forward and reverse primer specific for different genes of the kappa light chain families and joining regions of Rattus norvegicus germline sequences.An outer reverse primer was used for the overlap extension PCR for the assembly as scFv phagemid library.The primer may include an ambiguous DNA nucleotide (Y, R, W, S, K and M) and Locked Nucleic Acids (LNAs) were incorporated in specific conserved residues of the variable chains' genes facilitating the generation of short primers to avoid extension into the frame, minimizing mismatches in other regions of the primer.Five reverse primer were designed to cover all the joining regions of the VLK.CAR engineered T cells were functional and able to specifically kill CD160 positive cells.We next sought to demonstrate the therapeutic potential of CD160 binders.For this we converted the 5 different CD160 scFvs into CAR format.A third generation CAR construct with CD28 and OX40 costimulatory endodomains 20 and with a CD8STK as spacer moiety 21 was selected as the format for CAR comparison.Normal donor, peripheral blood T cells were transduced to express CAR constructs using gamma retroviral vectors.Transduced T cells were then co-cultured at different effector to target ratios (E:T) with either SupT1 cells (which do not express CD160) and SupT1 cells engineered to express CD160.One day post co-culture we observed already a drastic control in the growth of the SupT1 cells expressing CD160, with complete killing of the CD160 target cells by 72 h (Fig. 4a) at all the E:T tested.Furthermore, the 5 different CARs showed no nonspecific killing with complete recovery of the NT target cells at both time point. The 5 different CD160 CARs were further evaluated for the secretion of pro-inflammatory cytokines interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) at 72 h.All CARs tested showed high levels of cytokine production (Fig. 4b).No background cytokine release was observed to SupT1 cells.No differences were observed at any of the effector to target ratio tested, suggesting equal potency of all CARs generated.An interesting feature of all CARs tested was the high levels of IL2 secretion observed.This particular feature could be beneficial in the improvement of CAR T cells persistence in vivo. ",
"section_name": "Characterization of identified binders revealed high affinity kinetic clones. Alignment of the",
"section_num": null
},
{
"section_content": "Phage display has been successful in generating a large number of antibodies used in a variety of application including therapeutics 22 .Immune phage libraries combine advantages of both phage display and hybridoma technology.The in vivo selective expansion of high affinity B cell clones after immunization allows use of a small phage library and typically, multiple high affinity binders can be identified after limited rounds of bio-panning 23,24 . Immune libraries are typically derived from mice whose immune repertoire has been exhaustively studied 25 .Rats are rarely used for construction of such phage libraries, despite having advantages over mice as a host species to generate antibodies.For example, the propensity of rats to undergo rapid serum-conversion typically means that fewer animals need to be vaccinated 7 .Furthermore, being larger than mice, a tenfold greater amount of serum can typically be extracted, and larger secondary lymphoid organs allow the isolation of a greater number of lymphoid tissues.The use of rats as vaccination host results in non-cross-reactive antibodies and species-specific epitopes, distinct from those raised in mice [26][27][28] . Primers designed for the amplification of mouse immunoglobulins 29,30 have been successfully applied for the generation of scFv based phage libraries from immunized sources 31,32 .However, few primer sets that specifically amplify immunoglobulin variable regions from rats have been described.In 2008, Sepulveda and Shoemaker 6 compared rat EST sequences and mouse VL/VH coding sequences and designed a primer set which amplifies 65 heavy and 77 light chains-predicted variable domains.From the curated IMGT database, we currently know that in spite of the close similarity to mouse, rats have a very high number of functional variable genes in the genome (232 VH and 164VLK) with IGVH1, IGVH2 and IGVH5 33 being the most abundant families.Sepulveda's primer set only covers family 1 and 2, lacking coverage of the highly abundant family IGVH5 and other families such as IGVH8 and IGVH11.The Sepulveda primer set covers only IGVLK-IGVLK6 families, which is only a small proportion of possible Kappa light chain variable sequences. In order to expand the possibilities of using phage display-based methods for antibody discovery from immunized rats, we have utilised the curated IMGT database to design a set of primers that covers all currently known VH and VLK germline sequences in rats 16 .We used two strategies to exploit the similarity shared within each family at the start of the VH genes to minimize the numbers of primers: First, we incorporated up to 3 ambiguous nucleotides in each primer and secondly, we incorporated LNAs to shorten primers within highly homologous regions.This allowed us to cover all the families with a small number of primers (29 for VH and 39 for VLK) while fully covering the most represented families, such as IGVH1, IGVH2 and IGVH5 33 .Indeed, these were the most expressed families in rats as shown by NGS, confirming that the design of the primer set specifically captures the diversity of rat's immunoglobulins response. Because the rat's immunoglobulin repertoire is relatively unstudied, we first used NGS sequencing to investigate the family's usage in rats.This confirmed the IMGT annotation 33 with family 5 being the most represented VH family followed by an equal frequency of family 1 and 2, while for VLK we identified family 22 and 12 as the most commonly used genes in naïve rats.We compared these data with NGS of pooled VH and VLK amplified with our primer set to determine if our primer library resulted in any bias.Families containing fewer subgroups were also identified at lower frequencies and were covered proportionally in both the data sets.A similar profile of J region associated with each VH gene also confirms lack of bias introduced by the PCR with our primer set.Finally. the analysis of the CDR3 distribution in both the 5′RACE and primer set-amplified products demonstrated an almost identical pattern of diverse CDR3 length in both the VH and VLK genes, again confirming faithful amplification. To validate the performance of the primer set we used CD160 as a test antigen for the generation of a rat immune library.CD160 is a small protein expressed either as a GPI-anchored protein, with physiological expression restricted to NK cells and a subset of T cells.CD160 is a promising target for a number of clinical applications.The protein is expressed in neovasculature 13 , and has the potential to be used for vasculature targeting.It has been found in exhausted HIV-specific CD8+ T cells 34 and aberrantly expressed on B cell lymphoproliferative disorders 14 . We generated a phage display immune library for human CD160 from DNA immunized rats.We selected the immune phage display library with CD160 fused to the Strep-tag II peptide at the carboxy-terminus of the antigen.This construct facilitated direct capture to magnetic beads from the supernatant of transfected cells, avoiding the necessity of purification, biotinylation or passive absorption of the target molecule.Further, exploiting the higher affinity of biotin to Strep-Tactin compared with Strep-tag II, the addition of biotin allowed the gentle desorbing on phage from the beads through competitive elution.Sequence analysis identified fifteen unique scFvs with five different HCDR3 from either family 2 or 5 of the rat germline VH genes.Furthermore, characterisation of the obtained binders showed specific binding to the target with binding affinities from the mid-nanomolar to picomolar range. CAR T cells have demonstrated success in the treatment of lymphoid malignancies targeting CD19, and more recently BCMA 35,36 .To demonstrate the utility of the discovered scFv, we derived CARs from 5 of the scFv with unique HCDR3.Primary human T cells engineered to express these CARs showed specific cytokine release and cytotoxicity and in response to cells expressing CD160.Interestingly, there was limited difference in the cytotoxic capacity or secretion of cytokine despite measurable differences in the binding affinities of the different binders. Here, we describe a method of antibody isolation using phage display libraries based on a novel set of primers which amplifies the entire rat variable VH and VLK repertoires.We demonstrated the utility of such libraries in combination with genetic immunization and a simple display technique to generate antibodies, with a range of affinities to the target CD160.We anticipate this primer set will be of use in generating immune libraries from rats, in addition to other applications such as single B-cell antibody discovery methods.Finally, we hope that the overall methodology described here, combining immunization and panning strategy, opens up rapid and simple antibody discovery to laboratories with limited experience and resources for antibody engineering for CAR T and other applications. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Rat immunizations, RNA extraction and cDNA synthesis.Three adult Wistar rats were immunized with the target human CD160 (accession number: NM_007053.3).Genetic vaccinations were carried out using DNA encoding coated gold nanoparticles at Aldevron, GmBH.Briefly, the gene coding for human CD160 was cloned into a pVAC2 vector for the rats vaccination.A GeneGun system was used to deliver plasmid coated gold nanoparticles intramuscularly.Homogenized lymph nodes and spleens from the vaccinated animals were preserved in RNA later (QIAGEN).RNA was extracted from 7 × 10 6 lymph node cells using RNAeasy kit (QIA-GEN).RNA from multiple aliquots of cells was pooled together for downstream RT-PCR.The cDNA reaction was performed with 7 µg of RNA using QuantiTect Reverse Transcription kit (QIAGEN).The success of the reaction and potential genomic contamination was assessed by PCR amplification followed by gel electrophoresis of the housekeeping gene GAPDH using a 1:5 working dilution of the cDNA. All animals were used under protocols approved by UCL Animal Welfare and Ethical Review Body (AWERB) and authorization granted by the UK Home Office under the Animal Scientific Procedures Act 1986 (licence number: 12570).5′ RACE amplication of Ig genes naïve rats.Three 8-10-week-old wild-type Wistar rats (2 female and 1 male) were purchased from Charles River, UK.The animals were sacrificed and RNA extracted from homogenised spleens.Applying the Simple Method for Amplifying RNA Targets (SMART) 37 , Superscript II Reverse Transcriptase (Invitrogen) was used to generate cDNA; the reaction was primed to incorporate a specific tail for subsequent PCR.The resulting cDNA from each animal was then pooled and used to amplify the total VH and VLK gene set.The PCR was performed at 68 °C using KOD polymerase (Thermo Scientific) and a forward primer (5-cgacgtggactatccatgaacgca-3′) specific for the tail in the cDNA with a reverse primer specific for either the CH1 portion of the rat IgG's constant region (5-ccagactgcaggacagctgg-3′) or the kappa constant region (5′-atgatgtcttatgaacaacctcacaggtatagag-3′).Successful amplification was confirmed on an agarose gel by the presence of product between 600 and 700 bp.The DNA was subsequently purified by ethanol precipitation and resuspended in 20 μl of nuclease-free dH 2 0. The amplified fragments for VH and VLK chains were used for NGS sequencing.Biopanning of the scFv phage display library on Streptactin magnetic beads.(a) Schematic representation of the biopanning process employing the StreptagII-Streptactin system.CD160 fused with Twin streptag was capture directly from cell supernatant on the surface of Streptactin magnetic beads.Phages were incubated with the antigen' coated beads and bound phages were subsequently recovered by eluting with a 30 mM biotin solution and used to amplify the library for the next biopanning round.(b) The process of coating and elution of the beads throughout the biopanning process was assessed by flow cytometry analysis.These were stained during the several steps of the procedure (coating, blocking with 3%BSA and elution) using commercial anti CD160-PE.(c) Enrichment of the phage library for CD160 was determined after three rounds of biopanning on beads.The bacterial library from the different rounds of selection was induced to express the soluble scFv fragment in the supernatant.This was used to stain SupT1 cells overexpressing human CD160, due to the presence of a myc-tag at the C-terminus of productive scFvs we were able to detect binding using anti myc antibody.2xTY media + anti myc-tag-DL549 and the commercial anti-CD160-PE Ab were used as controls.(d) The 15 individual bacterial colonies identified in the third round of biopanning carrying unique combination of CDR1,2 and 3 were screened by against SupT1 CD160 positive and negative cells (NT). ◂ NGS sequencing and analysis.The total pool of VH and VLK products were sequenced using an Illumina MiSeq platform with 2 × 300 bp paired-end configuration (GENEWIZ).A quality FASTAQ report was delivered and used as input files in sequence reconstruction.The software for adaptative immune profiling MiXCR 38 was employed to overlap the paired-end sequencing and the rat's germline database obtained from IgBlast 39 was used as reference sequences for the alignment of the V, D and J segments.Output of each processing stage was converted to tab-delimited text files for manual inspection.Gene segments were identified using IMGT's nomenclature, including functional and open reading frame-defined gene segments.All productive VH and VLK segments were analysed and the percentage of usage was determined by dividing sequencing reads corresponding to each gene segment by the total number of gene segments.V(D)J pairing was only assessed from productive VH and VLK segments.Undetermined or unproductive sequences were excluded.Total counts from V-J pairings for heavy-and light-chains were tabulated and chord diagrams were generated using Circos Online 40 .Amplification of VH and VLK chains.The primary amplification of the two variable chains was performed with oligonucleotides designed on the Rattus norvegicus germline sequences of heavy variable chain (VH) and kappa light chain (VLK) genes; primers were designed to have the annealing portion with a melting temperature around 60 °C (Table 1).A PCR master mix was prepared using High Fidelity Phusion polymerase and buffer (NEB) in a 50 µl reaction with 1 µl of a 25 nM working dilution of each primer and 1 µl of the cDNA pool.An individual reaction for each forward primer containing 1 µl of an equimolar pooled mix of the reverse primers was performed.The samples were heated at 98 °C for 2 min, followed by 35 cycles of 98 °C for 30 s, 60 °C for 40 s, and 72 °C for 40 s with a 10 min final extension at 72 °C.Each product at approximately 400 bp was purified from the agarose gel. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "The amplified primary PCR products of VH and VL were pooled together for the overlap extension reaction.The outer primers were design to anneal 5′ end of the VH gene and the 3′ end of the VL gene for fusing into scFv format linked by a (Gly 4 Ser) 3 repeats as linker.The reaction mixture was amplified as above using 100 ng of each pool.After the initial denaturation for 2 min at 98 °C the PCR was cycled as follows: 98 °C for 30 s, 58 °C for 40 s and 72 °C for 1 min with a final extension at 72 °C for 10 min.The PCR product was run on agarose gel and the 800 bp band was extracted and purified using QIAquick PCR clean up kit (QIAGEN).The reaction was scaled up for the subsequent enzymatic digestion. Generation of the scFv library. 1 µg of purified fused product was digested with SfiI and NotI restriction enzymes (NEB) and cloned in to the phagemid vector pHEN1.The generated ligation mixture of plasmid DNA was cleaned-up by ethanol precipitation and re-suspended in 80 µl of dH 2 O prior to the electroporation into TG1 E. coli bacteria (MicroPulser, Bio-Rad).The transformed bacteria were recovered in 2xTY medium at 37 °C shaking at 225 rpm for 1 h before spreading on a Bio-Assay Dish (Fisher Scientific) containing 200 ml of 2xTY-agar, 100 µg/ml ampicillin and 1% glucose (w/v).Following overnight incubation, the dish was harvested Figure 4. Functional assays as chimeric antigen receptor.Four different healthy donors PBMCs were transduced with lentivirus to express the five different scFvs fused in a third generation (CD28-OX40ζ) CAR structure and tested in a cytotoxic assay.T cells were incubated with SupT1 CD160 positive and negative cells (NT) at different effector to target ratio (1:2, 1:4 and 1:8).The residual number of target cells for each donor was normalized against non-engineered PBMCs and all the five CARs demonstrated excellent killing at both 24 h and 72 h (a).Total cytokine production was assessed at 72 h and showed high levels of interferon-gamma (IFNγ) and IL-2 (b) with no background on target-negative cells.The mean from each donor was plotted individually and analysed using two-way ANOVA with Dunnett's post-test for comparison between CARs within the same target group.***p < 0.0001.and the library re-suspended in 10 ml of 2xTY and 15% glycerol (v/v) for storage in 500 µl aliquots at -80 °C.The successful generation and estimation of the library size was performed by counting the colonies plated on serially diluted 2xTY-agar/Amp/Gluc petri-dishes; a number of single colonies were grown in liquid culture and the plasmid DNA was screened by colony PCR using M13 primers that generated a ~ 1000 bp DNA in the presence of the full-length scFv insert (~ 800 bp).The library glycerol stock was then used to inoculate the starting culture of the phage production for the first biopanning round. Phage production.To produce phage-scFv particles for the biopanning steps, a 2xTY/Amp/Gluc starting culture (OD 600nm : 0.1) was inoculated with the library glycerol stock and grown to OD 600nm : 0.4-0.5, before infection with 100 µl of M13KO17 helper phage (NEB) followed by incubation for 40 min at 37 °C without shaking.The bacteria were then centrifuged and re-suspended in 100 ml 2xTY containing 100 µg/mL ampicillin, 50 µg/ mL kanamycin without glucose overnight at 30 °C shaking at 225 rpm. Next day, the culture was centrifuged at 3300×g for 20 min at 4 °C to remove bacterial cells, 18 ml of ice-cold 20% PEG-6000/2.5 M NaCl was added to the phage-containing supernatant and the solution was incubated 4 °C for 1 h.After centrifugation at 3300×g for 20 min at 4 °C, the supernatant was discarded and the phage-containing pellet was re-suspended in 1.5 ml of dH 2 O.The solution was then centrifuged at ≥ 14,000×g in a microfuge for 5 min to pellet any remaining bacterial cells and the supernatant was transferred into a clean 1.7 ml tube.0.25 ml of ice-cold 20% PEG-6000/2.5 M NaCl added, the solution was incubated at 4 °C for 1 h then centrifuged at ≥ 14,000×g for 10 min; the supernatant was discarded and the final phage pellet was re-suspended in 1 ml of dH 2 O.To assess the titre of phage particles, 10 µl were kept aside for E. coli infection and the remaining solution was divided into two ~ 500 µl aliquots which were blocked with 1 ml of PBS, 4% BSA (w/v) for 1 h at room temperature and subsequently used for the rounds of biopanning. ",
"section_name": "Overlap extension PCR.",
"section_num": null
},
{
"section_content": "CD160-containing supernatant from HEK293T cells and mouse IgG2a Fc purified protein were run by SDS-PAGE alongside a purified commercial source of CD160-His (Sino Biological). 1 µg of purified protein or 5 µl of supernatant were run on to a TGX 4-20% gel according to the manufacturer's protocols (Bio-Rad).Samples were prepared as follows: 5 µL of sample loading buffer, 2 µL of reducing buffer (not included under non-reducing conditions), made up to 20 µL final volume with PBS.Following SDS-PAGE, the gel was covered with Coomassie R-250 stain solution (Sigma-Aldrich) and stained for 60 min with gentle agitation on a rotating plate shaker.Staining solution was decanted and the gel was destained (dH 2 O, methanol, and acetic acid in a ratio of 50/40/10 (v/v/v)) until the protein bands became visible.Images of the proteins gel were acquired using QuantStudio imager (GE Lifesciences). ",
"section_name": "SDS-PAGE.",
"section_num": null
},
{
"section_content": "Protein stability was analysed on a Prometheus NT.48 (NanoTemper Technologies GmbH, Munich, Germany) as previously described 41 . ELISA.Antigens (CD160-Fc HEK supernatant 1:10 dilution; CD160-Fc HEK purified 1 µg/ml, 0.5 µg/ml, 0.25 µg/ml; CD160-Fc CHO purified 1ug/ml, 0.5 µg/ml, 0.25 µg/ml; CD160-His Sino biologicals) were coated in Nunc Maxisorp plates in coating buffer (Biologend, 50 μl/well) O/N at 4C. Plate were washed four times with PBS 0.05% Tween20, and blocked in 2% BSA (200 µl) for 1 h at RT.Primary antibody (BY55-humanFc HEK supernatant) was incubated with 1:3 serial dilutions in PBS 0.5% BSA for 1 h at RT. Plates washed in PBS 0.05% Tween20 4 times and incubated with anti-human HRP (Jackson Immunotools) at 1:3000 dilutions in PBS 0.5% BSA for 1 h at RT. Plates washed four times with PBS 0.05% Tween20, developed using one step TMB ultra (Thermo) 45 μl and stopped with 1 M H 2 SO 4 45 µl.The absorbance of the plate was read at 450 nm and used to determine the EC50 of the different formats of CD160 protein. Biopanning on magnetic beads.The biopanning rounds were performed using Strep-Tactin type II (IBA Lifesciences) magnetic beads coated with the target of interest.Human CD160 was genetically engineered to be fused with a mouse IgG Fc and a Strep-tagII (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) and cloned as a secreted protein into a SFG expression plasmid as described elsewhere 42 .HEK293T cells cultured in complete DMEM medium (Gibco) were transfected with 12.5 µg plasmid DNA using GeneJuice transfection reagent (MerckMillipore).After 48 h, the cell supernatant was harvested and 0.2 µm filtered prior to the coating of magnetic beads.5 µl of beads suspension were incubated with 1.5 ml of filtered cell supernatant for 1 h at room temperature shaking.Supernatant from non-transfected HEK293T cells was used to coat magnetic beads as a negative control during phage selection.The beads were magnetized and washed three times with PBS.The presence of CD160-mIgGFc-Strep-tag II fusion protein on the beads was assessed by flow cytometry of a representative aliquot of NT-supernatant coated beads and CD160-coated beads stained with anti-CD160 antibody (BY55-PE, BD Pharmigen).20 µl of beads coated with NT-supernatant and CD160-supernatant were respectively incubated with the blocked phage particles for 1 h at room temperature shaking.The bead-phage complex was magnetized and washed five times with PBS 0.1% Tween20 and five times with PBS.Phage particles were eluted from the beads by re-suspending the washed beads in 1.5 ml of DMEM 30 mM biotin and incubating for 2 h at room temperature shaking.The beads were removed using a magnet and the supernatant used to infect a 10 ml culture of TG1 E. coli grown to OD 600nm : 0.4-0.5 in 2xTY medium.Every aliquot of bacteria infected with phage was split; a portion was serially diluted and plated on 2xTY-agar/Amp/Gluc plates to assess phage titre.The remaining bacteria were spread on a Bio-Assay dish for the generation of the bacteria glycerol stock for the next round of biopanning, as described in the previous section.Before transduction, T cells were depleted of CD56-expressing NK cells using EasySep human CD56 Positive Selection Kit II (STEMCELL) according to the manufacturer's instructions.The PBMCs were transduced 72 h after stimulation with anti-CD3/anti-CD28 antibodies (Miltenyi) and 100 U/mL human IL-2 (GeneScript) and cultured in complete media RPMI1640, 1% glutamax and 10% FCS.The cells were transduced on retronectin (Takara) coated 6-well plates at 1.2 × 10 6 cells/well in complete RPMI1640 media, 100 U/mL human IL-2, 4 mL of lentiviral supernatant and were spun at 1000 g for 40 min. 5 × 10 4 SupT1 wild-type (NT) or SupT1 CD160-positive cells were used as target cells and co-cultured with CAR-engineered T cells to achieve an effector:target ratio of 1:2, 1:4 and 1:8 in 200 μL of complete media.CARmediated cytotoxicity was assessed by flow cytometry at 24 h and 72 h timepoints.T cells were identified from target cells by staining for human CD3 (UCHT1, Biolegend) and human CD2 (RPA-2.1,Biolegend) and target cells were gated by their lack of huCD3 and huCD2 expression.Viability was assessed with live/dead cell exclusion dye eFluor780 (Thermo Scientific).Viable target cells were enumerated for each co-culture condition by acquiring a fixed amount of counting-beads (CountBright, Thermo Scientific) and the percentage cytotoxicity was calculated by normalising the number of viable target cells to that recovered from co-cultures carried out with non-transduced T cells. Supernatants from the 72 h timepoint were harvested and used to measure the total amount of interferongamma (IFN-γ) and IL-2 produced.Cytokine concentrations were measured using human IFN-γ ELISA MAX kit (Biolegend) and human IL2 ELISA MAX kit (Biolegend) following the manufacturer's protocol. Statistical analysis was performed by two-way ANOVA with Dunnett's post-test for comparison between CARs within the same target group.*p < 0.1, **p < 0.001 and ***p < 0.0001. ",
"section_name": "Differential scanning fluorimetry.",
"section_num": null
}
] |
[
{
"section_content": "MP is supported by the University College London Hospital Biomedical Research Centre funded by the UK National Institute of Health Research.This work was funded by CRUK (515707). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "M.P., F.D. and K.C. designed the primers.M.P. and F.N. devised the panning strategy.F.N. analysed sequences and performed Biacore.F.N. performed the experiments.LM prepared and tested the rat vaccination.Y.B. and B.M. performed primer validation.M.R. and J.Y. helped with the phage library generation.F.P. and P.W. helped with the phage display selection.M.F.performed the protein quality experiments.S.O., C.R., K.C. and M.P. supervised the study, contributed to scientific discussion, data interpretation and manuscript preparation.M.P. and F.N. wrote the manuscript.K.C. introduced the rat immunization concept, provided experimental protocols and contributed to the manuscript.All authors approved the manuscript. The authors declare no competing interests. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "M.P., F.D. and K.C. designed the primers.M.P. and F.N. devised the panning strategy.F.N. analysed sequences and performed Biacore.F.N. performed the experiments.LM prepared and tested the rat vaccination.Y.B. and B.M. performed primer validation.M.R. and J.Y. helped with the phage library generation.F.P. and P.W. helped with the phage display selection.M.F.performed the protein quality experiments.S.O., C.R., K.C. and M.P. supervised the study, contributed to scientific discussion, data interpretation and manuscript preparation.M.P. and F.N. wrote the manuscript.K.C. introduced the rat immunization concept, provided experimental protocols and contributed to the manuscript.All authors approved the manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1158/0008-5472.22376621.v1
|
Supplementary Table 1 from <i>In vivo</i> Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow
|
<jats:p>Supplementary Table 1 from <i>In vivo</i> Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow</jats:p>
|
[
{
"section_content": "",
"section_name": "CLL",
"section_num": null
}
] |
[] |
10.18632/oncotarget.15316
|
Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors
|
Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.
|
[
{
"section_content": "Bruton tyrosine kinase (BTK) inhibitors like ibrutinib have demonstrated high clinical response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL) including refractory patients to conventional therapy or patients with tumor protein p53 (TP53) mutations [1][2][3][4][5].Patients who develop resistance to ibrutinib therapy typically have mutations in either BTK or phospholipase c γ 2 (PLCγ2) [1,6].Mutations in BTK at the C481S hotspot alter the BTK binding site rendering it reversible to binding ibrutinib which results in a loss of therapeutic activity.Alternatively, mutations in PLCγ2, which is immediately downstream of BTK in the B-Cell receptor signaling pathway, result in a gain of function and BTK independent B-Cell Receptor activation [6][7][8] While the emergence of these mutations has been reported to be associated with resistance to therapy, little is known about the development of these resistance mutations throughout the course of therapy.In clinical trials of CLL patients on BTK inhibitor (BTKi) therapy, whole exome sequencing with next-generation sequencing (NGS) has typically been used to detect specific mutations in BTK or PLCγ2 genes [1,6].Therefore, accurate, high-sensitivity assays that can be run in large volumes in a clinical setting are a necessity to further understand the relationship between the appearance of a mutation and the development of resistance to therapy and clinical progression. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Wild-type blocking polymerase chain reaction (WTB-PCR) followed by Sanger sequencing has demonstrated high sensitivity and versatility in the detection of low frequency mutations [9,10].By adding a short (10-12mer) inaccessible [locked or bridged nucleic acid (LNA or BNA)] oligonucleotide, complementary to wild-type hotspot loci, amplification of the wild-type (WT) allele is inhibited, leading to experimentally driven positive selection for mutant alleles.Because a single nucleotide mismatch in the LNA/BNA-DNA hybrid greatly decreases its melting temperature, only mutant template DNA is free to complete its extension.Therefore, WT DNA is amplified linearly but mutant DNA is amplified exponentially [9].BNA is a third generation nucleic acid analog with excellent mismatch discriminating power and is considered more potent in blocking.Its strong nuclease resistant properties coupled with a 3′ phosphate also prevents amplification of the wild-type DNA and selectively amplifies mutant DNA [11,12].The resulting WTB-PCR product can then be sequenced by traditional Sanger sequencing methods.We also theorized that the same principle could be applied to NGS library preparation. While WTB-PCR/Sanger sequencing or WTB-PCR/NGS can provide accurate, high-sensitivity mutation analysis, spatial sampling bias in patients with lymphomas or CLL with few circulating tumor cells and lymph node or organ involvement could potentially lead to false negatives [13][14][15][16][17][18].This is particularly relevant when tumor heterogeneity is considered.The presence of a mutation in a subclone of the tumor cells can be easily missed if the subclone is not circulating or patchy in bone marrow-if bone marrow aspiration is used.In patients with hematologic diseases, the peripheral blood (PB) plasma has been demonstrated to be enriched for tumor-specific DNA, RNA, and proteins [19][20][21][22].This is especially true for the DNA of the more aggressive subclone.Testing cell-free DNA (cfDNA) from plasma or serum may therefore provide greater sensitivity for detecting resistance mutations than cellular DNA from PB. In this study we describe the development of highly sensitive Sanger and next-generation sequencing strategies for detecting mutations in BTK and PLCγ2 based on WTB-PCR.Using this technology, we demonstrate the development of multiple resistant clones in patients with CLL treated with ibrutinib as they develop resistance to therapy. ",
"section_name": "Research Paper",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Using the HS assay with WTB-PCR greatly increased sequencing sensitivity when compared to the conventional assay with T-PCR.The conventional assay was able to detect approximately 15-20% mutant allele in a background of WT allele, while WTB-PCR was able to detect as low as 0.1% (Figure 1A).Similar sensitivities were obtained for the PLCγ2 sequencing, however, some loci were more amenable to positive selection by WTB-PCR than others.Sensitivities for exon 19, 20, and 24 of PLCγ2 were 0.2, 1, and 1%, respectively, mutant allele in a background of WT by HS assay (data not shown). ",
"section_name": "Significant improvement of sensitivity in detecting BTK and PLCγ2 mutations using HS assay",
"section_num": null
},
{
"section_content": "Using HS sequencing we tested samples from 44 patients with newly diagnosed CLL or after therapy with FCR.None of these patients showed mutations in BTK exon 15 or PLCγ2 Exon 19, 20 and 24 genes. ",
"section_name": "Lack of mutation in BTK and PLCγ2 in patients with BTKi-naïve CLL",
"section_num": null
},
{
"section_content": "We tested 63 samples from 16 patients with CLL treated with ibrutinib who were suspected of resistance or who showed evidence of progression while on therapy.Using conventional Sanger sequencing only 21% of tested samples showed mutations in BTK, while the HS testing showed mutations in 43% of tested samples (P < 0.00001).Mutations in PLCγ2 were detected in 5% of tested samples using conventional Sanger sequencing and in 33% of samples using HS testing (P < 0.00001).The mutations detected included BTK: C481S and C481R; PLCγ2: R665W, L845F, S707Y, P664S, P664L, Ser707TyrdelAlaTyr (6NT deletion).Without HS testing 56% of positive samples would have been missed for BTK (N = 27) and 85% of PLCγ2 (N = 20) would have been missed.No mutations detected by the conventional assay were missed by the HS assay. ",
"section_name": "Mutations in BTK and PLCγ2 after BTKi detected by HS",
"section_num": null
},
{
"section_content": "Overall, of the 16 patients on therapy with ibrutinib and suspected resistance or disease progression, 11 (69%) had a mutation in either BTK or PLCγ2, 6 (37.5%) patients had mutations in both genes, and 2 (12.5%) patients had three or more mutations that were detected by HS assay.By comparison, using conventional assay only 6 (37.5%) patients had mutations in either BTK or PLCγ2, 1 (6.3%) had mutations in both genes, and 1 (6.3%) patient had three or more mutations.More than half of the patients with mutations (55%, N = 11) had multiple drug resistant mutations that are detectable by the HS assay and two patients had 5 separate mutations (Table 1).The fact that we were able to see three separate subclones (as determined by NGS; Figure 1B) in at least one patient (Patient # 4) suggests that these other mutations also exist in separate subclones.Without HS testing, 83% of the additional clones would have been missed. Seven of the mutations in PLCγ2 (87.5%, N = 8) and three mutations in BTK (33%, N = 9) that were detectable by HS assay at progression were undetected by conventional assay.Median percentage of CLL cells in these samples as tested at progression was 58% (N = 8, range = 7-93%) as determined by flow cytometry. ",
"section_name": "Multiple subclones with BTK and PLCγ2 mutations in BTKi resistant patients detected using HS sequencing",
"section_num": null
},
{
"section_content": "In general, resistance mutations in BTK or PLCγ2 were detected by NGS in all tested samples, except for two samples: Patient # 5, who had a very low frequency PLCγ2 Exon 20 6NT deletion and patient # 3, who had two low frequency PLCγ2 Exon 19 R665W and Exon 20 S707Y mutations.The addition of BNA/LNA oligonucleotides enriched for BTK and PLCγ2 hotspot mutations (Table 2 and Figure 1B).In addition, NGS showed that when multiple mutations were detected in one sample, these mutations were not in tandem and were therefore present in different strands of DNA (Figure 1B).In particular, a sample from patient # 4, in which three BTK mutations were detected, the three mutations were completely independent events existing in separate DNA strands, thus suggesting different subclones. ",
"section_name": "Next-generation sequencing and improvement of sensitivity using blocking oligonucleotides",
"section_num": null
},
{
"section_content": "We performed parallel HS sequencing of 9 temporally matched pairs of plasma cfDNA and cellular DNA.Of these 9 pairs, 4 parallel cfDNA isolated from serum were also tested.Of the 9 plasma cfDNA samples, 7 (78%) showed mutations in BTK and 4 (44%) showed mutations in PLCγ2.The cellular DNA showed mutations in BTK in 7 (78%) samples, but only 2 (22%) mutations were detected in the PLCγ2 gene.Of the 4 serum cfDNA samples, only 1 (25%) showed a mutation in BTK, and 1 (25%) had a mutation in PLCγ2 (Table 3A). All mutations detected by either cellular DNA or serum cfDNA were also detected by plasma cfDNA. 2 out of 4 (50%) PLCγ2 mutations identified by plasma cfDNA were not detected by cellular DNA in the total 9 paired samples. 2 out of 3 (67%) of both BTK and PLCγ2 mutations identified by plasma cfDNA were not detected by serum cfDNA in the total 4 paired samples (Table 3B).It is difficult to quantify the mutation percentage using Sanger or WTB-PCR and sequencing, but overall, mutant peak was relatively significantly stronger in plasma samples as compared with cell samples in almost positive cases. The number of patients is too small and we cannot determine if plasma positivity correlated with more enlarged lymph node or lower number of circulating lymphocytes, since most patients with progression also had enlarged lymph nodes.Some of the patients with detectable mutations in cells also had low percentage of circulating CLL cells. ",
"section_name": "Testing using cell-free DNA",
"section_num": null
},
{
"section_content": "Given the association of BTK and PLCγ2 mutations with resistance to ibrutinib therapy, an accurate, highly sensitive assay-capable of being run in large volume-is a necessity.Using WTB-PCR with Sanger sequencing or NGS has multiple advantages in the clinical setting.Increased sensitivities of up to 0.1% mutant allele in a background of wild-type (Figure 1A) could allow clinicians to detect the presence of resistance mutations early on during the course of therapy.Knowing early on when mutations conferring resistance to therapy emerge and that the majority of the time they co-develop alongside additional sub-clones with resistance mutations (Table 1) may be very helpful in a strategy to overcome evolving resistance by, for example, adding additional therapeutic agents. WTB-PCR/Sanger or WTB-PCR/NGS testing allows broad coverage of mutation hot-spots and the detection of undiscovered mutations; they also provide adequate internal controls for ruling out false positives.Its additional utility in revealing low frequency mutant populations, especially in plasma cfDNA is invaluable and will guide future research. Because we know the limit of detection for the conventional assay is approximately 15%, mutations that are detectable by HS assay but not by conventional assay are therefore present in only a small fraction of CLL cells at progression despite the relatively high CLL cellularity in the tested samples (Median = 58%).The low percentage of CLL with the resistance mutations at time of progression implies that these mutations may have secondary effects on CLL cells lacking BTKi resistance mutations perhaps via tumor microenvironment resulting in loss of therapeutic activity [23][24][25].We are analyzing peripheral blood samples and there is a possibility that lymph nodes may contain more significant number of cells with mutation.However, testing lymph nodes might not be a practical approach, especially when these lymph nodes are deep and not easily accessable.Furthermore, the pattern of multiclonal BTK inhibitor resistance is unique, perhaps because of the chronic nature of the disease.In more acute malignancies, clonal evolution is typically Mutational status of BTK and PLCγ2 was determined by conventional and high-sensitivity (HS) Sanger sequencing.\"% CLL/WBC\" indicates the percentage of CLL cells of white cell count in the tested samples as determined by flow cytometry.Abbreviation: Pat = Patient, HS = high-sensitivity, WBC = white blood cells, WT = wild-type/unmutated. linear with one subclone outcompeting the others and giving rise to resistance [26,27].In these cases, however, we observed that 6 of the 16 patients at progression have multiple, persisting subclones (Table 1).Plasma cfDNA from PB is more sensitive for detecting resistance mutations than cellular DNA or serum cfDNA (Table 3, Figure 2).While no cellular DNA or serum cfDNA samples with mutant BTK or PLCγ2 were wildtype in their respective plasma cfDNA samples, 50% of the PLCγ2 mutations detected in plasma DNA were not detected in their respective cellular DNA samples.Furthermore, 67% of both BTK and PLCγ2 mutations detected in plasma were not detected in serum.Unfortunately no more plasma samples from patients with progression were available for testing.Although more testing of more plasma samples is needed for confirmation, this approach may be very useful in screening patients for resistance mutations, especially in patients with lymphomas or CLL with few circulating tumor cells and lymph node or organ involvement.Because the resistance mutations allow these cells to proliferate despite BTKi therapy, one plausible explanation for this result is that the increased proliferative rate and consequent improper processing of the cellular contents results in plasma enriched with DNA derived from CLL cells possessing resistance mutations.In serum, the coagulation process may unintentionally lyse fragile cells like granulocytes, which has the effect of diluting the serum with non-tumor cfDNA. In conclusion, our data indicates that incorporating WTB-PCR into Sanger sequencing or NGS is a highly sensitive and invaluable tool in screening and monitoring patients on ibrutinib or other BTKi therapy for resistance mutations.Additionally, plasma from peripheral blood may be more sensitive than serum and even cells in detecting the presence of these resistance mutations.Although the number of cases is small and further confirmation is needed, using these tools we show that multiple low-frequency subclonal populations of CLL with resistance mutations in BTK and PLCγ2 are quite common in patients who progress on therapy with ibrutinib.Using this information we can monitor, with increased accuracy, patients on BTKi therapy and make more informed therapy decisions when we detect the presence of mutations known to result in a loss of therapeutic activity.All samples used were from patients with suspected progression.High-sensitivity NGS includes BNA/LNA oligonucleotides in library preparation and conventional NGS does not. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Samples were collected from ibrutinib-naive CLL patients as well as from patients treated with ibrutinib as a part of the single-arm, phase-2 study of single agent ibrutinib in CLL with and without 17p deletions conducted at the NIH (NCT01500733).We tested 44 DNA samples from BTK inhibitor naïve patients with CLL by the high-sensitivity (HS) assay for mutations in BTK exon 15 and PLCγ2 Exon 19, 20, and 24.This included samples from PB, bone marrow aspirate, and fresh lymph node tissue.We also tested 16 patients with CLL that were on ibrutinib therapy and had suspected resistance or disease progression.The clinical characterstics of these patients is described in details in reference [28].Briefly, progression was defined as ≥ 50% increase in sum of the product of (A) High-sensitivity testing of 9 temporally matched plasma and cellular samples from the same patients of which 4 serum samples were also available indicates that plasma may be enriched for tumor specific DNA more so than serum and cells.(B) Comparison of mutation status between paired sample-types.the diameters of representative lymph nodesor ≥ 50% increase in absolute lymphocyte count, confirmed in two consecutive assessments and with an absolute B-cell count > 5,000/uL [28].These duration of ibrutinib therapy in these patients varied between one and three years [28]. Prior treatment data was available for 15 of the 16 patients.Median time from CLL diagnosis until beginning ibrutinib therapy was 5.1 years (range: 0.6-14.9). 3 patients were treatment naïve and 12 were relapsed/refractory (RR) CLL; median number of prior therapies for RR patients was 3.5 (range: 1-6).From these 16 patients we tracked the emergence of resistance mutations in BTK or PLCγ2 by both the HS and conventional assay using a total of 63 samples collected over a 43-month period.This also included samples from PB cells (N = 39), plasma (N = 10), serum (N = 11), and bone marrow aspirate (N = 3).Samples were either de-identified and tested according to IRBapproved protocol or tested after obtaining a consent form. From these samples we also performed HS sequencing on 9 temporally matched pairs of plasma cfDNA and cellular DNA.Of these 9 pairs, 4 parallel cfDNA samples isolated from serum were also tested. DNA extraction: We extracted DNA from PB cells, bone marrow aspirate, and fresh tissue using the QIAamp DNA Mini Kit (Qiagen; Venlo, Netherlands) in both manual and automated (QIAcube) extractions according to manufacturer's instruction.Extracted DNA was then quantified using a Nanodrop 2000 (Thermo Fisher Scientific; Waltham, MA, U.S.A.) instrument and adjusted to approximately 50-100 ng/µL with H 2 O. Total nucleic acid was extracted from PB plasma and serum via the NucliSenS EasyMAG automated platform (BioMerieux; Marcy-l'Étoile, France).DNA was then quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific; Waltham, MA, U.S.A.) and adjusted accordingly. ",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "The BTK inhibitor resistance assays were developed to amplify exon 15 of BTK and exon 19, 20 and 24 of PLCγ2.0.25 µL FastStart Taq DNA Polymerase, 5 U/µl in storage and dilution buffer Fast Start Taq DNA polymerase (Roche; Basel, Switzerland), 2.5 µL PCR reaction buffer 10× w/ 20 mM MgCl 2 , 250 µM dNTPs (Invitrogen; Waltham, MA, U.S.A.), 0.4 µM forward primer, 0.4 µM reverse primer (IDT; Coralville, IA, U.S.A.) (Table 4), and 2 µL genomic DNA (50-100 ng/µL) were added to DNAse, RNAse-free, ultra-pure H 2 O to create a final solution volume of 25 µL per reaction.All PCR primers were designed with a 5′-M13 sequence (M13forward: tgt aaa acg acg gcc agt; M13-reverse: cag gaa aca gct atg acc) to allow for annealing of complementary sequencing primers.The HS assays were identical to their conventional counterparts except for the addition of BNA (Bio-Synthesis; Lewisville, TX, U.S.A.) or LNA (Exiqon; Woburn, MA, U.S.A.) oligonucleotides A3, B3, C3, and D3 (Table 4) being added to the master mixes of BTK and PLCγ2 Exon 19, 20, and 24, respectively.A3 was added to the BTK master-mix at 4 µM; B3 to PLCγ2 Exon 19 at 4 µM; C3 to PLCγ2 Exon 20 at 40 nM; D3 to PLCγ2 Exon 24 at 4 µM.The LNA oligos were designed to feature a 3′ inverted dT to inhibit both extension by DNA polymerase and degradation by 3′ exonuclease.The BNA oligos were designed with a 3′ phosphate for the same reason.All reactions were subjected to identical thermocycler settings; initial denaturation at 95°C for 6 minutes; 40 cycles of denaturation at 95°C for 30 seconds, primer annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute 20 seconds; this was followed by a final extension at 72°C for 10 minutes.PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter; Brea, CA, U.S.A.), bi-directionally sequenced using a BigDye Terminator v3.1 Cycle sequencing kit (Life Technologies; Waltham, MA, U.S.A.), and subjected to ethanol precipitation.The precipitated DNA was then resuspended in 10 µL Hi-Di Formamide (Life Technologies; Waltham, MA, U.S.A.), denatured at 95°C for 3 minutes, and run on the ABI 3730XL sequencer.Sequencing data were base-called by sequencing software and analyzed by ABI Prism ® SeqScape software. In order to determine the sensitivity and limit of detection, dilution series experiments with genomic or amplicon DNA were carried out.Genomic DNA taken from samples that tested positive for BTK (C481S) or PLCγ2 (R665W) or amplicon DNA with PLCγ2 (S707Y, L845F) mutations were quantified using a Qubit dsDNA high-sensitivity assay kit (Invitrogen; Waltham, MA, U.S.A.).This DNA was serially diluted with WT DNA of the same type. ",
"section_name": "High-sensitivity and conventional sanger DNA sequencing",
"section_num": null
},
{
"section_content": "We applied the WTB-PCR principle to custom SureSelect QXT Target Enrichment (Agilent; La Jolla, CA) and Nextera Rapid Capture (Illumina; San Diego, CA) panels with the addition of the BNA/LNA oligonucleotides (A3, B3, C3, D3; Table 4) in order to increase our limit of detection for the hotspot mutations in hybrid-capture based NGS.A3, B3, and D3 were added to library preparation at a working concentration of 2 µM and C3 was added at 40 nM.Both panels cover 315 genes that include the BTK and PLCγ2 genes.One sample that was positive for resistance mutations in BTK and PLCγ2 was tested by Nextera Rapid Capture based assay with and without WTB-PCR in order to determine if mutant enrichment could be achieved in the NGS setting.One additional sample with resistance mutations was tested by the same assay without WTB-PCR.The SureSelect QXT Target Enrichment based assay with WTB-PCR was used on 3 additional samples with resistance mutations. ",
"section_name": "Next-generation DNA sequencing",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": ": Work for a diagnostic company offering testing for BTK and PLCγ2.Adrian Wiestner: Receives research funding from PCYC.GRANT SUPPORT IA, MF, and AW are supported by the Intramural Research Program of the NHLBI and NCI. ",
"section_name": "",
"section_num": ""
}
] |
10.1186/s13045-015-0157-x
|
Targeting neoplastic B cells and harnessing microenvironment: the “double face” of ibrutinib and idelalisib
|
Tyrosine kinase inhibitors (TKIs) targeting signaling molecules downstream B cell receptor (BCR) are powerfully spreading in the therapeutic landscape of B cell lymphoproliferative disease, due to a manageable toxicity profile and encouraging clinical effectiveness. In particular, ibrutinib, previously called PCI-32765, is a potent inhibitor of Bruton tyrosine kinase (Btk), recently approved for the treatment of relapsed mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Moreover, idelalisib (formerly GS-1101 and CAL-101) is a selective reversible inhibitor of the p110δ isoform of phosphoinositol 3 kinase (PI3K) approved for the treatment of patients with relapsed follicular lymphoma (FL) and CLL. These agents directly affect the neoplastic clone, disrupting the supportive platform provided by BCR signaling cascade and by other microenvironmental mutualistic interactions, and also interfering with chemokine gradients and adhesive properties of neoplastic B cells. In the present review, we describe the clinical efficacy of ibrutinib and idelalisib in CLL and B cell non-Hodgkin lymphoma (B-NHL), then focusing on the mode of action (MOA) of these TKIs towards the neoplastic B cell compartment. At last, the review would further expand the view on potential additional targets of ibrutinib and idelalisib belonging to other microenvironmental cellular elements.
|
[
{
"section_content": "Signaling from the B cell receptor (BCR) is essential for normal B cell development and controls several cellular functions such as proliferation, apoptosis, differentiation, and cell migration [1].Constitutive activation of BCR is a common hallmark of B cell malignancies, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and diffuse large B cell lymphoma (DLBCL), observation that has led the design of a novel panel of inhibitors targeting kinases responsible for BCR signal transduction.In particular, Bruton tyrosine kinase (Btk) and phosphoinositol 3 kinase (PI3K) δ are pivotal signaling molecules early positioned downstream the BCR cascade.Ibrutinib, previously called PCI-32765, is a potent (IC 50 0.5 nmol/L) inhibitor of Btk that is inactivated through irreversible covalent binding to Cys-481 in the ATP-binding domain of the kinase.Idelalisib (formerly GS-1101 and CAL-101) is a selective reversible inhibitor of the p110δ isoform of PI3K.The encouraging results of clinical trials using these tyrosine kinase inhibitors (TKIs) envision a possible shift towards a nonchemotherapy treatment era in B cell lymphoproliferative diseases. In the present review, we focused on ibrutinib, approved by the Food and Drug Administration (FDA) in November 2013 for the treatment of relapsed MCL and in February 2014 as a second-line treatment for CLL and idelalisib, which was approved by FDA in July 2014 for the treatment of patients with relapsed FL or relapsed small lymphocytic lymphoma (SLL) and CLL, pointing the attention to the clinical efficacy in CLL and B cell non-Hodgkin lymphomas (NHL), the antitumor mode of action (MOA) and the possible effects of these drugs on non-malignant cells in tumor microenvironment. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Clinical efficacy of TKIs",
"section_num": null
},
{
"section_content": "Since the first evaluation of ibrutinib in a phase 1 trial in a wide setting of B cell malignancies, it was demonstrated that this orally administered molecule was well tolerated and induced clinical objective responses (60 %) across B cell histologies, with highest response rate in MCL and CLL patients [2].Ibrutinib treatment was then investigated in relapsed CLL patients in a phase 1b/2 multicenter clinical trial conducted on 85 patients (33 % with 17p13 deletion), reporting an overall response rate (ORR) of 71 %, with two complete responses (CR), and further 18 % with partial response (PR) with lymphocytosis.At 26 months, the progression-free survival (PFS) was 75 % [3].The response to ibrutinib did not vary according to traditional high-risk prognostic factors, with 68 % of response in 17p13-deleted setting, even if most events associated with disease progression occurred in patients with high-risk cytogenetics (PFS = 57 %) (Table 1).The most common adverse events were diarrhea, fatigue and upper respiratory tract infections.The events of infection mainly occurred in the early phase of treatment, but collectively, the incidence of grade 3 infections did not result increased as compared to rates reported after traditional therapies.It was also reported that the levels of IgG and IgM remained relatively stable, whereas IgA increased throughout treatment [3].The efficacy of ibrutinib in previously treated CLL was also confirmed in a phase 3 multicenter study in which 391 patients were randomized to receive ibrutinib or anti-CD20 antibody ofatumumab [4].The ORR was 43 % (all partial responses) with additional 20 % PR with lymphocytosis in ibrutinib-treated patients as compared to only 4 % in the ofatumumab arm.Ibrutinib in comparison to ofatumumab significantly prolonged the duration of PFS and the rate of OS, regardless of high-risk features.The frequencies of any grade or grade >3 infections in the ibrutinib group were 70 and 24 %, respectively [4].Very recently, Byrd and colleagues reported updated observations indicating that ibrutinib treatment is well tolerated in CLL patients for an extended period (3-year follow-up) and responses are durable and improves in quality (median time to CR, 21 months), being disease progression uncommon, and primarily occurring in the high-risk patient settings with 17p or 11q deletions in leukemic clone [5].Ibrutinib is currently investigated in combination with both chemotherapy and monoclonal antibodies to reduce the extent of lymphocytosis and to achieve higher frequencies of complete responses.Burger and colleagues reported the results of a phase 2 study conducted on 40 high-risk CLL patients treated with ibrutinib plus rituximab [6].The ORR was 95 %, with 87 % PR and 8 % CR, and the PFS at 18 months was 78 % for all patients and 72 % for 17p-deleted/mutated patients.Infections were the most common adverse effects.Overall, the combination strategy was associated with a shorter duration of lymphocytes redistribution as compared to ibrutinib alone, and complete remissions were achieved in more patients.A randomized study comparing ibrutinib versus ibrutinib plus rituximab is ongoing (NCT02007044).As first-line monotherapy, a phase 1b/2 multicenter trial conducted on 29 elderly symptomatic CLL patients reported encouraging results with 71 % ORR (13 % CR) and rare hematological toxicity and infections, indicating that ibrutinib treatment is well tolerated and effective in elderly setting.Median serum levels of IgA increased during treatment, no changes in IgM and IgG over time [7].As upfront strategy, two phase 3 trials are ongoing comparing the efficacy of chlorambucil or ibrutinib in elderly CLL (RESONATE-2, NCT01722487) and the efficacy of bendamustine-rituximab versus ibrutinib-rituximab versus ibrutinib (NCT01886872).In a very recent phase 1b study evaluating the safety and efficacy of ibrutinib in combination with chemoimmunotherapy (CIT) (bendamustine, rituximab (BR); fludarabine, cyclophosphamide and rituximab (FCR)) in patients with relapsed/refractory CLL, the ORR in the BR-ibrutinib group was 93 % with 40 % CR with an expected toxicity profile and PFS 70 % at 36 months; all three patients receiving FCR-ibrutinib achieved CR.The treatment-related lymphocytosis was reduced, but not completely absent when ibrutinib was combined to BR [8].A phase 3 randomized trial combining ibrutinib with BR in relapsed/refractory CLL/ SLL (NCT01611090) and a phase 2 study combining ibrutinib with FCR in untreated, young CLL patients (NCT02251548) are ongoing.Another recent report of a phase 2 study conducted on 51 treatment naïve and relapsed/refractory CLL patient harboring TP53 aberrations reported activity of single-agent ibrutinib in this high-risk subset with ORR at 24 weeks of 92 % (50 % PR and 42 % PR with lymphocytosis) and PFS at 24 months of 82 % [9].Rapid disease control was achieved in all tissue compartments and durable responses were reported, but deep remissions were rare, even in previously untreated patients.Noteworthy, subclones carrying 17p13 deletion seemed equally sensitive to ibrutinib as those without the deletion [9]. Ibrutinib also showed antitumor activity in several types of NHL as single agent or in combination [2,10].Wang et al. reported the results of a phase 2 study conducted on 111 patients with relapsed or refractory MCL treated with a daily dose of 560 mg of single-agent ibrutinib.The treatment showed durable efficacy with ORR of 68 % (21 % CR) and PFS of 14 months [11].There was an increase of MCL cells in blood 10 days after treatment initiation in 34 % of patients, with a subsequent decline in these cells to near baseline by day 28 [11].In patients with relapsed DLBCL, ibrutinib showed preferential activity against tumors with the activated B cell-like (ABC) subtype with a response of 40 % [12].In a phase 1b study, 32 patients with B-NHL received ibrutinib plus rituximab, cyclophosphamide, doxorubicin, 1).In addition, idelalisib was well tolerated, not leading to myelosuppression or an increase in risk of infection as compared to the level already reported in the heavily pretreated CLL population [14].The combination of idelalisib plus rituximab was inspected in 220 relapsed CLL in a phase 3 multicenter randomized trial that reported acceptable safety profile and improvement in ORR (81 vs. 13 %, all PR), in PFS at 6 months (93 vs. 46 %) and in OS at 12 months (92 vs. 80 %) in the idelalisib group as compared to the placebo group [15].As in the case of ibrutinib, the addition of rituximab to idelalisib blunted and shortened the duration of treatment-related lymphocytosis. Idelalisib was also evaluated in two phase 1 studies [16,17], enrolling 40 patients with relapsed/refractory MCL and 64 patients with relapsed indolent NHL, respectively.In MCL, the ORR was 40 % with 85 % of patients having a reduction in lymph node size and 5 % of CR.Treatment-related lymphocytosis was infrequent in MCL setting and the median PFS was 3.7 months [16].The response rates reported in MCL treated with idelalisib are comparable to those obtained with other single-agent treatments, including bortezomib, lenalidomide and temsirolimus, but the response duration seems particularly brief.Idelalisib is well tolerated and active also in heavily pretreated, relapsed/refractory patients with indolent NHL, including FL, SLL, marginal zone lymphoma (MZL), and lymphoplasmacytic lymphoma (LPL), showing ORR of 47 % and median PFS at 7.6 months [17].Gopal et al. reported the results of a phase 2 trial conducted on 125 patients with relapsed indolent NHL treated with single-agent idelalisib confirming the antitumor efficacy (ORR = 57 %, with 6 % CR and median PFS of 11 months) and an acceptable safety profile with low rates of discontinuation due to toxicity and a low incidence of severe adverse events in this setting [18].Phase 3 trials of idelalisib in combination with rituximab (NCT01838434) and bendamustine/rituximab (NCT01569295) are underway. ",
"section_name": "Ibrutinib",
"section_num": null
},
{
"section_content": "",
"section_name": "Ibrutinib and idelalisib targeting neoplastic B cells",
"section_num": null
},
{
"section_content": "Uncontrolled BCR signaling plays a major role in the development and progression of B cell NHL and CLL.Btk is required for intracellular transduction of BCR signaling.In fact, Btk is activated by the upstream Srcfamily kinases Blk, Lyn, and Fyn, and in turn, phosphorylates phospholipase-Cγ (PLCγ) leading to calcium mobilization and activation of nuclear factor κB (NF-κB), and mitogen-activated protein kinase (MAPK) pathways.Moreover, Btk mutations cause the inherited disease X-linked agammaglobulinemia (XLA) in human and X-linked immunodeficiency (Xid) in mice, implying lack of peripheral blood B cells and low levels of serum immunoglobulins [19,20].So, the primary deficit associated with Btk disruption is B cell specific.In addition, Btk is involved in signaling of other cell-surface receptors, such as chemokine receptors and adhesion molecules that are essential for B cell trafficking and homing.Btk belongs to tyrosine kinase expressed in hepatocellular carcinoma (Tec) kinase family also including Bmx, inducible T cell kinase (Itk), resting lymphocyte kinase (Rlk) and Tec, preferentially expressed in the hematopoietic system.In particular, Tec/Btk and Itk/Rlk are mediators of BCR and T cell receptor (TCR) signaling, respectively.Itk is also expressed in mast cells and natural killer (NK) cells. Btk shows constitutive activity in CLL cells and is a critical kinase for CLL development and expansion [21].Ibrutinib exerts a direct effect by disrupting the BCR and NF-κB signaling.In particular, ibrutinib MOA comprises (i) modest induction of apoptosis dependent on caspase pathway activation, (ii) inhibition of proliferation, (iii) prevention of CLL response to survival stimuli provided by microenvironment, and (iv) impairment of migration and adhesion [22][23][24] (Fig. 1).Treatment with ibrutinib also mediates apoptotic stimuli on normal B cells but at lower extent in respect to CLL cells.CLL cells have been shown to effectively increase the survival in response to several microenvironmental stimuli such as CD40L, B cell activating factor (BAFF), TNFα, IL6 and IL4 or contact with stromal cells and nurse-like cells (NLCs).Ibrutinib significantly counteracted the protection mediated by such stimuli on CLL cells [23,22].Noteworthy, CpG-and NLC-induced CLL proliferation was also inhibited by treating cells with ibrutinib.In NOD/SCID/γchain (NGS) mouse xenograft model, Herman et al. [25] recapitulated ibrutinib-mediated inhibition of BCR and NF-κB signaling, attenuation of CLL proliferation and survival, and reduction of tumor burden in vivo [25].Concordantly, ibrutinib was shown to down-regulate BCR and NF-κB signaling, decrease the expression of surface activation markers CD69 and CD86, and reduce proliferation of CLL cells from both peripheral blood and tissue compartments in treated patients [26,27].In addition, treatment with ibrutinib impaired actin polymerization and migration in response to tissue-homing chemokines (CXCL12, CXCL13) and also down-regulated the expression of BCR-dependent chemokines (CCL3, CCL4).The ability of ibrutinib to cause transient early lymphocytosis and profoundly inhibit CLL progression was modeled in an adoptive transfer TCL1 mouse setting, resembling the transient surge in peripheral lymphocyte count characteristically observed in CLL patients treated with ibrutinib [22].In fact, in ibrutinib-treated CLL patients, a fraction of leukemic cells equal to 23 % of tissue disease burden redistributes into the blood, leading to a rapid resolution of enlarged lymph nodes [28].Overall, it was clearly demonstrated that ibrutinib impairs BCR-or chemokine-controlled integrinmediated adhesion or migration, implying attenuated retention and homing of CLL cells in tissue compartment, thus explaining transient peripheral lymphocytosis seen in treated patients. Davis et al. [29] firstly identified Btk in a siRNA screen as an essential kinase for survival of ABC-DLBCL, indicating ibrutinib as an agent able to promote apoptosis [29]. In spontaneous canine lymphoma model, ibrutinib was reported to efficaciously inhibit BCR signaling downstream of Btk and induce objective clinical responses [30].It was clearly demonstrated that BCR and MYD88 signaling pathways together with sustained expression of IRF4 promote ABC-DLBCL survival by inducing NF-κB.Treatment of ABC-DLBCL cells with ibrutinib decreased IRF4 protein, reduced NF-κB signaling, increased interferon (IFN) β production, and also synergized with lenalidomide in killing lymphoma cells in vitro and in mouse model [31].Furthermore, Btk is robustly expressed in malignant plasma cells from >85 % of multiple myeloma (MM) patients and in lymphoplasmacytic cells from Waldenström macroglobulemia (WM).In MM, ibrutinib was shown to decrease CXCL12-mediated migration, down-modulate CCL3, and affect MM cell growth and survival triggered by IL6 or contact with stromal cells [32,33].Similar inhibitory effects on proliferation and survival, CCL3/CCL4 secretion, and CXCL12 signaling occur in hairy cell leukemia (HCL) treated with ibrutinib [34].The δ isoform is one of the four catalytic isoforms of PI3K class I, also comprising p110α, p110β, and p110γ.The p110α and p110β are ubiquitously expressed, whereas the p110δ isoform is generally restricted to hematopoietic cell type and highly expressed in lymphoid cells.B cell defects, comprising lack of B1 lymphocytes, reduced number of mature B cells and impaired antibody production were reported in mice deleted or mutated PI3Kδ, whereas knockout mice for PI3Kγ showed predominantly T cell defects [36]. PI3Kδ is homogenously expressed in CLL cells and also present in normal B cells, even if it shows higher intrinsic in leukemic cells.It is also expressed in normal T cells and NK cells [37,38].Pre-clinical studies demonstrated that idelalisib exerts a dose-dependent cytotoxicity on CLL cells primarily by induction of caspase-dependent apoptosis, instead showing less effect on normal B cells.In CLL cells, idelalisib abrogates the protective effect of many microenvironmental signals including CD40L, BAFF, TNFα, ET1, fibronectin adhesion as well as contact with stromal cells and NLC [38][39][40] (Fig. 1).The PI3Kδ signaling is essential for B cell response after BCR stimulation [41,42].Accordingly, when CLL cells were treated with idelalisib, the prosurvival effect of BCR stimulation was abrogated [39].Clinical observations in idelalisib-treated CLL patients evidenced a redistribution of CLL cells from tissue to peripheral blood, suggesting a possible interference of idelalisib with leukemic cell migration and adhesion.In vitro studies demonstrated that idelalisib inhibits CLL chemotaxis and adhesion to stromal and endothelial layers [39,43].Noteworthy, it was demonstrated that idelalisib sensitizes CLL to the cytotoxic action of several drugs, such as fludarabine, bendamustine and dexamethasone, and also prevents some lenalidomide effects on CLL, i.e., cell activation, internalization of CD20 and secretion of pro-angiogenic factors [44,39].Moreover, idelalisib dramatically increased apoptosis mediated by histone deacetylase inhibitors in NHL and CLL cells [45].MCL and MM cells show constitutive activation of Akt that is dependent on PI3Kδ signaling.Thus, idelalisib induced apoptosis on MCL and overcame MM growth conferred by IL6, insulin-like growth factor 1 and co-culture with marrow stromal cells [46,47]. ",
"section_name": "Ibrutinib",
"section_num": null
},
{
"section_content": "",
"section_name": "Ibrutinib and idelalisib harnessing microenvironmental cells",
"section_num": null
},
{
"section_content": "NK cells are innate effectors that recognize and eliminate virally infected and transformed cells, by secreting preformed granules containing perforin and granzymes and also by inducing apoptosis of target cells throughout activation of death receptors.Btk is not only critical for the B development and differentiation but also regulates NK cell innate function.NK cells up-regulate Btk expression during maturation and activation, and NK cells lacking Btk show reduced toll-like receptor (TLR)-triggered immune response with low expressions of IFNγ, perforin, and granzyme B and impaired cytotoxic activity [48].Btk has also a critical role in regulating NK activation in response to antigen-presenting cells [49].In addition, Itk mediates calcium mobilization, granule release and cytotoxicity in Fc receptor (FcR)-stimulated NK [50].In line with these data, the inhibition of Btk and Itk by ibrutinib might impair NK function and counteract the mechanisms of action of therapeutic anti-CD20 antibodies (rituximab, obinutuzumab and ofatumumab) including FcR stimulation and antibody-dependent cell-mediated cytotoxicity (ADCC).Rituximab is currently approved for the treatment of B cell NHL and CLL in combination with chemotherapy, whereas ofatumumab and obinutuzumab are in clinical trials.All therapeutic anti-CD20 antibodies act throughout immune-mediated mechanisms, including complement-dependent cytotoxicity (CDC), ADCC and antibody-dependent phagocytosis (ADCP) by macrophages and neutrophils, and also through direct cell death in case of obinutuzumab.Ibrutinib was reported to inhibit rituximab-induced NK cell cytokine secretion, NK cytotoxic effect towards coated tumor cells and also to prevent FcR-stimulated NK cell degranulation.Thus, it seems that the concurrent administration of ibrutinib and rituximab determines an antagonistic effect in vitro, with the reduction of antitumor efficacy of rituximab as a result of ibrutinib-mediated inhibition of FcR-stimulated NK cell function, specifically ADCC [51] (Fig. 2).It is likely that the ibrutinib-mediated impairment of NK functions is mainly driven by Itk inhibition.As consequence, selective Btk inhibitors or alternative ibrutinib dosing schedule may overcome the observed antagonistic effect of these agents.Very recently, De Roit et al. demonstrated that pretreatment with ibrutinib does not impair direct cell death or complement-mediated lysis of leukemia cells by anti-CD20 antibodies but strongly inhibits all cellmediated mechanisms [52]. Idelalisib was reported to inhibit the immune cellmediated mechanisms of anti-CD20 antibodies even if at weaker extent as compared to ibrutinib [52].In addition, ibrutinib, fostamatinib and idelalisib were reported to down-regulate CD20 expression in some cell lines and in primary CLL and MCL samples in another study [53].Noteworthy, the PI3Kδ-specific signaling has a key role in NK maturation and cytokine production [54].In this contest, idelalisib lacks a direct cytotoxic effect on NK cells but decreases NK production of IFNγ [38].However, in another study, fosfamatinib and ibrutinib, but not idelalisib, were shown to affect rituximab-induced NK degranulation and cytotoxicity as well as NK secretion Fig. 2 Schematic view of the role of Btk and PI3Kδ molecules in non-malignant cells.The scheme summarizes the main cellular functions, in which Bruton tyrosine kinase (Btk) phosphoinositol 3 kinase (PI3K) δ molecules are involved in NK cells, T cells, macrophages, and osteoclasts.On the right section, some data concerning the effects of ibrutinib and idelalisib on these cells are indicated.NK natural killer, TLR toll-like receptor, CDC complement-dependent cytotoxicity, ADCC antibody-dependent cell-mediated cytotoxicity, IFNγ interferon γ, TCR T cell receptor, OC osteoclasts, MM multiple myeloma of TNF and IFNγ [53].Of interest, the dual PI3K p110δ and p110γ inhibitor, IPI-145, also affects NK cells by promoting cell death [55]. In this scenario, it has also to be considered that in vivo the egress of neoplastic B cells from tissues to periphery mediated by ibrutinib and other kinase inhibitors may cells to a more abundant and fully active complement compartment favoring the CDC induction by concurrent administration of anti-CD20 antibodies, mitigating the potential disadvantage of reduced ADCC.Indeed, encouraging clinical results of ibrutinib in combination with rituximab in high-risk CLL patients in phase 2 study [6], and idelalisib in combination with rituximab in a phase 3 study [15], are reported.However, studies testing different timing in the administration of the anti-CD20 antibodies relative to initiation of ibrutinib or idelalisib or modification of dosing schedules should be carried out to unravel the possible interactions between these drugs at molecular levels and thus maximize the clinical efficacy of the therapeutic combination. ",
"section_name": "NK cells",
"section_num": null
},
{
"section_content": "Tumor immune surveillance hinges upon the capacity to elicit a robust Th1 and CD8 T cell response that promotes cytotoxic effects with the production of IFNγ and IL2.CLL subversion of immune response comprises the disruption of T cell functions with impairment of immune synapse formation, alteration of actin rearrangement and signal transduction, and reciprocal induction of inhibitory receptors and ligands [56][57][58][59].Moreover, CLL cells secrete a panel of cytokines that promote the aberrant recruitment of a Th2-dominant response, thus interfering with an effective direct T cell cytotoxicity and also favoring the maintenance of a prosurvival niche for leukemic cells through IL4, IL10, and CD40 stimulation. In T cells, three Tec kinases are expressed, Itk, Rlk/ Txk, and Tec.Itk is expressed at highest amounts and plays the major role in regulating signaling from the TCR, starting from phosphorylation of PLCγ that leads a cascade of events comprising the activation of nuclear factor of activated T cells (NFAT), NF-κB, and MAPK pathways as well as the induction of calcium mobilization, reorganization of actin cytoskeleton, synapse formation, and cell adhesion.Ibrutinib is an irreversible inhibitor of Itk, leading to a decrease in PLCγ phosphorylation, a downstream inactivation of IkBα, JunB, and NFAT signaling, and a reduction of intracellular calcium flux in response to TCR stimulation in primary CD4+ cells and in Jurkat T cell line [60].In Th1 CD4 and CD8 T cells, Rlk plays a redundant role to Itk in response to TCR stimulation.As consequence, ibrutinib at clinical relevant doses is able to influence the Th1/Th2 polarization of CD4+ population by decreasing the Th2-dominant transcription factor JunB, whereas inducing the Th1-specific transcription factor T-bet.It turns out in a prevalence of IFNγ-producing Th1 in respect to IL4-producing Th2 cells.The Th1 induction was also sustained by the observation in CLL patients treated with ibrutinib, showing a decrease level in serum of Th2type cytokines such as IL4 and IL10 at day 28 of treatment compare to baseline.Overall, the study of Dubovsky and colleagues [60] provides evidence that ibrutinib may influence T cell function, by skewing T cells from a Th2-dominant to a Th1 and CD8+ cytotoxic population, due to a selective Itk inhibition and a compensatory mechanism mediated by Rlk that remains uninhibited in Th1 cells (Fig. 2).Accordingly, mice deficient in Itk showed impairment in Th2 polarization and Th2 cytokine production in response to Th2-inducing agents and infections in vivo [61,62]. Cytokine production by T cells is mediated by PI3K signaling.Idelalisib did not show any direct cytotoxic effect on T cells, but it could inhibit the production of several inflammatory cytokines such as IL6, IL10, TNFα, and CD40L [38].Similarly, the inhibition of both PI3K p110δ and p110γ by IPI-145 was reported to reduce the secretion of IL2, TNFα, and IFNγ by TCR-stimulated T cells [55].Few correlative observations are reported in published clinical trials.Burger et al. showed a reduction of CD3+CD4+ and CD3+CD8+ T cell count in the periphery of patients treated with ibrutinib and rituximab after 6 and 12 months of treatment [6].In phase 1 trials testing single-agent idelalisib on relapsed/refractory CLL or refractory indolent NHL, a correlative study did not show any significant changes in the absolute number of CD4+/CD3+ T helper cells, CD8+/CD3+ cytotoxic T cells or CD56+/CD16+/CD3-NK cells in peripheral blood of treated patients [14,17]. ",
"section_name": "T cells",
"section_num": null
},
{
"section_content": "Macrophages are involved in the essential process of engulfment and clearing of microbial pathogens.In particular, the ingestion of IgG-opsonized targets initiates with the engagement of FcγRs, activation of multiple downstream signaling pathways and actin polymerization, thus leading to the formation of phagocytic cup, the extension and fusion of pseudopods, and the internalization and maturation of phagosomes, at which the clearing of pathogens occurs. Among cytoplasmic tyrosine kinases of Tec family, macrophages express Btk, Tec and Bmx.The involvement of Btk in FcγR-mediated phagocytosis was first reported in monocytes from XLA patients and also in peritoneal macrophages from Xid mice lacking functional Btk [63,64].Furthermore, Btk seems irrelevant for macrophage functions associated with T cell activation such as antigen presentation, co-stimulation, or production of T cell-directed cytokines but instead plays a major role in \"innate effector\" functions such as phagocytosis, induction of reactive metabolites, production of proinflammatory cytokines and microbicidal activity. Btk and Tec kinases are required for optimal FcγRmediated phagocytosis.Both kinases are activated throughout phagocytosis in primary macrophages and inhibition of Btk by small interfering RNA markedly reduced Mac-1 activation and FcγR-induced phagocytosis [65].In particular, the first stage of Tec kinases activation rapidly occurs at nascent phagosomes with their ment to the membrane through their pleckstrin homology domain.Btk and Tec are also involved in the transition of integrin from a cytoskeleton-bound resting to a mobile state at phagosome cup.It is also likely a subsequent involvement of Btk and Tec in later stages of phagosome maturation with distinct roles.Thus, Btk function is involved in phagocytosis by sustaining the late and prolonged activation of PLCγ2 [65].In accordance with these results, recent studies indicated that pharmacological inhibition of Btk by ibrutinib is able to impair the phagocytosis of rituximab-coated CLL cells by macrophages at clinically relevant doses [52,66] (Fig. 2).Macrophages also expressed the ubiquitous isoforms of PI3K, p110α and p110β as well as the hematopoietic enriched p110δ isoform.PI3K has been demonstrated to regulate phagocytosis and migration of macrophages [67].The inhibition of PI3Kδ by idelalisib was reported to reduce rituximab-mediated CLL engulfment and also abrogate macrophage spreading and invasive capacity mediated by colony stimulating factor-1 [68,52]. In line with the T cell paradigm of Th1 and Th2 subsets, macrophages polarize in response to different types of microbial and environmental signals into two subpopulations, i.e., classic inflammatory M1 and alternative immunosuppressive M2 macrophages [69,70].The M1 phenotype is characterized by the expression of proinflammatory cytokine, high production of reactive nitrogen, stimulation of Th1 response, and potent activity against microbes and tumor cells.Conversely, M2 macrophages show efficient phagocytic activity, high expression of scavenging molecules, mannose and galactose receptors, and involvement in parasite containment, tissue remodeling and tumor promotion.M1 macrophage polarization is mediated by STAT1, the p65 subunit of NF-κB and IRF3, whereas STAT3 and STAT6 mediate M2 macrophage polarization.Btk is a critical signal transducer downstream of TLR4 and interferon γ receptor (IFNGR) by regulating NF-κB and IRF3 activation.As consequence, Btk is involved in macrophage lineage commitment to inflammatory profile.Accordingly, Btk(-/-) macrophages show an impaired ability to polarize into M1 (inflammatory) macrophages through inefficient activation of NF-κB p65 following lipopolysaccharide and IFNγ stimuli, instead showing enhanced induction of M2-associated markers [71].As an M2 phenotype characterizes tumor-associated macrophages (TAMs) leading to low tumoricidal activity, promotion of angiogenesis and poor prognosis for patients in several tumoral settings [72,73], it will be of great interest to evaluate the effects of ibrutinib on macrophage polarization.In particular, NLCs are round or fibroblast-shaped adherent cells differentiated from peripheral blood-derived monocytes in vitro and also detected in lymph nodes (LN) of CLL patients [74][75][76].NLCs share several features with tumor-associated macrophages and deregulated expression of genes involved in immunocompetence [77,75,78,79].Lenalidomide was shown to modify the balance of NLCs phenotype from a M2-skewed immunosuppressive towards a M1-skewed inflammatory profile [80].In this scenario, studies evaluating the effect of ibrutinib on NLCs and the combination with lenalidomide would be useful. ",
"section_name": "Macrophages",
"section_num": null
},
{
"section_content": "Osteoclasts (OC) originate from bone marrow-derived monocyte/macrophages precursor cells and are strictly regulated by the receptor activator of nuclear factor KB ligand (RANKL), which cooperates with immunoreceptor tyrosine-based activation motif (ITAM) signals to mediate calcium release and then induces the master transcription factor for osteoclastogenesis, the nuclear factor of activated T cells c1 (NFATc1).Btk and Tec are highly expressed in OC and bridge the RANKL and ITAM signals, thus leading osteoclast differentiation [81].In line with the role of Btk in osteoclast function, studies on Btk mutation disease (XLA in humans and Xid in mice) clearly revealed that Btk-deficent osteoclasts do not completely differentiate into mature multinucleated osteoclasts, leading to defective bone resorption activity [82,83]. Analogously, the pharmacological blockage of Btk by ibrutinib impairs the RANKL signaling in OC and NFATc1 induction, thus reducing the bone resorption.Noteworthy, ibrutinib treatment significantly inhibits MM-induced osteolysis of implanted human bone chips in SCID mice and ameliorates the bone loss in a RANKL-induced osteoporosis mouse model [32,84] (Fig. 2).Myeloma cells have a high capacity to induce osteolytic bone lesions in patients, especially in the advance stages, due to the presence of overactive osteoclasts and inactive osteoblasts.In this scenario, ibrutinib may have a potential role in counteract MM-induced osteoclast iperactivation and bone lysis as well as in disruption of mutualistic interaction between MM cells and osteoclasts, by interfering with crucial cytokine and chemokine signals [32,85].Ibrutinib generated abnormal giant osteoclasts with defective bone erosion activity and induced OC apoptosis in vitro in long-term culture.Moreover, Btk inhibition by ibrutinib leads to an aberrant organization of actin cytoskeleton critical for bone resorption [32].It has to be considered that OC obtained from XLA patients showed an impaired bone resorption activity in vitro, but conversely, any evidence of alteration in bone density was detected in patients.This effect seems to be derived from a compensatory mechanism mediated by the up-regulation of several inflammatory cytokines (IL6, IL1β, and TNFα) in patients that re-equilibrates OC function [83].Ibrutinib was reported to block the secretion of these multiple inflammatory cytokines in MM microenvironment, leading to the potential interruption of the cytokine-mediated compensation of OC activity [32]. ",
"section_name": "Osteoclasts",
"section_num": null
},
{
"section_content": "Several novel molecules acting as inhibitors of pivotal tyrosine kinases downstream the BCR signaling cascade and/or involved in B cell trafficking and homing are rapidly spreading in the landscape of therapeutic options in B cell malignancies, in particular in CLL and MCL settings where current standard therapeutic strategies induce substantial toxicity and are not curative, with nearly all patients relapsing.In this scenario, the Btk inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib have demonstrated good safety profile and promising clinical efficacy in different B cell malignancies, clearly affecting the survival of neoplastic B cells by destabilizing the multifactorial platform of microenvironment signals that commonly sustain the malignant clone.These agents also delocalize a consistent fraction of the tumor B cell clone from the protective tissue compartment to the periphery interfering with pathogenetic mechanisms of recirculation and homing.However, it had been immediately clear the limited capacity of these agents to completely eradicate the neoplastic clone, even if an improvement in quality of responses is seen when patients are treated for an extended period.Noteworthy, the molecular targets of ibrutinib and idelalisib are not restricted to B cell compartment but regulate key functions in other cellular elements, i.e., NK, T cells, macrophages and osteoclasts.Neoplastic B cells deeply modify the architecture of infiltrating tissue compartment, skewing the functions of surrounding nonmalignant cellular components to generate a protective and nurturing microenvironment.Moreover, they also modify the attitude of cells devoted to natural and adoptive immune response, inducing an immunosuppressive and immune-tolerant behavior.Deepening the effects of TKIs on the normal cellular components in the microenvironment of lymphoproliferative disorders might be of extreme relevance to better understand some unexplained phenomena observed in the clinical setting, first among all, why TKIs are so efficient, able to quickly reduce lymphoid mass, but they can obtain a complete response only in a small minority of cases.Moreover, specific TKI toxicity could find a good explanation. In conclusion, shedding light into the effects of these TKIs to non-malignant cellular compartment will help to define how they modify the nurturing and protective niches of B cell clone into tissues, giving information for a more efficient and rational use of TKIs and, importantly, suggesting a rational approach for association with other agents as the classical cytotoxic drugs, monoclonal antibodies and immune-modulating agents. ",
"section_name": "Conclusive remarks",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC IG10621-RMar, SF) Milan, and Ricerca Finalizzata Giovani Ricercatori 2011-2012, Ministero della Salute (GR-2011-02349282, RMaf), Rome, Italy. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "ABC: activated B cell-like subtype; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent phagocytosis; BCR: B cell receptor; BR: bendamustine, rituximab; Btk: Bruton tyrosine kinase; CDC: complementdependent cytotoxicity; CIT: chemoimmunotherapy; CLL: chronic lymphocytic leukemia; CR: complete responses; DLBCL: diffuse large B cell lymphoma; FcR: Fc receptor; FCR: fludarabine, cyclophosphamide and rituximab; FDA: Food and Drug Administration; FL: follicular lymphoma; HCL: hairy cell leukemia; IFN: interferon; IFNGR: interferon γ receptor; IL2: interleukin 2; ITAM: immunoreceptor tyrosine-based activation motif; Itk: inducible T cell kinase; LN: lymph nodes; LPL: lymphoplasmacytic lymphoma; MAPK: mitogenactivated protein kinase; MCL: mantle cell lymphoma; MM: multiple myeloma; MOA: mode of action; MZL: marginal zone lymphoma; NFAT: nuclear factor of activated T cells; NFATc1: nuclear factor of activated T cells c1; NF-κB: nuclear factor κB; NHL: non-Hodgkin lymphoma; NK: natural killer; NLCs: nurse-like cells; OC: osteoclasts; ORR: overall response rate; PFS: progression-free survival; PI3K: phosphoinositol 3 kinase; PLCγ: phospholipase Cγ; PR: partial response; RANKL: receptor activator of nuclear factor KB ligand; R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; Rlk: resting lymphocyte kinase; SLL: small lymphocytic lymphoma; Tec: tyrosine kinase expressed in hepatocellular carcinoma; TKIs: tyrosine kinase inhibitors; TLR: toll-like receptor; TNF: tumor necrosis factor; WM: Waldenström macroglobulemia; Xid: X-linked immunodeficiency; XLA: X-linked agammaglobulinemia. The authors declare that they have no competing interests. RMaf conceived and wrote the manuscript.SF, SM, LP, ML and RMar drafted the manuscript and approved the final version. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "ABC: activated B cell-like subtype; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent phagocytosis; BCR: B cell receptor; BR: bendamustine, rituximab; Btk: Bruton tyrosine kinase; CDC: complementdependent cytotoxicity; CIT: chemoimmunotherapy; CLL: chronic lymphocytic leukemia; CR: complete responses; DLBCL: diffuse large B cell lymphoma; FcR: Fc receptor; FCR: fludarabine, cyclophosphamide and rituximab; FDA: Food and Drug Administration; FL: follicular lymphoma; HCL: hairy cell leukemia; IFN: interferon; IFNGR: interferon γ receptor; IL2: interleukin 2; ITAM: immunoreceptor tyrosine-based activation motif; Itk: inducible T cell kinase; LN: lymph nodes; LPL: lymphoplasmacytic lymphoma; MAPK: mitogenactivated protein kinase; MCL: mantle cell lymphoma; MM: multiple myeloma; MOA: mode of action; MZL: marginal zone lymphoma; NFAT: nuclear factor of activated T cells; NFATc1: nuclear factor of activated T cells c1; NF-κB: nuclear factor κB; NHL: non-Hodgkin lymphoma; NK: natural killer; NLCs: nurse-like cells; OC: osteoclasts; ORR: overall response rate; PFS: progression-free survival; PI3K: phosphoinositol 3 kinase; PLCγ: phospholipase Cγ; PR: partial response; RANKL: receptor activator of nuclear factor KB ligand; R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; Rlk: resting lymphocyte kinase; SLL: small lymphocytic lymphoma; Tec: tyrosine kinase expressed in hepatocellular carcinoma; TKIs: tyrosine kinase inhibitors; TLR: toll-like receptor; TNF: tumor necrosis factor; WM: Waldenström macroglobulemia; Xid: X-linked immunodeficiency; XLA: X-linked agammaglobulinemia. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "RMaf conceived and wrote the manuscript.SF, SM, LP, ML and RMar drafted the manuscript and approved the final version. ",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.7759/cureus.21823
|
An Unusual Case of Chronic Lymphocytic Leukaemia Involving the Cervix
|
Chronic lymphocytic leukaemia (CLL) is a malignant monoclonal expansion of B lymphocytes, with accumulation of abnormal lymphocytes in the blood, bone marrow, spleen, lymph nodes and liver. It is mainly a disease of the elderly population. Though extra-nodal involvement is common, cervical and vulvovaginal involvement by CLL is particularly uncommon. In this case report, we discuss the case of cervical involvement of CLL in an 84-year-old patient who presented to rapid-access gynaecological clinic following concerns of persistent postmenopausal bleeding. Previously the patient was known to haematology with a well-controlled diagnosis of CLL since 2007. The initial examination was significant for an enlarged, irregular cervix, whereby a punch biopsy was then obtained. Histological analysis revealed evidence of CLL within the cervix.
|
[
{
"section_content": "Chronic lymphocytic leukaemia (CLL) is a neoplastic disease characterised by the malignant monoclonal proliferation and accumulation of small, mature, long-living lymphocytes leading to lymphadenopathy, organomegaly and systemic abnormalities [1].CLL displays classical immunophenotyping alongside a routine histological infiltration within blood, bone marrow, liver and lymphoid tissue [2]. Though extra-nodal involvement is also common, cervical and vulvovaginal involvement by CLL is particularly uncommon, which can be highlighted by the limited volume of previously published material.In this case report, we discuss the interesting case of cervical involvement of CLL in an 84-year-old patient who presented to rapid-access gynaecological clinic following concerns of persistent postmenopausal bleeding. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "An 84-year-old lady with a past medical history of ischaemic heart disease, chronic kidney disease stage 3, chronic obstructive pulmonary disease and gout presented to the rapid-access gynaecology clinic at a district general hospital.She was referred via the rapid-access pathway by her general physician due to complaints of postmenopausal bleeding, intermittent night sweats and weight loss.The patient had been regularly followed up by her local haematology team with a known diagnosis of CLL, which has been stable for the prior 14 years.As a consequence, the patient had still been on the watch and wait regime from haematology, whereby a prior computerised tomography (CT) scan four months prior to the patient's attendance to gynaecology clinic had confirmed stable CLL. The patient was assessed within the rapid-access gynaecology clinic.She is Para 3 with all normal vaginal deliveries.The patient is unaware of when she had her last smear test.A bimanual and speculum examination revealed a small retroverted uterus.Additionally, an enlarged and friable cervix was noted with an abnormal appearance.Two lumps were identified at 1 and 4 o'clock position during examination.Subsequently, a punch biopsy was obtained from the largest lump at 4 o'clock position on the cervix. A transvaginal ultrasound was performed following attendance in clinic.This displayed a small mass in the anterior myometrium measuring 3.2 × 2.6 × 3.4 cm, which represented a calcified fibroid (Figure 1).Endometrial thickness was 3.5 mm and a normal adnexa was present (Figure 2).Subsequent results of the biopsy through histological and immunophenotypic analysis is in keeping with small lymphocytic lymphoma (SLL)/CLL as can be seen in Figure 3. ",
"section_name": "Case Presentation",
"section_num": null
},
{
"section_content": "Additional few foci of ill-defined, indistinct paler-looking areas, possibly represent proliferation centres. Immunohistochemistry of cells displayed diffuse positive expression with B-cell markers CD20, CD79a and PAX5.There was an additional positive staining with CD21 and CD23, whilst there was a negative staining with CD3, CD10 and cyclin D1.Additionally, co-expression of CD5 and CD43 on B-cells was displayed, whilst LEF1 highlighted patchy focal area with very weak nuclear staining.BCL6 also showed patchy weak staining considered negative.MUM-1 and C-MYC highlighted the paraimmunoblasts.Finally, Ki67 showed a proliferation index of 20-30%. Following the findings of SLL/CLL within the cervix, a repeat CT neck, thorax, abdomen and pelvis with IV contrast was arranged by haematology two months after the patient's attendance to the gynaecology clinic.This was following communication between both specialties through a multidisciplinary meeting regarding the patient's care.The CT scan displayed evidence of disease progression with increased lymphadenopathy within the mediastinum, left axilla and para-aortic regions with splenomegaly (15.4 cm) (Figures 4567). ",
"section_name": "FIGURE 3: Microscopic findings highlighting diffuse infiltrates of smallto medium-sized lymphoid cells admixed with singly dispersed larger cells with immunoblast-like morphology (present within the circled areas of the images).",
"section_num": null
},
{
"section_content": "A month following the CT scan, the patient was seen within the haematology outpatient clinic.Despite the patient's multiple co-morbidities, her performance status was Eastern Cooperative Oncology Group (ECOG) score 1. Haematologists subsequently commenced the tyrosine kinase inhibitor, acalabrutinib, for the patient's progressive disease.The intention of this treatment was as a curative therapy.The patient responded well to this over the next two months, denying any B symptoms and the per vaginal (PV) bleeding had spontaneously resolved.However, the following month the patient was admitted for a total of eight days for an episode of neutropenic sepsis, although she responded nicely to granulocyte-colony-stimulating factor (GCSF).During this inpatient admission, acalabrutinib was omitted.Whilst the patient felt reasonably well upon discharge, she was noted to have a small degree of oxygen requirement and moderate fatigue.The patient was reviewed within the haematology outpatient clinic two weeks following her inpatient admission.Since the omission of acalabrutinib, the patient noted the return of some, albeit small, lymph nodes in the neck, but denied any B symptoms.Following this clinic review, the patient is now being regularly reviewed within the outpatient haematology clinic, with considerations being made to recommence acalabrutinib.Decisions are to be made as to whether starting acalabrutinib as a lower dose, with intermittent administrations of GCSF, will help minimise her chances of future readmission with neutropenic sepsis. ",
"section_name": "cm (orange arrow).",
"section_num": null
},
{
"section_content": "Within this particular case, the patient presents with a long history of CLL, which has been stable throughout until detection of a secondary cervical deposit that displays a histological profile associated with CLL/SLL.For close to three decades the World Health Organization have considered CLL and SLL to be neartissue equivalents, which has been demonstrated within the 2016 revision of the World Health Organization classification of lymphoid neoplasms [3].Differences may be demonstrated on location of cancer or expression of chemokines [2,4].Nevertheless, the management of such entities is near the same.This can be through the utilisation of immunochemotherapy agents, either systemic or targeted, as well as possible allogenic haematopoietic stem cell therapy [5][6][7].However, new and novel agents are being developed on a more frequent basis. Gynaecological lymphoblastic infiltration secondary to haematological disorders is rare; however, it is more frequently seen in lymphomas rather than in leukaemias.Thus, we have found the literature available for CLL gynaecological deposits to be scarce [8].Typically, CLL can have involvement of extra-lymphoid sites within the body, with the skin being the most common site, accounting for approximately 8% of cases [9,10].Regardless of this, deposits within the female genital tract resulting from primary lymphomas are still particularly rare, resulting in <1% of all extra-nodal lymphomas; however, frequent gynaecological sites tend to be either the ovary or cervix [11,12]. The median age of diagnosis for CLL is 72 years; however, case reports identified through a literature search have displayed a wide age range for patients.Shelke et al. presented a case of a 57-year-old patient with known stage III CLL who presented to the gynaecological department with postmenopausal bleeding [13].Later analysis revealed a uterine cervix metastasis.In contrast, Seoud et al. demonstrated an evidence of an 82-year-old woman with a known six-year history of stable and non-medicated CLL present with postmenopausal bleeding secondary to CLL deposits with her upper vagina and cervix [14].Additionally, Magley et al. revealed the case of a 70-year-old lady with a known two-year history of stage IV CLL who was primarily referred to gynaecology following an abnormal cervical smear, which later demonstrated cervical CLL infiltrates [15].Presented data highlight the evidence that extra-nodal CLL deposits within the cervix can vary with the patients' age, staging of CLL, treatment regime and particularly prognostic factors. Prior literature has demonstrated the risk of developing a second neoplasm in patients with known CLL to be increased 2.2-fold when associated against a healthy population [16].In conjunction to this, Schollkopf et al. investigated 12,373 patients with known CLL from 1943 to 2003 for evidence of secondary cancers.Within the cohort, 1,105 secondary cancers occurred, of which 20 involved the female genital tract [17].Four of the 20 cases originated within the uterine cervix.Despite the rarity of this condition, it is key for the gynaecologists to have a keen awareness of patients presenting with primary gynaecological symptoms with a known haematological malignancy and its possible association to secondary gynaecological metastasis. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, this case demonstrates a rare presentation of CLL with secondary cervical involvement causing postmenopausal bleeding.A definitive diagnosis can be made only by histological analysis and immunophenotyping.The case is presented due to its rarity, and to highlight the awareness of the association between haematological and gynaecological malignancies. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "Human subjects: Consent was obtained or waived by all participants in this study.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 Disclosures",
"section_num": null
}
] |
10.3389/fimmu.2023.1134123
|
The potential value of Notch1 and DLL1 in the diagnosis and prognosis of patients with active TB
|
<jats:sec><jats:title>Objectives</jats:title><jats:p>The Notch signaling pathway has been implicated in the pathogenesis of active tuberculosis (TB), and Th1-type cell-mediated immunity is essential for effective control of mycobacterial infection. However, it remains unclear whether Notch signaling molecules (Notch1, DLL1, and Hes1) and Th1-type factors (T-bet and IFN-γ) can serve as biomarkers for tracking the progression of active TB at different stages along with peripheral blood white blood cell (WBC) parameters.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>A total of 60 participants were enrolled in the study, including 37 confirmed TB patients (mild (n=17), moderate/severe (n=20)) and 23 healthy controls. The mRNA expression of Notch1, DLL1, Hes1, T-bet and IFN-γ in the peripheral blood mononuclear cells (PBMCs) of the subjects was measured by RT-qPCR, then analyzed for differences. Receiver Operating Characteristic curve (ROC) was used to assess the effectiveness of each factor as a biomarker in identifying lung injury.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>We found that mRNA expression levels of Notch1, DLL1, and Hes1 were upregulated in active TB patients, with higher levels observed in those with moderate/severe TB than those with mild TB or without TB. In contrast, mRNA levels of T-bet and IFN-γ were downregulated and significantly lower in mild and moderate/severe cases. Furthermore, the combiROC analysis of IFN-γ and the percentage of lymphocytes (L%) among WBC parameters showed superior discriminatory ability compared to other factors for identifying individuals with active TB versus healthy individuals. Notably, Notch pathway molecules were more effective than Th1-type factors and WBC parameters in differentiating mild and moderate/severe cases of active TB, particularly in the combiROC model that included Notch1 and Hes1.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Our study demonstrated that Notch1, Hes1, IFN-γ, and L% can be used as biomarkers to identify different stages of active TB patients and to monitor the effectiveness of treatment.</jats:p></jats:sec>
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[
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"section_content": "Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis, with the lungs being the most commonly affected organ.Despite numerous efforts to combat the disease, it persists as a serious global public health challenge.The World Health Organization's Global TB Report 2021 (1) showed that prior to the coronavirus (COVID-19) pandemic, TB was the leading cause of death from a single infectious agent, surpassing even HIV/AIDS.Although extensive research has been conducted on the pathogenesis of TB, there is currently no cost-effective and reliable hematological method available for monitoring the progression of active TB and the effectiveness of treatment. Upon infection with TB, the host mounts both adaptive and innate immune responses aimed at eliminating the bacteria.Lymphocytes are key immune cell types involved in this process.Previous studies have shown that IFN-g secretion by Th1 cells (a type of T-cell subset) is beneficial for activating macrophages and mononuclear cells to control TB infection in the early stages, enhancing host autophagy and bacterial clearance (2)(3)(4).However, the persistent secretion of IFN-g by T cells in patients with latent TB can lead to the development of active TB within four years (5).This suggests that a strong association between IFN-g secretion by Th1 cells and disease progression of TB. Of note, the role of Notch signaling in disease has received considerable attention in recent years.Studies have reported that this pathway can protect colonic epithelial cells from proinflammatory responses (6), promote immune molecules expression and exacerbate cytokine disorders (7), and also involves in Th1 differentiation and IFN-g secretion (8,9).The Notch signaling pathway is a highly conserved pathway that mediates cell-cell communication through various Notch signaling molecules.These molecules are present ubiquitously on the cell surface and play a crucial role in determining the fate of the cell.In mammals, the binding of the Notch receptor (Notch1-4) to Notch ligand proteins (Delta-like 1, 3, 4, Jagged1, and Jagged2) leads to the activation of CSL transcription factors (CBF-1, Suppressor of Hairless, Lag1) and downstream target genes (such as Hes1), forming signal transduction pathways.For example, Notch signaling was activated after binding of Notch receptor with ligand DLL1, which influences the direction of cell differentiation and regulates cell fate (10,11). In addition, recent studies have confirmed that Notch signaling is involved in the progression of active TB (12)(13)(14).Blockade of Notch signaling by DAPT restores theTh1/Th2 imbalance in TB patients (13).DLL1 levels in cerebrospinal fluid (CSF) and serum of patients with tuberculous meningitis (TBM) were higher than those in the bacterial meningitis, viral meningitis/encephalitis and nondiagnosed groups, indicating that DLL1 may be a new biomarker for TBM diagnosis (12).Based on these findings, we hypothesized that Notch signaling molecules (Notch1, DLL1 and Hes1) and Th1-type response factors (T-bet and IFN-g), along with differences in white blood cell (WBC) parameters may serve as potential biomarkers for predicting disease progression and monitoring therapeutic effect in patients with active TB. In the current study, the mRNA levels of Notch1, Hes1 and DLL1 were upregulated in PBMCs from active TB patients, while the expression of T-bet, and IFN-g were downregulated.IFN-g was superior to Notch pathway molecules in identifying active TB patients from healthy controls, particularly the model combined with L% had the best discriminatory power.Interestingly, Notch pathway molecules could better differentiate mild and moderate/severe cases of active TB than Th1-type factors and WBC parameters.Especially, the combiROC model with Notch1 and Hes1 was more effective.These may provide novel insights into the use of Notch signaling molecules as biomarkers of progression and efficacy in active TB. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
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{
"section_content": "Study participants were obtained at Xinhui District People's Hospital affiliated with Southern Medical University from September 2021 to July 2022, and were aged 18 years or older.The patients with confirmed active TB who had not yet started anti-TB treatment and a control group of apparently healthy individuals from the same hospital's medical examination center underwent physical examinations.This study had been approved by the Ethics Committee of Xinhui District People's Hospital affiliated with Southern Medical University, Jiangmen (Medical Research Ethics Audit 2019 No. 027), and written informed consent was obtained from the participants. Individuals enrolled in this study were described in detail as follows.Negative chest x-ray and IFN-g release assay results further confirmed apparently healthy control subjects.Patients with active TB were diagnosed on the basis of clinical manifestations, positive results of at least one laboratory test (TB DNA test, acid-fast bacilli smear test, IFN-g release test), and characteristics of TB on chest radiograph, meeting the diagnosis criteria of Tuberculosis WS288-2017 (15).Then, they were classified as mild, moderate, and severe groups based on the lung injury and symptoms.In mild cases, symptoms are mild and imaging shows mainly plaques, nodules, and striae or tuberculomas or isolated cavities.In moderate cases, symptoms may include cough, sputum production, shortness of breath, or low-grade fever.Imaging may show scattered lesions of low to moderate density in one or both lungs, or even fusion, but the area of high-density lesions does not exceed one-third of the volume of a lung.In severe cases, symptoms include dyspnea, hemoptysis, or hyperthermia.Imaging may show lobar infiltrates, caseous pneumonia, multiple cavitations, and bronchial dissemination, even extrapulmonary dissemination occurs.The study excluded individuals who were either taking antibiotics or had medical conditions that could potentially affect the study outcomes.These medical conditions included severe heart disease, significant liver disease, active infectious diseases, hematologic disorders, autoimmune diseases, and receiving immunotherapy. ",
"section_name": "Study subjects",
"section_num": null
},
{
"section_content": "Approximately 7 mL of venous blood was collected from TB patients and healthy individuals via venous puncture.Blood samples were drawn into tubes containing EDTA-K2 (BD Vacutainer, New Jersey, USA).Of the 7 ml, 5 ml was used for the peripheral blood mononuclear cells (PBMCs) isolation, and the remaining 2 ml was used to register the complete blood count.PBMCs were separated by Ficoll-Hypaque density gradient centrifugation (TBD, Tianjin, China), which was centrifuged at 400 x g for 30 min at 20 ∘ C. ",
"section_name": "Blood sample preparation",
"section_num": null
},
{
"section_content": "Blood samples were analyzed for peripheral blood cell count using the Sysmex XN2000 automated hematocytometer (Sysmex, Tokyo, Japan).It provides analysis of peripheral blood cell components, cell volume, cell percentage and other classification parameters.Among these parameters, the number of total white blood cells, the percentage and the number of granulocytes, lymphocytes and monocytes are widely used in the monitoring of infectious diseases.Complete blood count is performed under strict quality control procedures.These include daily high and low internal quality control, bi-weekly quality control and annual quality assurance as the important parts of the Clinical Laboratory QC Program. ",
"section_name": "Complete blood count",
"section_num": null
},
{
"section_content": "According to the manufacturer's protocol, the extraction of total RNA of PBMCs from individuals was used Trizol method (TRizol reagent, Takara Biotechnology, Dalian, China) and treated with RNase-free DNase (Solarbio, Beijing, China).And then reverse transcription was accomplished by a High-Capacity cDNA Reverse Transcription synthesis kit (Vazyme, Nanjing, China). ",
"section_name": "RNA extraction and RT-PCR",
"section_num": null
},
{
"section_content": "Quantitative real-time PCR was performed using SYBR Green PCR mixture (Vazyme Nanjing, China) to validate the differential mRNA levels of Notch1, Hes1, DLL1, T-bet and IFN-g under the following conditions: 45 cycles of 95°C for 30 seconds, 95°C for 10 seconds and 60°C for 40 seconds in an Applied Biosystems instrument.The human primer sequences were shown in Table 1.The relative fold change in mRNA expression of all genes was measured by the 2 -DDCt method ( 16) using b-actin as the internal reference gene, according to the following calculation: DDCt = experiment group (Ct target -Ct internal reference)control group (Ct target -Ct internal reference).Means and SDs were computed from triplet datasets. ",
"section_name": "Quantitative real-time PCR",
"section_num": null
},
{
"section_content": "Statistical analysis and graphs were performed by R language (mainly R package GGploT2 [version 3.3.3])(used for visualization) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA; www.graphpad.com).Categorical variables were shown as number (%), and analyzed by c 2 test.Continuous variables were shown as mean ± SD and analyzed by Student's t-test if normally distributed, or as median (interquartile range) and analyzed by Mann-Whitney U test otherwise.For non-normally distributed independent samples, Spearman method correlation analysis was used for assessing the relationship between two genes.One-way analysis of variance (ANOVA) and Kruskal-Wallis's test were used for multivariate comparisons among groups.ROC curve analysis was used to evaluate the diagnostic efficiency of each factor.Differences were considered statistically significant if p-value was < 0.05.* p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001, ns, not significant. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "The percentage of lymphocytes in peripheral blood of active TB patients were lower than those of healthy controls A total of 60 participants were enrolled in this study, of which 23 healthy individuals were negative controls and 37 TB patients were in the positive group.The main clinical information of all ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Human primer sequences 5'-3' participants was shown in Table 2.The classification parameters of the WBC were obtained through the complete blood count conducting with the Automatic Hematology Analyzer Sysmex XN2000.No significant difference was observed in age, sex, total WBC count, neutrophil count, monocyte count and percentage (M %) between the positive and healthy groups.However, the percentage of lymphocytes (L%) was lower in the TB patients than that in healthy controls, while the percentage of neutrophils (N%), the ratio of neutrophils/lymphocytes (N%/L%), and the ratio of monocytes/lymphocytes (M%/L%) in TB individuals were higher than in the healthy controls.These results confirmed that the lymphocytes were more attrition in the progression of active TB compared to the healthy controls, which was similar with the conclusions of previous studies (17,18).The increase in N% was likely due to a decrease in L%, as we term this phenomenon a \"increased passivity\".This is because neutrophils, lymphocytes, and monocytes are the main components in peripheral blood leukocytes, with the normal reference range for N% being 50%-70%, and L% being 20%-40%.However, our study showed a true decrease in both percentage and absolute number of lymphocytes. The mRNA expression of Notch1, DLL1 and Hes1 were upregulated in PBMCs of TB patients compared to uninfected individuals To investigate the mRNA expression of Notch1, DLL1 and Hes1 in PBMCs of TB-infected and uninfected individuals, Q-PCR was performed for comparison.It was observed that the mRNA expression of Notch1, DLL1 and Hes1 were upregulated in the PBMCs of TB patients compared to uninfected individuals.(Figures 1A-C).These findings demonstrated that the expression of Notch pathway molecules was up-regulated during TB infection. ",
"section_name": "Gene",
"section_num": null
},
{
"section_content": "The mRNA expression of Notch1, DLL1 and Hes1 of active TB patients (n=37) and healthy controls (n=23) were detected by RT-QPCR.The data were standardized to b-actin and presented as the mean ± SD of triple independent experiments.Mann-Whitney U test (Wilcoxon rank sum test) was used for statistical analysis.(A-C) respectively showed that the mRNA expression of Notch1, DLL1, and Hes1.*p < 0.05, ****p < 0.0001.The levels of Notch signaling molecules were higher in moderate/ severe TB patients than that in mild and uninfected individuals To further explore the levels of Notch signaling molecules at different stages of TB infection, participants were divided into healthy (n=23), mild (n=17), and moderate/severe (n=20) groups based on the lung injury and symptoms meeting the diagnosis criteria of Tuberculosis WS288-2017.As shown in Figures 2A-C, the mRNA expression of Notch1, DLL1 and Hes1 were significantly higher in patients with moderate/severe TB compared to those with mild TB or no infection.However, there was no difference observed between the mild and healthy participants.These results suggested that the expression of Notch signaling molecules may be associated with the severity of TB infection. ",
"section_name": "B C A",
"section_num": null
},
{
"section_content": "To explore the mRNA expression of T-bet and IFN-g in PBMCs of TB infected and uninfected individuals, Q-PCR was performed for comparison.It was observed that the mRNA expression of T-bet and IFN-g were down-regulated in the PBMCs of TB patients compared to uninfected individuals (Figures 3A,B).These results demonstrated that the expression of Th1-type factors were downregulated during TB infection. ",
"section_name": "The mRNA expression of T-bet and IFN-g were down-regulated in PBMCs of TB patients compared to uninfected individuals",
"section_num": null
},
{
"section_content": "To further explore the levels of T-bet and IFN-g at different stages of TB infection, participants were divided into healthy, mild and moderate/severe groups (the number of participators respectively were 23, 17, 20) based on the lung injury and symptoms that met the diagnosis criteria of Tuberculosis WS288-2017.As shown in Figures 4A,B, the mRNA expression of T-bet and IFN-g were lower in mild and moderate/severe TB patients than that in uninfected individuals and the difference were statistically significant.No difference was observed between the mild and moderate/severe groups.These results suggested that a decrease in the frequency of Th1 cells, and the immune response mediated by Th1-type factors is tends to be impaired in the progression of TB patients. The combiROC of IFN-g and L% contribute to discriminate the active TB patients from healthy individuals, while the combiROC of Notch1 and Hes1 was favored for distinguishing mild TB patients from moderate/severe individuals The results presented above proved that the Notch signaling molecules and Th1-type factors had changed differently at different stages of TB patients, as well as the WBC parameters.To further verify the diagnostic efficiency of Notch1, DLL1, Hes1, T-bet, IFN-g and the percentage of lymphocytes (L%) in discriminating the severity of the disease provoked by TB from healthy individuals, the ROC analysis method was used (Figure 5). The results showed that IFN-g and L% were more effective than other indicators in aiding the diagnosis of active or inactive TB, with an AUC of 0.946 and a cut-off point of 0.26 for IFN-g, and an AUC of 0.894 and a cut-off point of 20.95 for L% (Figure 5A).And then the accuracy of their predictions was achieved the best diagnostic efficacy in the combined model of both (Figure 5B), which was with the AUC value of 1.00 and cut-off point of 0.136.On the other hand, the molecules of the Notch pathway (Notch1, DLL1 and Hes1) were superior to the factors of Th1 cells (T-bet and IFN-g) and the WBC parameters in terms of diagnostic efficacy for discriminating mild from moderate/severe TB, with AUC values of 0.897, 0.853 and 0.753, for Notch1, DLL1, and Hes1, respectively (Figure 5C).It is worth noting that the diagnostic efficacy of Notch1 and Hes1 was close, with an AUC value of 0.906 and cut-off point of 0.066 for the combined model (Figure 5D).And a sensitivity of 0.800, specificity Comparison of the mRNA expression of Notch1, DLL1 and Hes1 in healthy individuals (n=23) and active TB patients with mild (n=17) or moderate/ severe (n=20) disease were compared by using Kruskal-Wallis one-way analysis of variance by ranks.The standardization of data were used the housekeeping gene b-actin and shown as the mean ± SD of three times experiments.(A-C) presented the mRNA expression of Notch1, DLL1, and Hes1 in healthy controls and patients with different degrees of disease, respectively.**p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.of 0.882, and CI in the range of 0.809-1.000,this model has high accuracy in discriminating mild from moderate/severe TB patients. Consequently, the diagnostic efficiency of the combined ROC analysis of IFN-g and L% had the highest accuracy in discriminating the active TB patients from healthy individuals.Additionally, the combiROC of Notch1 and Hes1 was favored for distinguishing mild TB patients from moderate/severe individuals.These findings highlight the potential diagnostic utility of Notch pathway molecules as biomarkers for management active TB, particularly in identifying disease severity. ",
"section_name": "The levels of Th1-type factors were lower in mild and moderate/severe TB patients than that in uninfected individuals",
"section_num": null
},
{
"section_content": "Despite advancements in TB prevention and treatment, the disease remains a significant global health threat with high morbidity and mortality rates.Hyperreactive TB can cause chronic inflammation, leading to irreversible damage in older adults with subtle symptoms.Post-TB sequelae can significantly add to the overall burden of pulmonary TB by reducing lung function and quality of life (19).Early diagnosis and monitoring of TB progression can decrease the risk of long-term sequelae.Identifying biomarkers to monitor TB progression, including post-TB, is clinically significant. This study analyzed the expression of Notch pathway molecules (Notch1, DLL1 and Hes1) and Th1-type response factors (T-bet and IFN-g) in TB patients, along with routine WBC parameters.The results of WBC parameters showed a significant reduction of lymphocytes and a corresponding increase in the proportion of neutrophils during active TB progression.This phenomenon is typically seen in chronic bacterial infections or mild viral infections.It has been reported that an elevated neutrophils ratio is significantly associated with a decline in lung function, while the opposite is true for the lymphocytes frequency (20).Additionally, the results of lymphocytes were consistent with findings from previous studies indicating their crucial role in mycobacterial clearance (17,18).Other report also considered that the frequency of lymphocytes in peripheral blood is one of the valuable parameters for predicting the risk of active TB (21).Therefore, monitoring the frequency of lymphocytes in peripheral blood is particularly important during the progression of active TB.The results of this study confirmed that the mRNA expression of Notch1, DLL1 and Hes1 were higher in TB group compared to the control group, particularly in patients with moderate/severe disease.These results were consistent with previous researches (12,14), indicating that the Notch signaling pathway is aberrantly activated in TB patients with moderate/severe.In contrast, the expression of Th1-type factors (T-bet and IFN-g) were downregulation compared to the healthy controls, with no significant difference between mild and moderate/severe patients.These results concurred with previous studies (22,23), but rather, they contradicted earlier studies (24,25).It may be attributed to the fact that earlier studies mostly focus on the transitional response between latency and activity.In the early stages of TB infection, Th1 cells activation and IFN-g secretion play an immunoprotected role (2,3).But as the disease proceeds, there is a severe depletion of T cells and suppression of innate and adaptive immune function, particularly the reduction of IFN-g-secreting Th1 cells (22).This suppression is accompanied by an increase in other proinflammatory cells and cytokines (23,26,27), factors accelerate the progression of TB patients to severe disease.The reduction of Th1 response is associated with poor prognosis in patients with TB infection (28).In this study, the decreased frequency of Th1 cells may predict that Th1-type-mediated protective immune responses tend to be impaired as TB disease progress.This requires further in vitro validation or disease follow-up studies.Meanwhile, this highlights the need for new treatments for TB that boost Th1 immune responses.On the other hand, we further analyzed the correlation between Notch signaling molecules expression and the WBC parameters, as well as the Th1-type factors, but no correlation was found (data not shown).Other complex mechanisms may regulate the Notch pathway and WBC in blood separately.However, the Notch pathway may still be involved in regulating other pro-inflammatory cells or pathways that impact the TB infection process.Although the literature on this topic is scarce and inconclusive, our findings suggest that further exploration in this area is necessary. Remarkably, there have been new discoveries of Notch signaling molecules in disease treatment.Several studies have shown that therapeutic targeting of molecules in the Notch pathway can be effective in the prevention and diseases treatment (29)(30)(31).Thus, it is clear that the Notch signaling is involved in cell differentiation and offers new ideas for the treatment of some inflammatory diseases.However, less research has been reported on its usefulness as a biomarker for monitoring disease progression.In this paper, the potential monitoring efficacy of Notch signaling molecules was evaluated using ROC curves, along with Th1-type response-related factors and routine WBC parameters at different stages of TB disease progression. The results showed that both the L% of WBC parameters and the level of IFN-g were superior to other genes in aiding the diagnosing active TB.The combination of both was the most effective model.Currently, IFN-g is widely used as an aid in monitoring the presence or absence of TB infection.However, it is not yet ideal for monitoring the progression of TB disease.Previous studies (18,21) have also found that lymphocyte parameters, such as L%, can be used to predict the risk of active TB disease.At present, the application of the lymphocyte parameter L% in the monitoring active TB is not mentioned in the domestic diagnostic criteria (15).This study showed that the combined model of IFN-g and L% was helpful in identifying individuals with active TB.The blood cell count is widely used in clinical practice, easy to operate and inexpensive.Therefore, Combining IFN-g and L% may aid in the diagnosis of active TB.This may benefit more patients, especially in suspicious cases with negative sputum test for bacteria or nucleic acid of the pathogenic bacteria. On the other hand, the study found that Notch1, DLL1, and Hes1 molecules exhibit superior diagnostic efficacy in discriminating mild from moderate/severe TB patients compared to Th1-type factors and routine WBC parameters.The combined model of Notch1 and Hes1 had high accuracy in diagnostic efficacy.These findings suggest that Notch1 and Hes1 molecules are potential biomarkers for mild and moderate/severe TB, providing a basis for adjunctive diagnosis and monitoring of disease efficacy.Previous research has identified Notch3 as a diagnostic marker for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (32, 33), while DLL1 has shown utility in diagnosing tuberculous meningitis (12).Although no other Notch signaling molecules have been identified as biomarkers for other diseases, the regulation of immune cells function and cytokines secretion by Notch signaling molecules can affect the outcome of TB disease.Consequently, using Notch signaling molecules as biomarkers of TB disease progression can provide a more accurate reflection of the direction of disease progression.This paper proposes for the first time that Notch1 and Hes1 can be used as potential biomarkers to assist in the monitoring of tuberculosis-induced moderate/severe lung disease, providing novel ideas and directions to support clinical monitoring and therapeutic effect observation. In addition, there are still several limitations of this study.Firstly, more studies are needed to support the potential clinical monitoring value, including more data on the comparison of monitoring efficacy from randomized trials containing patients of different ages and with complex TB-associated disease.Also, preand post-treatment testing of different treatment regimens.Secondly, the expression of Notch1 and Hes1 should be tested in post-treatment of patients with TB infection, which would provide comprehensive data for a longitudinal study of Notch1 signaling.Finally, more comprehensive studies are needed to fully understand the function of Th1 cells and the mechanisms between Notch signaling pathway and other cellular and factor alterations of immune responses in active TB patients. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "The Medical and Health care science and technology projects of Jiangmen, China (Nos.Jiangke[2019]107, 2019H013), supported this study. ",
"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": "This study involving human participants was reviewed and approved by The Ethics Committee of Xinhui District People's Hospital affiliated with Southern Medical University, Jiangmen.The patients/participants provided their written informed consent to participate in this study. This project was designed and directed by GL and JX.JX, YC, SC, WZ and HL conducted the data of participators collection and analysis, as well as the experimental data.The manuscript was written by GL and JX.All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "This study involving human participants was reviewed and approved by The Ethics Committee of Xinhui District People's Hospital affiliated with Southern Medical University, Jiangmen.The patients/participants provided their written informed consent to participate in this study. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "This project was designed and directed by GL and JX.JX, YC, SC, WZ and HL conducted the data of participators collection and analysis, as well as the experimental data.The manuscript was written by GL and JX.All authors contributed to the article and approved the submitted version. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ",
"section_name": "Publisher's note",
"section_num": null
}
] |
10.3389/fonc.2022.945060
|
Case report: Long-lasting SARS-CoV-2 infection with post-COVID-19 condition in two patients with chronic lymphocytic leukemia: The emerging therapeutic role of casirivimab/imdevimab
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<jats:p>Post-coronavirus disease 2019 (post-COVID-19) condition, previously referred to as long COVID, includes a post-acute syndrome defined by the presence of non-specific symptoms occurring usually 3 months from the onset of the acute phase and lasting at least 2 months. Patients with chronic lymphocytic leukemia (CLL) represent a high-risk population for COVID-19. Moreover, the response to SARS-CoV-2 vaccination is often absent or inadequate. The introduction of monoclonal antibodies (mAbs) in the treatment landscape of COVID-19 allowed to reduce hospitalization and mortality in mild–moderate SARS-CoV-2 infection, but limited data are available in hematological patients. We here report the effective use of casirivimab/imdevimab (CI) in the treatment of two CLL patients with persistent infection and post-COVID-19 condition. Full genome sequencing of viral RNA from nasopharyngeal swabs was performed at the time of COVID-19 diagnosis and before the administration of CI. Both patients experienced persistent SARS-CoV-2 infection with no seroconversion for 8 and 7 months, respectively, associated with COVID symptoms. In both cases after the infusion of CI, we observed a rapid negativization of the nasal swabs, the resolution of post-COVID-19 condition, and the development of both the IgG against the trimeric spike protein and the receptor-binding domain (RBD) of the spike protein. The analysis of the viral genome in the period elapsed from the time of COVID-19 diagnosis and the administration of mAbs showed the development of new mutations, especially in the <jats:italic>S</jats:italic> gene. The genome variations observed during the time suggest a role of persistent SARS-CoV-2 infection as a possible source for the development of viral variants. The effects observed in these two patients appeared strongly related to passive immunity conferred by CI treatment permitting SARS-CoV-2 clearance and resolution of post-COVID-19 condition. On these grounds, passive anti-SARS-CoV-2 antibody treatment may represent as a possible therapeutic option in some patients with persistent SARS-CoV-2 infection.</jats:p>
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[
{
"section_content": "Although most of the patients affected by the coronavirus disease 2019 (COVID-19) fully recovered within a few weeks, a large proportion of them, up to 76% at 6 months, reported at least one symptom of a post-COVID-19 condition (1).Post-COVID-19 condition (previously referred to as long COVID) occurs in individuals with a history of probable or confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection usually 3 months from the onset of COVID-19, with symptoms that last for at least 2 months and cannot be explained by an alternative diagnosis.The common symptoms include fatigue, shortness of breath, and cognitive dysfunction among others and generally have an impact on everyday functioning.The symptoms may be new onset following initial recovery from an acute COVID-19 episode or persist from the initial illness, and these symptoms may also fluctuate or relapse over time (2). Patients with hematological malignancies represent a highrisk population for COVID-19 because of immunodeficiency related to their disease (e.g., hypogammaglobulinemia, dysfunction of the innate and adaptive immune system), immunosuppressive therapies (3), and frequent hospital admissions.A recent meta-analysis on 3,337 hematologic patients with COVID-19 demonstrated a death risk of 34% for adult patients and 4% for pediatric patients.Additionally, the pooled risk of death for lymphoma and chronic lymphocytic leukemia (CLL) patients was 32% and 31%, respectively (4).Furthermore, adult patients with cancer, including hematological patients, are likely to develop post-COVID-19 complications, particularly respiratory symptoms and residual fatigue, in about 15% of the cases with high rates of cessation or modification of anticancer treatment and subsequent impairment of prognosis and survival (5). Based on these data, hematological patients represent a highpriority population for vaccination in order to mitigate COVID-19 morbidity and mortality.Unfortunately, the experiences of these patients resulted in suboptimal antibody responses following COVID-19 vaccination.For instance, the serologic response to the BNT162b2 mRNA COVID-19 vaccine was about 40% in CLL patients with a lower response rate in patients actively treated with Bruton's tyrosine kinase inhibitors (BTKs) or venetoclax +/-anti-CD20 antibodies (6). Since the beginning of the COVID-19 pandemic in 2020, the therapy for COVID-19 has focused on the acute viral phase of SARS-CoV-2 infection.The availability of an effective antiviral is even more crucial for unvaccinated patients as well as for immunocompromised patients not responding effectively to vaccination.Various therapeutic options have been explored for patients with COVID-19, including convalescent plasma and immunomodulators, with contrasting results (7)(8)(9)(10).In the last year, the therapeutic strategies for COVID-19 have been enriched.With the aim to reduce viral load, prevent hospitalization, and ameliorate the symptoms of COVID-19, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) had issued the emergency use authorization (EUA) for monoclonal antibodies (mAbs) targeting the SARS-CoV-2 spike protein.In particular, the combination of bamlanivimab and etesevimab, casirivimab and imdevimab (REGN-COV2), and sotrovimab has been shown to prevent hospitalization and mortality in high-risk outpatients with mild-to-moderate COVID-19 (11)(12)(13).Though data regarding the use of mAbs in early COVID-19 cases are more robust (11)(12)(13), limited data on their use in immunocompromised patients with prolonged SARS-CoV-2 infection are available (14,15).Here, we present our experience on the use of mAbs in the treatment of two patients affected by CLL and post-COVID-19 condition with long-term persistently positive SARS-CoV-2 infection. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "On 17 January 2021, a 66-year-old man was admitted to the emergency department due to fever, asthenia, ageusia, anosmia, diarrhea, and dyspnea.This patient had a history of type 2 diabetes on metformin therapy and was followed by our hematological department since June 2019 for an untreated CLL associated with severe hypogammaglobulinemia.At diagnosis, the biologic characterization of CLL cells showed unmutated heavy chain variable (IGHV), and the fluorescence in situ hybridization (FISH) analysis was positive for trisomy of chromosome 12 with no further alterations of chromosomes 11, 13, and 17 and TP53 wild type.A nasal swab was positive for molecular testing [reverse transcription-polymerase chain reaction (RT-PCR)] of SARS-CoV-2.Arterial blood gas showed hypoxia with pO 2 equal to 57 mmHg in ambient air, and chest X-ray and pulmonary computed tomography (CT) revealed a bilateral interstitial pneumonia.The patient was admitted to the Infectious Diseases (ID) unit for oxygen supply (e.g., high-flow nasal oxygen, HFNC), dexamethasone, and enoxaparin prophylaxis.At admission, his blood tests were as follows: white blood cells 44 × 10 9 /L, neutrophils 2 × 10 9 /L, lymphocytes 41 × 10 9 /L, hemoglobin 87 g/L, platelet count 256 × 10 9 /L, C-reactive protein 106.8 mg/L, D-dimer 2,597 ng/ ml, total proteins 60 g/L with severe hypogammaglobulinemia (gamma globulin 6.9%, IgG 4.45 g/L), and stable mild paraproteinemia IgG lambda (1 g/L).During the subsequent period of hospitalization, the patient required a course of antibiotic therapy with piperacillin/tazobactam and gentamycin for hospital-acquired pneumonia.He improved progressively, and 30 days after the admission, the patient was discharged with nasal swabs RT-PCR-positive for SARS-CoV-2.During the following 5 months, the patient continued to complain of dysgeusia, anosmia, dyspnea, asthenia, and foot paraesthesia with a slight impairment in maintaining an erect posture and in walking.Such symptoms were attributable to the post-COVID-19 condition and required a multidisciplinary approach.Nasopharyngeal swab RT-PCR was repeated monthly and gave persistent positive results in detecting SARS-CoV-2 infection.Full genome sequencing was performed on two different patients' samples collected on 25 February (hCoV-19/Italy/FVG-TS-36474928/2021) and on 24 May 2021 (hCoV-19/Italy/FVG-TS-64028678/2021), and these samples were stored at -80°C until processed, together, in the same analytical session [cycle threshold value (Ct) was S 18, N 19, Orf1ab 18 for the first sample and S 17, N 18, Orf1ab 17 for the second sample].In both cases, the B.1.177variant (Pango v.3.1.202022-02-28) was identified (for the materials and method, see Supplemental).Interestingly, careful inspection of the sequences identified 21 polymorphisms including the amino-acid T95I (nt 21846) in the spike gene that has been observed in several variants of concern (VOC) including BA.1 (Omicron) (Table 1).A reverse mutation was observed in two positions (nt 17333 and 27826).In 13 sites, the frequency of mutated nucleotide ranged from 20% to 59%, suggesting the emergence at different times.In six sites, a polymorphism was detected in both samples, but the proportion of the mutant nucleotide increased in the second sample from 32% to 72%.Excluding the two reverse mutations, the non-synonymous/ synonymous mutation ratio was 13/6, suggesting an ongoing positive selection pressure, and this was higher in the S gene (7/ 1) than the M gene (2/1) or ORF1ab (4/2).The second swab was also inoculated in Vero E6 cells, and viable virus was recovered demonstrating the infectivity of the sample. Due to the persistence of SARS-CoV-2 infection, in this patient, we investigated the tissue reservoir harboring the infectious virus.Cells obtained by a nasopharyngeal brush in May 2021 were subjected to single-cell RNA sequencing (scRNAseq).A total of 2,838 cells were recovered from the analysis, which were clustered and classified based on their expression patterns, as shown in Figure 1.The most represented cellular types were epithelial cells (N = 2,128) and immune cells, including neutrophils (N = 304), T cells (N = 165), B cells (N = 111), macrophages (N = 42), and monocytes (N = 21).As shown in Figure 1, viral RNAs were identified mostly in epithelial cells (red dots, N = 14/16), with residual positivity also in macrophages and T cells (N = 1 each, respectively). The patient did not develop an anti-SARS-CoV-2 humoral response on plasma collected 6 months after the primary infection; more specifically, no IgG anti-S1-RBD was present in July 2021, while only low levels of IgG against the trimeric spike protein were detected.Due to the persistence of upper respiratory tract SARS-CoV-2 infection and symptoms consistent with post-COVID-19 condition, off-labeled anti-SARS-CoV-2 casirivimab/imdevimab (1,200 mg/1,200 mg) was administered intravenously in the outpatient service of the ID unit on July 28.This therapy was well-tolerated without any adverse reaction.After mAb infusion, the patient had a rapid clinical improvement with the resolution of all post-COVIDassociated symptoms in the subsequent 3 weeks.On August 6, the patient had positive anti-spike IgG antibodies, confirmed by two independent immunochemiluminescent tests (see Supplemental).Anti-trimeric spike protein IgG was positive with 2,080 BAU/ml, as well as IgG anti-S1-RBD with 185,311 AU/ml values.Furthermore, two successive RT-PCR nasal swabs performed on September 4 and 8 did not detect SARS-CoV-2, and the virus could not be isolated in Vero E6 cells.The patient has been doing well during the next 4-month follow-up period.A timeline including the clinical and virological courses is presented in Figure 2. ",
"section_name": "Case 1",
"section_num": null
},
{
"section_content": "On 17 May 2021, a 67-year-old man presented to the emergency room of our hospital with fever, dyspnea, asthenia, skin, and mucous hemorrhagic manifestations.The patient had a previous diagnosis of CLL (unmutated IGHV gene, positive for 11q deletion with no further alterations of chromosomes 12, 13, and 17 and TP53) that was treated with six cycles of fludarabine, cyclophosphamide, and rituximab (FCR) achieving a complete response in April 2016.Because of progressive symptomatic disease in 2018, he started second-line therapy with ibrutinib with a persistent partial response.During the last 5 years, the patient experienced several bacterial and viral infectious episodes (viral stomatitis, varicelliform herpes zoster with residual neuritis, bacterial infection of the oral cavity, and Pseudomonas aeruginosa infection of the nasal cavities).Furthermore, he was vaccinated for COVID-19 with the second dose performed on 9 May 2021 (BNT162b2 mRNA vaccine).At the time of admission to the emergency room, a nasal swab confirmed SARS-CoV-2 infection; blood tests showed moderate anemia (hemoglobin 87 g/L), leukopenia (white blood cells 2.59 × 10 (9)/L, neutrophils 1.08 × 10 (9)/L, lymphocytes 1.45 × 10 (9)/L), severe thrombocytopenia (platelet count 17 × 10 (9)/L), increased acute-phase reactants (C-reactive protein 192.1 mg/L, procalcitonin 2 ng/ml, and D-dimer level 41,791 ng/ml FEU).Oxygen saturation was 92% and a chest X-ray and a pulmonary CT confirmed interstitial pneumonia.The patient was admitted to the ID unit; ibrutinib therapy was stopped, while treatment with amoxicillin and clarithromycin was started together with supplemental oxygen, methylprednisolone therapy, and red blood cell (RBC) and platelet transfusions.During hospitalization, respiratory failure 2).A reverse mutation was observed in two positions (nt 22337 and 23009), and excluding the reverse mutations and deletion, the non-synonymous/synonymous mutation ratio was 7/3.The second swab was also inoculated in Vero E6 cells, and viable virus was recovered demonstrating the infectivity of the sample. On 6 December 2021, the patient was treated with casirivimab/imdevimab (600mg/600 mg) with good tolerance; a nasal swab for SARS-CoV-2 performed 4 days later was negative.Because of progressive disease, on 30 December 2021, he resumed ibrutinib therapy with no response; on March 2022, BTK and phospholipase Cg2 (PLCG2) mutations of acquired resistance to ibrutinib therapy were detected.Currently, in April 2022, the patient is in fair clinical conditions, and he started venetoclax therapy.A timeline including the clinical and virological courses is presented in Figure 2. ",
"section_name": "Case 2",
"section_num": null
},
{
"section_content": "Data from the COVID-19 pandemic have clearly indicated that patients with hematological malignancies are associated with a higher risk to develop multiorgan complications and a significant increased mortality rate upon SARS-CoV-2 infection (3,4).Because of their immune-incompetent status, secondary to the characteristic of hematological disease and/or treatments adopted to cure it, these patients frequently fail to develop anti-SARS-CoV-2 antibodies or cellular response to primary infection leading to prolonged viral replication (6).The same immune defect leads frequently to an impaired immune response to SARS-CoV-2 vaccination. Several experiences highlighted this issue; 19 patients with lymphoma treated with chemotherapy regimens, including anti-CD20 antibodies, have been found to show persistent SARS-CoV-2 infection (median duration 65 days, range 3 weeks-12 months), and most of them did not develop anti-SARS-CoV-2 antibodies (16).Similarly, a persistent PCR positivity (defined as SARS-CoV-2 RNA detection ≥30 days after initial positivity) has been observed in 51 (13.9%) of 214 lymphoma patients in a 1-year period of observation.In this series, the risk factors independently associated with prolonged infection were lymphopenia, treatment with anti-CD20 antibodies within 1 year, and cellular therapy including hematopoietic stem cell transplantation (HSCT) (17).Hueso et al. reported a small cohort of lymphoma patients (15) with profound B-cell lymphopenia and prolonged SARS-CoV-2 infection treated with convalescent plasma.The median duration of COVID-19 symptoms was 56 days (range 7-83), and all patients failed to develop SARS-CoV-2 antibodies (18).Other case series described prolonged SARS-CoV-2 infection associated with clinical relapse of COVID-19; for instance, in a patient diagnosed with mantle cell lymphoma (MCL), a blastoid variant was described.He was previously treated with two cycles of bendamustine, cytarabine, and rituximab and experienced persistent SARS-CoV-2 viremia associated with four clinical relapses of COVID-19 effectively treated with remdesivir (19). On these grounds, the long-term persistent positivity of the SAR-CoV-2 swab observed in our two CLL patients was not surprising.Even though case 1 did not receive any chemotherapy, he had severe hypogammaglobulinemia with the inability to produce neutralizing antibodies (15,16).As a matter of fact, immunodeficiency in CLL is multifactorial and mediated by T-cell defects, suboptimal complement activity, neutrophil and natural killer cell dysfunction, and altered normal B-cell activity (20).The second patient has been treated with multiple regimens including FCR and, subsequently, ibrutinib due to the progressive disease.Both FCR and ibrutinib are two well-known therapeutic regimens associated with profound immune depression and a higher risk to develop common or opportunistic infections in CLL patients. In particular, patients receiving ibrutinib or, similarly, other BTK inhibitors, which nowadays are more and more adopted as first-line or salvage therapy in CLL, have been associated with a very high risk of COVID-19 complications and mortality (6,21,22).The second patient was vaccinated with two doses of mRNA vaccine, the last one on 9 May 2021 before the onset of COVID-19 but unfortunately without achieving active immunization. Both cases had long complained of symptoms with multiple organ impairment following COVID-19 pneumonia; they did not develop anti-SARS-CoV-2 plasma humoral response and showed persisting positive nasopharyngeal RT-PCR for SARS-CoV-2 for 236 and 199 days, respectively.As suggested by Proal and colleagues (23), SARS-CoV-2 may cause chronic symptoms because it persists in different tissue reservoirs after acute infection as confirmed by the identification of SARS-CoV-2 inert viral RNA and/or proteins.The cell culture obtained by a nasal brush of case 1 detected viral RNA predominantly in epithelial cells but, at a lower extent, also in macrophages and T cells.These findings confirm that SARS-CoV-2 preferentially infects nasal epithelial cells, as already demonstrated by the high levels of angiotensinconverting enzyme 2 (ACE2) expression in this cell type (24).While SARS-CoV-2 infection has been reported also for macrophages by scRNA-seq analysis (25), viral infection of T cells on the contrary has not been reported. Although data about SARS-CoV-2-acquired mutations in hematological patients are scarce, preliminary experiences demonstrated that persistent viral infection may promote intrahost viral evolution as a consequence of several acquired mutations, particularly in the spike gene.This effect may lead to the emergence of SARS-CoV-2 variants (26, 27) that could negatively impact patients' clinical outcome, particularly in immunosuppressed hosts.In our two patients, indeed, nasopharyngeal swab samples obtained 2 and 3 months apart showed an increasing number of mutations suggestive of an ongoing positive selection pressure, especially in the S gene. To the best of our knowledge, this is the first analysis reporting the successful outcome of persisting SARS-CoV-2 infection associated with post-COVID-19 condition in hematologic patients treated with anti-SARS-CoV-2 mAbs.F o l l o w i n g t h e r a p y w i t h c a s i r i v i m a b / i m d e v i m a b , nasopharyngeal RT-PCR for SARS-CoV-2 became quickly negative in both patients; at the same time, post-COVID-19 condition symptoms progressively improved until completely disappearing in both patients. There are two case reports in the literature on SARS-CoV-2 persistent infection treated successfully with mAbs; however, such patients were not found to be affected by long COVID (14,15).On the contrary, despite the proven effectiveness of monoclonal antibodies, secondary acquired mutations of SARS-CoV-2 following monoclonal antibody therapy are emerging as an immune escape resistance mechanism in patients with B-cell malignancies (28). At present, there are no evidence-based guidelines indicating how to manage persistent COVID-19 infection in asymptomatic as well as post-COVID-19 patients.Although we cannot exclude that the virological and clinical cure of our patients was just a matter of time, the strong time relationship between mAb infusion and viral disappearance is consistent with the favorable effect of mAb therapy in these patients. In conclusion, our data show that the recovery of our patients might be due to passive immunity conferred by mAb treatment permitting SARS-CoV-2 clearance and resolution of post-COVID-19 condition.Controlled studies are needed to confirm this therapeutic strategy in immunocompromised patients with persisting viral infection and post-COVID-19 condition.Moreover, the variations observed in the sequences obtained in the two samples collected at 2-and 3-month intervals from both patients suggest a role of persistent SARS-CoV-2 infection as a possible source for the development of viral variants. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We thanked Fondazione Cassa di Risparmio di Trieste for the purchase of the Illumina MiSeq sequencer (Chromium 10x Genomics testing) and Generali SpA for the support. ",
"section_name": "Acknowledgments",
"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": "This study was reviewed and approved by Regional Ethics Committee (Unique Regional Ethical Committee, Friuli Venezia-Giulia 16 April 2020), No. CEUR 2020-OS-072.Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. LB and OS collected the patient data and wrote the manuscript.PDA analyzed NGS sequencing data and critically revised the manuscript.LS and RK performed SARS-CoV-2 real time PCR and NGS sequencing.PMO performed viral isolation.EO, FD, SDM performed single-cell RNA sequencing.AMa and DL collected and analyzed data and revised the manuscript.FZ and RL critically revised the manuscript and approved the final version of the paper.All the other authors contributed to the article and approved the submitted version. Dr. LB received honoraria for giving lectures at medical meetings from AbbVie.Dr. FZ received advisory board fees or honoraria for giving lectures at medical meetings from Roche, Celgene, Janssen, Sandoz, Gilead, Novartis, AbbVie, Amgen, Sobi, Argenx, Grifols, Takeda, and BeiGene. 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. 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/fonc.2022.945060/full#supplementary-material ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "This study was reviewed and approved by Regional Ethics Committee (Unique Regional Ethical Committee, Friuli Venezia-Giulia 16 April 2020), No. CEUR 2020-OS-072.Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "LB and OS collected the patient data and wrote the manuscript.PDA analyzed NGS sequencing data and critically revised the manuscript.LS and RK performed SARS-CoV-2 real time PCR and NGS sequencing.PMO performed viral isolation.EO, FD, SDM performed single-cell RNA sequencing.AMa and DL collected and analyzed data and revised the manuscript.FZ and RL critically revised the manuscript and approved the final version of the paper.All the other authors contributed to the article and approved the submitted version. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "Dr. LB received honoraria for giving lectures at medical meetings from AbbVie.Dr. FZ received advisory board fees or honoraria for giving lectures at medical meetings from Roche, Celgene, Janssen, Sandoz, Gilead, Novartis, AbbVie, Amgen, Sobi, Argenx, Grifols, Takeda, and BeiGene. 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. ",
"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/fonc.2022.945060/full#supplementary-material ",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.3389/fimmu.2022.867302
|
Abrogation of Notch Signaling in Embryonic TECs Impacts Postnatal mTEC Homeostasis and Thymic Involution
|
<jats:p>Notch signaling is crucial for fate specification and maturation of thymus-seeding progenitors along the T-cell lineage. Recent studies have extended the role of Notch signaling to thymic epithelial cells (TECs), showing that Notch regulates TEC progenitor maintenance and emergence of medullary TECs (mTECs) in fetal thymopoiesis. Based on immunohistochemistry studies of spatiotemporal regulation of Notch activation in the postnatal thymus, we show that <jats:italic>in vivo</jats:italic> Notch activation is not confined to fetal TECs. Rather, Notch signaling, likely mediated through the Notch1 receptor, is induced in postnatal cortical and medullary TECs, and increases significantly with age in the latter, in both humans and mice, suggesting a conserved role for Notch signaling in TEC homeostasis during thymus aging. To investigate the functional impact of Notch activation in postnatal TEC biology, we used a mouse model in which RPBJκ, the transcriptional effector of canonical Notch signaling, is deleted in epithelial cells, including TECs, under the control of the transcription factor Foxn1. Immunohistochemistry and flow cytometry analyses revealed no significant differences in TEC composition in mutant (RPBJκ-KO<jats:sup>TEC</jats:sup>) and wild-type (WT) littermate mice at early postnatal ages. However, a significant reduction of the medullary region was observed in mutant compared to WT older thymi, which was accompanied by an accelerated decrease of postnatal mTEC numbers. Also, we found that organization and integrity of the postnatal thymic medulla critically depends on activation of the canonical Notch signaling pathway, as abrogation of Notch signaling in TECs led to the disruption of the medullary thymic microenvironment and to an accelerated thymus atrophy. These features paralleled a significant increase in the proportion of intrathymic non-T lineage cells, mostly B cells, and a slight decrease of DP thymocyte numbers compatible with a compromised thymic function in mutant mice. Therefore, impaired Notch signaling induced in embryonic development impacts postnatal TECs and leads to an accelerated mTEC degeneration and a premature thymus involution. Collectively, our data have uncovered a new role for Notch1 signaling in the control of adult mTEC homeostasis, and point toward Notch signaling manipulation as a novel strategy for thymus regeneration and functional recovery from immunosenescence.</jats:p>
|
[
{
"section_content": "T lymphocytes, unlike the rest of blood cell lineages derived from multipotent hematopoietic progenitor/stem cells (HPCs), develop in a specialized organ distinct from the bone marrow or the embryonic liver; i.e. the thymus (1).Thymic epithelial cells (TECs) are the specific components of the thymus microenvironment that provide unique inductive signals for keeping early thymic progenitors on track to T-cell differentiation (2)(3)(4).Two molecularly and functionally distinct TEC subsets are sequentially involved in T-cell development, cortical (c) TECs and medullary (m) TECs, which are located at the thymus cortex and medulla, respectively.cTECs impose T-cell commitment and induce the differentiation, expansion and positive selection of developing thymocytes, by providing continuous activation of the evolutionary conserved Notch signaling pathway (5-7) through the expression of the nonredundant Delta-like 4 (DLL4) Notch ligand (8,9).Notch is a family of transmembrane receptors (Notch1 to Notch4 in mammals) with a major role in the regulation of critical processes such as cell fate specification, differentiation and proliferation/apoptosis in multiple cell lineages.Upon interaction with a membrane-bound specific ligand (Delta-like or Jagged in mammals), the intracellular domain of Notch (ICN) is proteolytically cleaved and released, entering the nucleus where it behaves as a transcriptional regulator of downstream genes, activating a particular genetic program (10,11).In the thymus, progenitors that interact with TECs in the cortex activate the T-cell maturation program and then migrate to the medulla where mTECs promote their terminal differentiation and participate in central tolerance induction (12)(13)(14). Despite its unique and crucial function in generating selfrestricted and self-tolerant functional T cells throughout life, the thymus is the first organ to undergo aged-related involution.This is an evolutionary conserved process beginning as early as birth and no later than the onset of puberty in humans and mice (15).Thymic involution mainly results from the degeneration of the epithelial component of the thymic stroma and is characterized by dramatic reductions in thymus size and TEC numbers, the expansion of adipocytes and fibroblasts, and the disorganization of the thymic architecture, leading to diminished thymocyte numbers and reduced naïve T cell output (16)(17)(18).These features characterize as well the thymic involution process induced under physiological stress conditions such as infection, pregnancy, and cancer treatments (reviewed in 18).While several molecular mechanisms have been proposed to be involved in stress-induced acute thymic atrophy, the underlying mechanisms of chronic age-related involution remain less clear.Recent studies have documented many changes of TEC biology throughout life, revealing a surprisingly dynamic population with a high turnover (17).Therefore, understanding how TEC maintenance and regeneration are regulated in the adult thymus is of critical relevance for understanding thymic involution. cTECs and mTECs arise early in ontogeny from a common thymic epithelial progenitor cell (TEPC) originated in the thymic primordium derived from the embryonic third pharyngeal pouch endoderm (19).This bipotent TEPC was identified in the fetal thymus (20)(21)(22) and its existence has been confirmed in the adult thymus (23)(24)(25)(26), although the physiological contribution of bipotent TEPC to adult TEC generation remains controversial (27).In the embryo, differentiation of TEPCs into cTEC and mTEC lineages and development of a functional thymus is critically controlled by the transcriptional regulator Foxn1 (23,28), which is induced in TEPCs by signals provided by other thymic components, including developing thymocytes (29)(30)(31).However, how cTEC/mTEC lineage specification and differentiation from the TEPC is induced has been a matter of intense debate.Studies showing that fetal TEPCs exhibit features and markers associated with the cTEC lineage (32,33), support a serial progression model of TEC differentiation, in which cTEC lineage represents a default pathway, whereas mTEC specification from the common TEPC requires additional specific cues (34).The potential mechanisms controlling this mTEC specification step and the emergence of separate mTEC-and cTEC-restricted progenitors have remained poorly understood, although independent evidence has begun to emerge suggesting that the Notch pathway may be involved.In fact, signaling provided by the DLL1 Notch ligand induces maturation of fetal mTECs leading to the organization of medullary areas in a FTOC (35), while mice deficient in Jagged2 have thymi with reduced medullary areas (36).In the adult thymus, however, TEC-specific overexpression of active Notch leads to inhibition of mTEC lineage development and reduced TEC cellularity (37), indicating that Notch expression by TECs might be temporally regulated.Recently, two groups have provided genetic evidence that Notch signaling plays a crucial role at multiple embryonic stages during TEC development, but may be dispensable in postnatal life (38,39).Importantly, they showed that Notch activation is required for maintenance/expansion of the undifferentiated TEPC and mTEC-restricted progenitor pools, and also for mTEC fate induction (38), while once the mTEC lineage was specified, further mTEC development was independent of Notch activity.Accordingly, repression of the Notch pathway was shown mandatory for progression of early mTECs to the mature mTEC stage (39), a fact that concurs with the downregulation of Notch activation in TECs after birth (35,37).Collectively, these data have revealed a critical role of Notch as a potent regulator of TEPC homeostasis and mTEC lineage fate during fetal thymus development, although Notch function in the epithelial compartment of the postnatal thymus remains to be investigated.This is an important issue, regarding the hypothetical contribution of TEPC to adult TEC turnover (17,(23)(24)(25)(26)(27), which may impact the dynamics of thymus involution and its consequences to immunosenescence. In this study, we have approached the potential contribution of Notch to postnatal TEC biology using two complementary strategies.First, we performed quantitative immunohistochemistry and confocal imaging approaches of in situ thymus Notch signaling (40) and provide evidence of a spatiotemporal regulation of in vivo Notch activation in both human and mouse postnatal TECs.Then, we made use of an in vivo genetic model of Foxn1-controlled conditional inactivation of Notch signaling in murine epithelial cells, including TECs, and reveal that lack of Notch signaling accelerates age-dependent loss of mTEC numbers and affects medulla integrity in the postnatal thymus.Therefore, we suggest a key role for Notch signaling in the control of postnatal mTEC homeostasis and age-dependent thymic involution. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Human thymus biopsies were obtained from male and female Caucasian pediatric patients aged 3-days to 15-years undergoing corrective cardiac surgery, after informed consent was provided, and in accordance with the Declaration of Helsinki and to the procedures approved by the Spanish National Research Council Bioethics Committee. Animal studies were reviewed and approved by the Animal Experimentation Ethics Committee of the Comunidad de Madrid, in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123).Mice were kept under specific pathogen-free conditions and used according to institutional regulations.C57BL/6J mice were obtained in-house from the departmental breeding facility.C57BL/6J RBPjk fl/fl conditional knockout mice generated by Prof. Tasuku Honjo (41) and C56BL/6J Rosa26 loxPLacZ reporter mice (Jackson Laboratory) were obtained from Dr. Jose Luis de la Pompa (CNIC, Madrid).The transgenic B6D2F1/J Foxn1-Cre line containing seven copies of the Cre transgene under the control of the Foxn1 promoter has been previously generated (42), and heterozygous mutants (Foxn1 Cre/+ ) were kept as a colony.Mice homozygous for a conditional deletion of RBPjk specifically in epithelial cells were obtained by crossing Foxn1 Cre/+ heterozygous to RBPjk fl/fl homocygous mice, followed by backcrossing of resultant Foxn1 Cre/+ RBPjk fl/+ F1 heterozygous to RBPjk fl/fl homocygous mice (Supplementary Figure 1A).Further selection of Foxn1 Cre/+ x RBPjk fl/fl mice was performed by PCR genotyping (Supplementary Material).These mice, referred to as RBPjk-KO TEC , displayed Cre-mediated RBPjk deletion exclusively in epithelial cells, including TECs, but not in other thymic cells.Mouse gender was not considered in any experiment.Foxn1 +/+ x RBPjk fl/fl littermates were used as wildtype (WT) controls.Selection of crossed mice was performed by PCR genotyping of genomic DNA obtained by proteinase K (Sigma) digestion of 3 weeks-old mouse ear discs tissue, as described in Supplementary Material. ",
"section_name": "Human and Mouse Thymus Samples",
"section_num": null
},
{
"section_content": "Tissue samples were fixed in 4% paraformaldehyde/phosphatebuffered saline (PBS) [PFA/PBS, Sigma-Aldrich] and paraffinembedded (Paraplast Plus, Sigma-Aldrich).Serial 8 mm sections were obtained from formalin-fixed paraffin-embedded (FFPE) slides that were mounted on poly-lysine-coated slides (SuperFrost UltraPlus, Thermo Fisher Scientific).Deparaffinised, rehydrated FFPE tissue slides were properly blocked as previously described (40).Tissue antigens were retrieved by boiling in sodium citrate (10 mM, pH 6.0) and endogenous peroxidase activity was quenched using 1% H2O2 100% methanol.For blocking of non-specific antibody binding sites, samples were incubated for 1h in blocking solution (3% bovine serum albumin, 20 mM MgCl2, 0.3% Tween 20, 5% fetal bovine serum in PBS), and permeabilized slides were incubated in blocking solution containing primary antibodies (Supplementary Table 1).Background and nonspecific staining was determined by incubating with Ig isotype-matched controls (Supplementary Figures 2,3).Before addition of secondary antibodies, tissue endogenous biotin was quenched with Avidin/ Biotin blocking solutions (Vector Laboratories).For Jag1 signal detection, tissue slides were incubated for 1 hour at RT with a horseradish peroxidase (HRP)-coupled anti-rabbit IgG secondary antibody (DAKO) and the signal was amplified using a Cyanine-3 Tyramide Signal Amplification (TSA-Cy3) Kit (NEL 744, 25 Perkin Elmer).For Notch1, Notch3, Notch4 and cleaved Notch1 (ICN1) signal detection, biotinylated anti-rabbit IgG secondary antibody (Vector Laboratories) was added before signal amplification with an Avidin/Biotin-HRP complex (Elite Vectastain ABComplex Kit, Vector Laboratories) and TSA-Cy3 Kit.For pan-cytokeratine (pCK) signal detection, Alexa Fluor dye-conjugated secondary antibodies were used (Thermo Fisher Scientific).The ABCamplified signal was developed by adding Alexa Fluor 488-or Alexa Fluor 555-conjugated streptavidin (Thermo Fisher Scientific).Nuclei were stained with Topro3 (Thermo Fisher Scientific) and slides mounted with Fluoromount-G (SouthernBiotech). Images were acquired using an LSM510 or an LSM900 laser scan confocal microscope (Zeiss) coupled to an Axio Imager.Z1 or an Axiovert 200 or an Axio Imager 2 (Zeiss) microscope using the following magnifications (Zeiss): 10× Plan-Neofluar (numeric aperture [NA] 0.3), 25× Plan-Neofluar [oil (NA 0.8)], 40×Plan-Neofluar [oil (NA 1.3)], 40xPlan-Apochromat [oil (NA 1.3)] and 63×Plan-Apochromat [oil (NA 1.4)].Images were processed using ImageJ.Brightness and contrast were adjusted equally in samples and controls when needed.For defining nuclear (Hes1, ICN1 and Topro) regions of interest (ROIs), Otsu algorithm was used to select positive cells by intensity threshold (43) For defining pCK + ROIs, Li algorithm (44) was used (Supplementary Figure 3).A median filter at 0.2 mm was used to remove noise before creating the selections. Quantitative analyses of Hes1 + or ICN1 + cell numbers in thymus cortical or medullary regions was performed by using image thresholding (45).As TECs, and particularly cTECs, form an extensive network of finely branched cell processes, numbers of individual TECs in this network are difficult to define (46).Therefore, no quantitative measurements of TEC frequencies, especially of Hes1 -/ICN1 -TECs, in the cortex vs the medulla could be performed.Rather, total numbers of Hes1 + /ICN1 + nuclei within pCK + ROIs were calculated relative to total Topro area or to pCK + area defined in the thymus cortex or the medulla (Supplementary Figure 3).To this end, pCK + ROIs were first defined as described above, and then used to create binary masks.Both nuclear (Hes1 or ICN1) and pCK binary masks where then processed on Image J's \"Image Calculator\" using the logic operator \"AND\".During image processing, a particular pixel intensity level (the threshold) is automatically defined by algorithms.Then, the number of pixels within the threshold is used to make a selection of ROIs, which exclusively contain the pCK-specific signal.The ROI is then used to calculate the total pCK + area of TECs (in mm) and the number of Hes1 + / ICN1 + nuclei within.Every cell out of the pCK ROI, including thymocytes positive for Notch activation markers, are systematically excluded and thus not considered in the analysis Histomorphometric measurements of thymic cortex and medulla (Supplementary Figure 4) were also performed in ImageJ by ROIs using Jag1 and/or Topro intensity level threshold (43). ",
"section_name": "Immunohistochemistry and Confocal Microscopy",
"section_num": null
},
{
"section_content": "Skin samples were fixed in 4% paraformaldehyde (PFA)/PBS solution (Sigma-Aldrich) and embedded in paraffin.Deparaffinised tissue slides were incubated for 3 min.in Harry's hematoxilin (Sigma), washed and quickly differentiated (10 to 15 sec) in acid alcohol solution (0.5% HCl; 70% ethanol).Next, they were incubated for 9 min.in 0.5% (w/v) Eosin solution (Sigma) and sequentially dehydrated in graded ethanol series.Tissue slides were briefly incubated in xylene, mounted with Entellan mounting medium (Merck, Millipore), and analyzed with an optical microscope (DM2500; Leica) equipped with a CCD camera (DFC420; Leica), with Leica Application Suite software (version 4.3.0). For b-galactosidase staining, thymic samples were fixed in 0.125% glutaraldehyde/PBS solution, washed (0.02% Nonidet-P40, 0.11% sodium deoxycholate, and MgCl2 2mM in phosphate buffer 0.1M, pH 7.3) and stained with X-gal staining solution (washing buffer supplemented with potassium ferricyanide 5mM, potassium ferrocyanide 5mM and 1mg/ml of X-gal resuspended in N,N-dimethyformamide).Samples were then washed, fixed in 4% PFA/PBS and paraffin-embed.Sections (8 mm) were mounted on poly-lysine-coated slides (SuperFrost Ultra Plus, Thermo Scientific) and deparaffinised as specified earlier.Cell nuclei were stained with Nuclear Fast Red (Vector Labs), sequentially dehydrated in graded ethanol series and xylene, and mounted with Entellan mounting medium (Merck, Millipore). ",
"section_name": "Hematoxilin/Eosin and b Galactosidase (LacZ) Staining",
"section_num": null
},
{
"section_content": "For flow cytometry TEC analysis, thymus samples from either RBPjk-KO TEC or Foxn1 +/+ x RBPjk fl/fl control littermates, no separated by gender and aged from 0.5-to 12-months, were dissociated in RPMI medium (1.25 mg/ml collagenase D (Roche) following three digestion steps of 15 min at 37°C.Isolated cells were then diluted in RPMI1640 medium with 10% FBS (Gibco) containing DNaseI (Roche; 0.05 mg/ml).After filtering cell suspension through 70 mm cell strainer (Filcon) to remove clumps, flow cytometry was performed using a sequential gating strategy (Supplementary Figure 5) on cells stained with DAPI (Beckman Coulter) to exclude dead cells, anti-CD45-FITC (eBioscience) and anti-TER-119-FITC (Biolegend) mAb, to exclude hematopoietic and erythroid-lineage cells.Anti-MHCII-PECy7 (eBioscience) and anti-EpCAM-APCCy7 (Biolegend) was used to electronically gate TECs.EpCAM-gated TEC cells were then analyzed for reactivity with the anti-Ly51-PE (eBioscience), and UEA-1 biotinilated (Vector Labs) cTEC and mTEC-specific mAbs, respectively, developed using Streptavidin-APC (Biolegend). For thymo cyte fl ow cy tome try, Fic oll-Hypaque (Lymphoprep, Axis-Shield PoC AS)-separated thymus cell suspensions were stained with the following mAbs: anti-CD8-FITC (Life Technologies), anti-CD4-PE (BD Biosciences), anti-CD19-PE (eBioscience), anti-CD90 (Thy1) (Biolegend), anti-CD11b-FITC (BD Biosciences), anti-B220-PE-Cy5 (Life Technologies), anti-NK1-APC (BD Biosciences).Anti-Ly5.1 and anti-Ly5.2mAbs (BioLegend) were used in adoptive transfer experiments.Acquisition and analysis was performed in a FACSCanto II (BD Biosciences).All flow cytometry data were analyzed using FlowJo Version 10.0.7.2. ",
"section_name": "Flow Cytometry",
"section_num": null
},
{
"section_content": "For the generation of BM chimeric mice, cell suspensions were isolated by Fycoll-Hypaque from BM samples obtained from femurs of 9 weeks-old RBPjk-KO TEC (Ly5.2 + ) mice, and BM cells (5x10 6 ) were resuspended in 100ml of sterile PBS and injected i.v.into 8 weeks-old C57BL/6 (Ly5.1 + ) hosts (n=4) subjected to lethal irradiation (10 Gys) the day before.Recipient mice were euthanized 4 months post-transplantation and thymus reconstitution by Ly5.2 + cells was analyzed by flow cytometry.As control, BM cells from Foxn1 +/+ x RBPjk fl/fl WT littermates (Ly5.2 + ) were injected into C57BL/6 (Ly5.1 + ) irradiated hosts (n=2). ",
"section_name": "Adoptive Cell Transfer",
"section_num": null
},
{
"section_content": "Statistical analysis was performed with GraphPad Prism 7.0 Software.The normal distribution of the data was tested using the Shapiro-Wilk normality test.When comparing two means of normal data, statistical significance (p) was determined by the unpaired two-tailed Student's t-test.When comparing twomeans of non-normal data, statistical significance (p) was determined by the unpaired Mann-Whitney test.When comparing more than two groups of normal data, one-way ANOVA was used, and for no normal data Klustal-Wallis was used.When comparing groups of two independent variables, two-way ANOVA was used.In all cases, the a-level was set at 0.05.Data in graphs are presented as mean ± SEM. ",
"section_name": "Statistics",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Detailed analyses of Notch activation in postnatal TECs are scarce in mice and remain to be performed in humans.We approached this issue by three-color immunohistochemistry and quantitative confocal microscopy of several postnatal human (≤ 6-years) and mouse (≤ 5-months) thymus samples labelled with a mAb recognizing the well-established target of canonical Notch signaling Hes1 ( 13), together with a TEC-specific anti-pCK mAb mix, and with Topro3 for nuclear staining.General examination of representative thymus sections stained with anti-Hes1 and Topro3 revealed a continuous pattern of nuclear Hes1 expression throughout the whole human thymus, which seemed more prominent at the medulla and was similar in the mouse postnatal thymus.Detailed analyses aided by the co-staining with anti-pCK, allowed the identification of Hes1 + pCK + TECs at the thymus cortex and medulla in both species (Figures 1A,C).Hes1 + cells lacking the pCK TEC maker, characterized in previous studies as developing thymocytes (40), were also identified distributed throughout the inner cortex in both human and mouse thymi; while, as shown previously (40), Hes1 + thymocytes seemed less abundant at the medulla, suggesting that Hes1 expression at the medulla occurs mostly in TECs.(Figures 1A,C).A significant fraction of such Hes1 + mTECs, which displayed the highest Hes1 expression levels, was found accumulated in Hassal's corpuscles (HCs) in the human thymus (Figure 1A).Therefore, these results indicate that Notch activation is conserved in postnatal cTECs and mTECs.Quantitative confocal analyses based on thresholding image approaches (45; Supplementary Figure 3), confirmed that measurable numbers of Hes1 + nuclei were distributed within the cortical and medullary pCK + areas analyzed in both human and mouse postnatal thymus samples, with Hes1 + cells being more abundant at the medulla in both species (Figures 1B,D).However, no frequencies of Hes1 + cTECs versus mTECs could be establish by this approach, as TECs, and particularly cTECs, have a complex morphology and display a high intrathymic cellular density (46), making it difficult to identify individual TECs and to define TEC numbers within particular ROIs.Collectively, these analyses provide the first direct evidence that Notch signaling is active in vivo in the human postnatal thymus, in TECs located both at the cortex and the medulla, and confirm that activation of Notch is also induced after birth in the mouse thymus, pointing to a conserved role for Notch signaling in postnatal TEC biology. ",
"section_name": "Notch Signaling Is Active In Vivo in Human and Mouse Postnatal TECs",
"section_num": null
},
{
"section_content": "While murine fetal TECs express several Notch receptors (35,38,39), genetic evidence has been provided that Notch1 is the receptor responsible for Notch activation in mouse embryonic TECs (39).To begin to decipher which Notch receptor/s is responsible for in vivo Notch signaling in the human thymus, we analyzed in situ Notch receptor expression in tissue sections of human postnatal thymus labeled with the anti-pCK mAb in combination with a mAb specific for either Notch1, Notch3 or Notch4.Immunohistochemistry and confocal microscopy showed that, as expected from previous studies (40), Notch1 is broadly expressed by pCK-negative thymocytes distributed mostly throughout the cortex.In addition, Notch1 was expressed by a minor population of pCK + cTECs and by a significant number of mTECs (Figure 2A).Notch3 displayed an expression pattern similar to Notch1, and was significantly expressed by cortical thymocytes, but only by few cTECs, while substantial numbers of mTECs coexpressed pCK and Notch3 (Figure 2A).In contrast, Notch4 expression was essentially confined to a non-epithelial pCK -population located at medulla, which has previously been characterized as dendritic cells (47), although rare Notch4 + mTECs could be identified as well (Figure 2A).Therefore, as shown before for mouse fetal TECs (39), Notch1 may be the preferential receptor that mediates Notch signaling in vivo in human postnatal TECs in both cortex and medulla, with a possible contribution of Notch3 in mTECs. To directly investigate the contribution of Notch1 to in vivo activation of Notch signaling in human postnatal TECs, we performed immunohistochemistry and confocal microscopy, using a mAb against the active intracellular form of Notch1 (ICN1) in combination with anti-pCK antibodies.These analyses confirmed Notch1 activation in situ in the human postnatal thymus, revealing nuclear expression of ICN1 in cells distributed throughout both the cortex and the medulla (Figure 2B).As shown before (40), we found that significant numbers of cells expressing active Notch1 in the cortex were pCK-negative hematopoieitic cells, although ICN1 + cTECs were also identified, while cells that display Notch signaling at the medulla seemed to be mostly pCK + mTECs (Figure 2B).Quantitative analyses of imaging data allowed to measure significant numbers of ICN1 + nuclei within the pCK + cortical and medullary areas (Figure 2C), supporting that both cTECs and mTECs activate Notch1 in vivo.Collectively, the observed ICN1 expression pattern suggests that the Notch1 receptor contributes significantly to in vivo activation of Notch signaling in human postnatal TECs. ",
"section_name": "The Notch1 Receptor Mediates In Vivo Activation of Notch Signaling in Human Postnatal mTECs",
"section_num": null
},
{
"section_content": "In the course of our studies on in vivo activation of Notch signaling, we noticed a consistent heterogeneity of ICN1 + cell numbers among human thymus samples at distinct postnatal ages from 1-month to 6-years.Considering that significant physiological changes occur in the human thymus during the first few years of life (15), we wanted to investigate the possibility that activation of Notch signaling could be regulated along time in the postnatal thymus.To this end, we performed quantitative immunohystochemistry and confocal microscopy of ICN1 expression in two groups of human thymus samples representative of early (≤1.5 years) and late (6-13 years) postnatal ages.The selected groups were expected to differ in age-dependent physiological features associated to thymic involution, as regression of the thymic epithelium can be observed early in life in humans, long before puberty (reviewed in 15).As current data in mice have shown that in vivo Notch activation during thymopoiesis is selectively induced in medullary-lineage TECs (38,39), age-dependent Notch activation was specifically analyzed in the thymus medulla.We thus performed detailed image analyses of ICN1 and pCK expression in mTECs and found that activation of Notch1 signaling was more prominent in the medulla of late compared to early human postnatal thymi (Figure 3A).Although morphologically heterogeneous, mTECs are less dense than cTECs, and therefore more easily defined as individual cells (46), allowing us to perform quantitative measurements of pCK + cells expressing nuclear ICN1, as shown in Supplementary Figure 3B.These analyses revealed that numbers of mTECs expressing ICN1 increased 50% on average in the late compared to the early human postnatal thymus (Figure 3B), supporting an age-dependent activation of Notch1 signaling in mTECs.Then, we investigated whether this progressive increase of mTECs expressing active Notch1 could be observed in mice.To this end, we performed quantitative analyses of in situ Notch1 activation in mTECs from mice aged 2-weeks to 9-months (Figures 3C,D).The results showed a slight, but not significant, decrease in the numbers of murine mTECs that expressed active Notch1 during the first weeks of life from 0.5 to 1.5 months of age (Figure 3D), coincident with the period of neonatal thymus growth (17).However, ICN1 + mTEC numbers increased significantly by 3 months, and up to 4-fold by 5 months (Figure 3D), confirming a highly significant age-dependent upregulation of Notch1 activation in postnatal mouse mTECs.Collectively, the observed age-associated activation of Notch1 signaling in the postnatal thymus of both humans and mice suggests a conserved role for Notch1 signaling in the biology of postnatal mTECs. ",
"section_name": "Activation of Notch Signaling Increases With Thymus Age in Postnatal mTECs",
"section_num": null
},
{
"section_content": "To better understand the contribution of the Notch pathway to postnatal mTEC biology, we next analyzed the impact of impaired Notch activation in TECs, by using a conditional loss-of-function mouse model, in which canonical Notch signaling was selectively abolished in epithelial cells by crossing Foxn1-Cre mice (42) to the Rbpj fl/fl conditional knockout mouse line (41) (Supplementary Figure 1A).Transgenic Cre expression in Foxn1-Cre mice parallels endogenous Foxn1 expression in epithelial cells and can be detected as early as E10.5 in the thymus primordium (42).Crossing Foxn1-Cre mice to the Rosa26 loxPlacZ reporter strain has revealed Foxn1 protein expression at E11.5, while Foxn1controlled b-galactosidase reporter expression detected by LacZ staining is induced at E12.5 (42), and can be observed in the postnatal thymus as well (Supplementary Figure 1B).Therefore, RBPjk in Foxn1-Cre x Rbpj fl/fl homozygous mice (hereafter referred to as RBPjk-KO TEC ) might not be abolished before E11.5-12.5, which corresponds to a time in development when TEC progenitors have been established and their progeny has contributed to an initial thymus primordium.At later stages, emerging TECs and skin epithelial cells (42), will be unable to activate the canonical Notch signaling pathway in mutant mice (41).Confirming Notch abrogation in skin epithelial cells, RBPjk-KO TEC mice developed macroscopic cutaneous lesions, which were evident at 8-months, when animals showed clear signs of disease including numerous lesions at the face, footpad, tail and ventral skin (Supplementary Figure 1C).Microscopic examination of these lesions revealed a clear disorganization of the skin with signs of inflammation, leukocyte infiltration, hair follicle hyperproliferation, and the generation of keratin cysts (Supplementary Figure 1D), consistent with previous observations in distinct mouse models of Notch-deficient skin epithelium (48,49). Having confirmed the loss of Notch activation in the skin of RBPjk-KO TEC mice, we next investigated specific abrogation of Notch signaling in mutant postnatal TECs (≥ 5-months), as compared to Foxn1 +/+ x RBPjk fl/fl WT littermate controls.To this end, we performed comparative immunohistochemistry of Hes1 expression as readout of canonical Notch activation.Consistent results showed a prominent expression of Hes1 in the medulla of WT postnatal thymi, which was drastically reduced in RBPjk-KO TEC mutant thymi, confirming abrogation of Notch signaling (Figure 4A).Detailed examination of the cortical and medullary TEC niches (Figure 4B) confirmed that, as shown above (Figures 1,3), TECs that display Notch signaling in vivo represent a conspicuous population in the medulla of WT thymi, and Hes1 + TECs were also detected in the WT cortex (Figures 4C,D).Quantitative measurements of Hes1 + nuclei distributed within pCK + ROIs (Supplementary Figure 3) revealed a significant reduction of nuclei expressing Hes1 in both the cortex and the medulla of mutant RBPjk-KO TEC thymi compared to WT thymi of mice aged 3-months (Figure 4C), and a similar reduction was maintained in mice of 5-9-months (Figure 4D), which was consistently more significant in the medulla than in the cortex (Figures 4C,D).Collectively, these results confirmed that Foxn1-controlled abrogation of RBPjk impairs canonical activation of the Notch pathway in a substantial population of mTECs and also in a subset of cTECs in the postnatal thymus of RBPjk-KO TEC mutant mice. ",
"section_name": "Foxn1-Controlled RBPjk Deletion Abrogates Canonical Notch Activation in Postnatal TECs",
"section_num": null
},
{
"section_content": "To investigate the impact of the specific abrogation of Notch signaling in the TEC compartment of the postnatal thymus, we next performed flow cytometry to analyze the TEC composition of thymi isolated from mutant RBPjk-KO TEC mice and Foxn1 +/+ x RBPjk fl/fl WT littermates at different postnatal ages.To this end, cell suspensions from collagenase-dissociated thymi were analyzed for expression of EpCAM and MHC-class II (MHC-II) TEC markers after electronic exclusion of hematopoietic and erythroid-lineage cells by gating off CD45 + and Ter119 + cells (Supplementary Figure 5).Absolute and relative cell counts of EpCAM + cells revealed no significant numerical differences of total TECs between RBPjk-KO TEC and WT thymi at early (4weeks) postnatal ages, while TEC proportions decreased significantly in late (8-months) postnatal thymi of RBPjk-KO TEC mutant mice (Figure 5A).As we found that active Notch is expressed in vivo in mTECs in an age-dependent manner, we assessed whether the observed decrease of TEC numbers in aged mutant mice was the result of a preferential loss of mTECs.Thus, we then quantified cTECs and mTECs among EpCAM + TECs by FACS analyses based on expression of the specific Ly51 and UEA1 markers, respectively (Figure 5B).No significant differences were observed in the proportions of either cTECs or mTECs in RBPjk-KO TEC compared to WT thymi at 4-weeks of age, while relative mTEC numbers decreased significantly in thymi from 8-months-old RBPjk-KO TEC mice compared to Foxn1 +/+ x RBPjk fl / fl control littermates (Figures 5B,C).Therefore, TEC-specific loss of Notch signaling results in a marked decrease in the proportions of TECs in late but not early RBPjk-KO TEC mutant thymi, which results in a preferential reduction of mTECs.To further assess the kinetics of mTECs loss, we performed quantitative flow cytometry analyses of cTEC and mTEC numbers in mutant and WT littermates aged from 2-to 26-weeks.We found no significant differences in relative TEC numbers between the two groups at young postnatal ages (2-and 4-weeks) However, TEC proportions decreased markedly at 9-weeks in RBPjk-KO TEC compared to WT littermate mice, and this decrease progressed steadily to 26weeks (Figure 5D).Importantly, we found that WT mice also displayed a progressive age-dependent decrease of relative TEC numbers, as previously reported (17), although loss of TECs in mutant mice followed accelerated kinetics compared to WT littermates (Figure 5D).Therefore, impaired Notch signaling in TECs results in a marked acceleration of TEC number loss in the postnatal thymus.Independent quantification of relative cTEC and mTEC numbers revealed a preferential decrease of mTECs along age in both WT and mutant mice, which led to a significant reduction of the mTEC:cTEC ratio in both mouse groups by 9weeks (Figure 5E).The mTEC:cTEC ratio was maintained to minimal levels up to 26-weeks in RBPjk-KO TEC thymi, and the decrease was less pronounced in the thymus of WT littermates (Figure 5E).Therefore, our results indicate that in both RBPjk-KO TEC and WT mice, the observed age-associated decrease of postnatal TEC numbers can be attributed to a preferential loss of mTECs.However, abrogation of Notch signaling in RBPjk-KO TEC mutant mice leads to an accelerated loss of postnatal mTECs, suggesting that Notch activation regulates mTEC homeostasis in postnatal life. ",
"section_name": "Foxn1-Controlled Abrogation of Notch Signaling Leads to an Accelerated Loss of Postnatal mTECs",
"section_num": null
},
{
"section_content": "Age-dependent mTEC loss occurs in normal thymus as part of the thymic involution process (17).It is thus possible that Notch signaling may contribute to the control of mTEC homeostasis and age-dependent thymus involution in postnatal life.As thymic regression results in loss of thymic structure and disorganization of thymic architecture (reviewed in 18), we next performed histomorphometric analyses aimed at establishing detailed comparisons between the cortical and medullary compartments of postnatal thymi from RBPjk-KO TEC mice and Foxn1 +/+ x RBPjk fl/fl WT littermates.Expression of the Notch ligand Jag1, which is selectively expressed on TECs located at the medulla (40), was used to define the medullary microenvironment (Supplementary Figure 4A).Cortical and medullary area measurements by confocal microscopy revealed no significant differences in size and morphology of the cortex and medulla of young (0.5-months) thymi from RBPjk-KO TEC mice, as compared with WT littermates.However, a significant reduction of the medullary area was evident at 3 and 5 months of age in RBPjk-KO TEC thymi (Figure 6A).Compared to the WT thymic medulla, the mutant medulla appeared disorganized and composed of small discrete islets (Figure 6A), suggesting that TEC-specific abrogation of Notch signaling leads to the disruption of the medullary thymic microenvironment.Accordingly, histomorphometric measurements of cortical and medullary areas revealed a significant decrease of the average medulla to cortex area ratio of RBPjk-KO TEC thymi compared to WT thymi from the 3-and 5-months-old mice analyzed 4) of postnatal thymi from WT Foxn1 +/+ x RBPjk fl/fl and mutant RBPjk-KO TEC mice in A).Coexpression of pCK and Jag1 (confined to the medulla) was used to calculate medullary areas.Cortical areas were identified as pCK + Jag1 -and nuclear staining by Topro3 (blue) defined total thymic area.Data are shown as mean area ratios ± SEM obtained from n ≥ 10 images per sample (n ≥ 3 thymus samples per age).p values were calculated using a two-tailed t-test.(C) Ratio of medulla: cortex area measurements derived from histomorphometric analyses as in (B) of thymi obtained from WT Foxn1 +/+ x RBPjk fl/fl and mutant RBPjk-KO TEC mice at the indicated ages.Data are shown as mean area ratios ± SEM obtained from n ≥ 10 images per sample (n ≥ 3 thymus samples per age).Two-way ANOVA table summarizing the statistical analysis is shown *p<0.05;**p<0.01.(D) Thymus atrophy in RBPjk-KO TEC mice mutant mice at 3-months of age. (Figure 6B).Importantly, kinetic studies based on histomorphometric measurements of postnatal thymi at increasing ages, from 0.5-to 12-months, revealed that the significant reduction of the medulla to cortex area ratio observed in RBPjk-KO TEC thymi at 3-months of age was progressive along life (Figure 6C).Also, macroscopic examination revealed that the observed medulla reduction correlated with postnatal thymus atrophy in mutant mice that was evident by 3-months (Figure 6D).Collectively, these data indicate that maintenance of the anatomical organization and integrity of the postnatal thymic medulla critically depends on the activation of the canonical Notch signaling pathway in mTECs. The above findings showing a reduced and disorganized medulla in RBPjk-KO TEC postnatal thymi is consistent with the possibility that specific abrogation of Notch activation in TECs results in a premature thymic involution and leads to an impaired thymus function.To investigate this possibility, we analyzed T-cell development and thymic output in RBPjk-KO TEC and WT Foxn1 +/+ x RBPjk fl/fl aged mice by flow cytometry.We found that thymocyte numbers were equivalent in young WT and RBPjk-KO TEC mice (not shown), but decreased significantly in mutant compared to WT mice along life, to up to 70% by 12-months (Figure 7A).The observed thymocyte decrease paralleled a weak but significant reduction of the CD4 + CD8 + double positive (DP) thymocyte subset in RBPjk-KO TEC mice (Figures 7B,C).This decrease could be attributed to a homeostatic defect in mTECs (18) and associated paracrine signaling axes (46), which may indirectly affect cortical epithelial cell function.Alternatively, it may directly result from a defective function of cTECs in mutant mice.In addition to the DP cell loss, we observed a marked increase of non-T lineage (Thy1 -) cells in mutant mice compared to WT littermates, which accounted for up to 20% of total thymic cells at 12-months (Figure 7D).Flow cytometry analyses using lineage-specific markers identified B cells as the major non-T cell type accumulating in the adult mutant thymus, but NK cells and myeloid cells were also significantly increased (Figure 7E).As increased frequencies of thymic B cells is a feature associated with thymic involution in aged mice (18), our results suggest that a defective thymic microenvironment rather than an intrinsic functional defect of developing thymocytes is responsible for the observed expansion of non-T lineage cells in RBPjk-KO TEC thymi.To assess this possibility, we performed adoptive transfer experiments consisting on intra-venous injection of total hematopoietic cells isolated from the BM of either RBPjk-KO TEC or WT Foxn1 +/+ x RBPjk fl/fl Ly5.2 + littermates into lethally-irradiated C57BL/6J Ly5.1 + normal mice.Flow cytometry analyses of cells recovered from the thymus of host mice at 4 months posttransplant revealed no differences in the reconstitution efficiency of BM progenitors from either WT or mutant mice, as indicated by the equivalent proportions of Thy1 + T-lineage cells and DP, double negative (DN) and single positive (SP) subsets present in the host thymi (Figure 7F).Therefore, we can exclude an intrinsic functional defect of T-cell progenitors derived from RBPjk-KO TEC mutant mice.Based on our results, we concluded that Foxn1-controlled impaired activation of canonical Notch signaling leads to an accelerated loss of mTECs accompanied by disruption of the medulla integrity in the postnatal thymus, which concurs with an aberrant increase in the proportion of thymic non-T lineage cells and a decrease in DP thymocyte numbers, compatible with a premature thymic involution. ",
"section_name": "Abrogation of Canonical Notch Signaling in TECs Leads to a Reduced and Disorganized Postnatal Thymic Medulla and Accelerates Thymic Involution",
"section_num": null
},
{
"section_content": "We have studied the potential contribution of the Notch pathway to postnatal TEC biology using two complementary strategies.First, we analyzed Notch activation in situ in the human postnatal thymus by performing quantitative immunohistochemistry and confocal imaging.Our results show for the first time that Notch activation is regulated in vivo in the human thymic epithelium in a spatio-temporal manner.We found that Notch signaling, mediated in particular through the Notch1 receptor, is induced in situ in postnatal human TECs mostly located at the medulla, and this activation pattern is conserved in the mouse.Importantly, numbers of mTECs showing Notch activation increase significantly with age in both human and mouse postnatal thymi, suggesting a conserved role for Notch signaling in TEC homeostasis during aging.To further investigate this possibility, we made use of an in vivo genetic model of Foxn1controlled conditional inactivation of Notch signaling in murine epithelial cells.The model revealed that impaired Notch signaling in mutant TECs leads to an accelerated age-dependent decrease of postnatal mTECs that results in the disruption of the medullary thymic microenvironment and in an accelerated thymus atrophy. The observation that Notch signaling is activated in situ in the epithelial compartment of the postnatal thymus was somehow unexpected, as preliminary studies in mice (35)(36)(37), recently confirmed by genetic approaches, pointed to a role of Notch signaling limited to embryonic stages of TEC development, while Notch activation has been shown to be downregulated afterwards disappearing in postnatal TECs (38,39).Accordingly, Notch signaling critically regulates mTEC-lineage fate specification of embryonic TEC progenitors, but further mTEC development is dependent on repression of Notch activation (39), a process that may rely on HDAC3 function (37).These results seem in conflict with our finding that Notch is active in vivo in postnatal TECs; particularly, in a significant population of TECs located at the medulla.However, an important question is whether such mTECs with active Notch are immature or fully mature mTECs.While our current results cannot give a definitive answer to this question, the first possibility seems very likely considering that, during embryonic TEC development, Notch signaling is critical not only for mTEC specification, but also for maintenance/expansion of the pool of undifferentiated TEPC and mTEC-restricted progenitors (38,39).Considering that both TEPC and mTEC progenitors have been identified in the adult murine thymus (23)(24)(25)(26)(27), an attractive explanation for our results would be that expression of active Notch in the postnatal thymus is restricted to the TEPC and/or mTEC progenitor pools (50), thus controlling the high turnover of mTECs and their maintenance and regeneration in the adult thymus (17).In fact, it is known that the TEC compartment has an extensive cell division in fetal and neonatal life, but postnatal TEC proliferation decreases significantly by 4 weeks (17), while medullary TECs display relatively high turnover rates also during the postnatal stage.An alternative possibility is supported by the finding that mTECs that display active Notch signaling accumulate in the postnatal human thymus in HC, a structure derived from terminally differentiated mTECs, suggesting that Notch activation could be induced in mature mTECs. Considering the high developmental and functional heterogeneity revealed for the TEC compartment (14,51), generation of conclusive results on the exact maturation stage of postnatal mTECs that activate Notch signaling in vivo demands further studies.Nonetheless, an interesting finding of our work is that postnatal mTECs activate Notch signaling in an age-dependent manner in both humans and mice, as revealed by quantitative analyses.Comprehensive kinetics in mice showed that numbers of mTECs with active Notch signaling increased by 3-months of age, immediately after achievement of maximal thymic cellularity and coincident with the initiation of thymic involution (17).It is thus possible that activation of Notch signaling is upregulated at early postnatal ages to counteract the loss of mTECs associated with thymic involution (17).Supporting such a role, our loss-of-function genetic approach has shown that abrogation of canonical Notch signaling results in decreased proportions of TECs, mostly of mTECs, during postnatal life, while normal mTEC numbers were found during the first month of life.These findings concur with the results shown by Blackburn and coworkers using a distinct Foxn1-Cre x Rbpjfl/fl mouse model (38), in which mTEC generation is impaired in embryonic life, but mTECs proportions were normalized at week 8 after birth.Given that transgenic Cre expression in FoxN1-Cre mice parallels endogenous Foxn1 expression in epithelial cells (E11.5), and Foxn1-controlled expression is induced one day later as indicated by b-galactosidase expression (42), Notch signaling could not be abolished before E12.5 in mutant mice, which corresponds to a time in development when TEC progenitors have been established and their progeny has contributed to an initial thymus primordium.Thus, a relatively late timing of RBP-Jk deletion could result in reduced numbers rather than total loss of mTEC progenitors that would be able to recover normal numbers of mTECs in mutant thymi early after birth.Importantly, we show that, after mTEC numbers are normalized, abrogation of Notch signaling in mutant mice results in a further age-dependent dramatic loss of mTECs.Whether mTEC loss results from the impaired maintenance/ expansion or the enhanced mortality of mTECs and/or mTEC progenitors remains to be determined; but itconcurred with a marked disorganization of the thymic medulla architecture, and a significant reduction in thymus size, together with diminished thymocyte numbers, decreased proportions of DP thymocytes and the accumulation of intrathymic B cells.As all these features are associated with age-dependent thymic involution (16)(17)(18), we concluded that abrogation of Notch signaling in postnatal TECs may accelerate thymus aging and impaired thymus function (49).Accordingly, DP thymocyte frequency is a readout of thymus functionality that correlates inversely with thymus involution and mTEC loss (18), and has been associated with apoptosis susceptibility of thymocytes (52).Although we cannot establish whether DP thymocyte loss is directly dependent on the homeostatic defect in mTECs, it is posible that defective mTECs located at the corticomedullary junction, where accumulation of ICN1 + TECs was observed, could impact viability of recently selected DP thymocytes migrating from the cortex to the medulla.Alternatively, a defective mTEC paracrine signaling axis may indirectly affect cortical epithelial cell function (46), or defective DLL4 expression on mutant cTECs (53) may affect DP thymocyte generation. Understanding how mTEC maintenance and regeneration are regulated in the adult thymus downstream of Notch signaling is of critical relevance for understanding thymic involution, but the effectors involved in such Notch-mediated function remain to be identified.In this regard, it is worth noting that Myc and cyclin D1, two well-known downstream targets of Notch signaling have been shown to contribute to TEC growth and to promote a dramatic increase of thymus size upon ectopic expression in TECs (18,54,55).Notably, as described for Notch activation (38,39) Myc transcription declines in TECs during embryonic development, and minimal levels have been described after birth, suggesting that regulation of Myc function is required to limit thymic growth in adult mice.As Myc expression in adult TECs drives proliferation and results in thymic regeneration (54), it is possible that Notch signaling controls mTEC maintenance and thymic involution through Myc.An important question is how Notch signaling is temporally regulated to control mTEC maintenance and thymus homeostasis.To answer this question, we have to consider that spatio-temporal regulation of Notch ligand expression defines particular Notch signaling microenvironments in the thymus (40,56).Manley and coworkers have shown tan Notch1 signaling in TEC development begins soon after the onset of Foxn1 expression, when Jag1 and DL4 Notch ligands are expressed (39).Notch1 could also be the receptor mediating Notch signaling in postnatal mTECs, given the coincident patterns of Hes1 and active intracellular Notch1 (ICN1) expression observed in both human and mouse postnatal thymi.Although we cannot ignore the expression of Notch3 in human mTECs, this receptor could be upregulated following Notch1-mediated signaling as reported in thymocytes (56).In the postnatal thymus medulla, a possible source of Notch1 ligand would be other mTECs, which express Jag1 (40), though Notch ligand presented by developing thymocytes could induce Notch1 activation as well.In this regard, it is important to note that crosstalk between developing thymocytes and TECs in one of the mechanisms that control TEC development and likely thymus involution (16)(17)(18).TECs depend on the presence of thymocytes for their differentiation and organization (57,58), and they reciprocally provide the signals that regulate T lymphocyte generation (59).Therefore, Notch activation could be negatively regulated in mTECs during thymopoiesis once a given cellular density of SP thymocytes has been reached at the medulla.In this regard, recent results by Blackburn's group provided evidence of a cross-regulatory relationship between Notch and Foxn1, the master regulator of TEC differentiation that is required to maintain the postnatal thymic microenvironment in a dosage-sensitive manner (60)(61)(62), suggesting a Foxn1-mediated repression of Notch activity that could be reinforced via its direct ligands (38).Conversely, Foxn1 downregulation during thymus involution (60-62) could trigger Notch activation to counteract mTEC loss and thymus aging. While further studies are required to reach a full understanding of mechanisms controlling postnatal mTEC turnover and thymic involution, our results point toward manipulation of Notch signaling as a novel and promising strategy for thymus regeneration during aging. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "We thank Dr. Danay Cibrian (Centro Nacional de Investigaciones Cardiovasculares.Instituo de Salud Carlos III.Madrid, Spain) and Prof. Francisco Sańchez-Madrid (Servicio de Inmunologıá, Hospital Universitario de la Princesa, UAM, IIS-IP, Madrid, Spain) for helpful discussions and technical support. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "This work has been supported by the European Union Seventh Framework Programme (FP7/2007-2013) collaborative project ThymiStem (602587 to MLT) and by Spanish Ministry of Science and Innovation.(Agencia Estatal de Investigacion/European Regional Development Fund, European Union, SAF2014-62233-EXP, SAF2016-75442-R and PID2019-105623RB-I00 to MLT).Institutional grants from the Fundacioń Ramoń Areces and Banco de Santander to the Centro de Biologıá Molecular Severo Ochoa are also acknowledged. ",
"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 studies involving human participants were reviewed and approved by Spanish National Research Council Bioethics Committee.Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.The animal study was reviewed and approved by Animal Experimentation Ethics Committee of the Comunidad de Madrid (PROEX 002.16/21). MLT conceptualized, designed and supervised the study, wrote the manuscript and acquired funding.MG-L, MM, CC, and JA collected and processed the samples, performed the experiments, analyzed the data, and prepared the figures.SZ ̌and GH developed the animal models and supervised the study.MG-L drafted the first version of 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/fimmu.2022.867302/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or 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": "The studies involving human participants were reviewed and approved by Spanish National Research Council Bioethics Committee.Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.The animal study was reviewed and approved by Animal Experimentation Ethics Committee of the Comunidad de Madrid (PROEX 002.16/21). ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "MLT conceptualized, designed and supervised the study, wrote the manuscript and acquired funding.MG-L, MM, CC, and JA collected and processed the samples, performed the experiments, analyzed the data, and prepared the figures.SZ ̌and GH developed the animal models and supervised the study.MG-L drafted the first version of 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/fimmu.2022.867302/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or 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": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.3324/haematol.2009.010173
|
Differential diagnosis of cyclin D2+ mantle cell lymphoma based on fluorescence in situ hybridization and quantitative real-time-PCR
|
Mantle cell lymphoma is characterized by the t(11;14) chromosomal translocation, resulting in the overexpression of cyclin D1 (CycD1). Recently, cases of mantle cell lymphoma negative for cycD1 but positive for cycD2 or cycD3 were identified by gene expression profiling and confirmed by immunohistochemistry. We analyzed 4 cases of cycD2(+) mantle cell lymphoma with a translocation involving the CCND2 locus, and its differential diagnosis from 35 mature B-cell non-Hodgkin's lymphomas based on immunohistochemistry, quantitative RT-PCR and FISH analysis. Bona fide cycD2(+) mantle cell lymphoma carried translocations involving the CCND2 gene, and IGH and IGK loci were identified as partners. As a result of this translocation, cycD2 mRNA was highly over-expressed when compared with normal lymphoid tissue and other B-cell non-Hodgkin's lymphomas, including chronic lymphocytic leukemia, making this technique ideally suited to identify cycD2(+)mantle cell lymphoma. In contrast, positive immunostaining for cycD2 was found in most B-cell non-Hodgkin's lymphomas, and therefore, it is not specific for a diagnosis of cycD2(+)mantle cell lymphoma.
|
[
{
"section_content": "Mantle cell lymphoma (MCL) is a distinct subtype of aggressive B-cell non-Hodgkin's lymphoma (NHL) with specific clinical and pathological features that accounts for approximately 6% of all lymphomas. 1The genetic hallmark of mantle cell lymphoma (MCL) is the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the immunoglobulin heavy chain (IGH) gene on 14q32 to the CCND1 gene on 11q13 resulting in the overexpression of cyclin D1 (cycD1) mRNA and protein. 1 Recently, a gene expression profiling study of MCL identified a small subset of tumors negative for cycD1 mRNA expression but morphologically, immunophenotypically, and by global expression profile otherwise undistinguishable from conventional MCL. 2 Interestingly, these cases instead expressed cycD2 or cycD3 mRNA, suggesting that any of these cyclins can functionally substitute for cycD1 in MCL. Accordingly, cycD1 negative MCL cases lacked the t (11;14) translocation by fluorescence in situ hybridization (FISH) analysis, 2 and were negative for cycD1 protein expression by immunostains. 3However, no evidence of chromosomal translocations involving the corresponding CCND2 and CCND3 gene loci were identified. 3The controversy surrounding cycD1 negative MCL was ended with the demonstration of bona fide cases of cycD2 positive MCL secondary to gene translocations involving the CCND2 locus on chromosome 12p13 with either the IGK locus on chromosome 2p12 t(2;12)(p12;p13), 4,5 or a t(12;14)(p13;q32) translocation juxtaposing the CCND2 gene next to the IGH locus. 6he diagnosis of cycD1 negative MCL is challenging because some low-grade B-cell lymphomas, such as chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL) and follicular lymphoma (FL), may mimic MCL both morphologically and immunophenotypically. Indeed, the dif- Mantle cell lymphoma is characterized by the t(11;14) chromosomal translocation, resulting in the overexpression of cyclin D1 (CycD1).Recently, cases of mantle cell lymphoma negative for cycD1 but positive for cycD2 or cycD3 were identified by gene expression profiling and confirmed by immunohistochemistry.We analyzed 4 cases of cycD2 + mantle cell lymphoma with a translocation involving the CCND2 locus, and its differential diagnosis from 35 mature B-cell non-Hodgkin's lymphomas based on immunohistochemistry, quantitative RT-PCR and FISH analysis.Bona fide cycD2 + mantle cell lymphoma carried translocations involving the CCND2 gene, and IGH and IGK loci were identified as partners.As a result of this translocation, cycD2 mRNA was highly over-expressed when compared with normal lymphoid tissue and other B-cell non-Hodgkin's lymphomas, including chronic lymphocytic leukemia, making this technique ideally suited to identify cycD2 + mantle cell lymphoma.In contrast, positive immunostaining for cycD2 was found in most B-cell non-Hodgkin's lymphomas, and therefore, it is not specific for a diagnosis of cycD2 + mantle cell lymphoma.ferential diagnosis is important and relevant for patient treatment and prognosis.Until now, the recognition of potential cycD1 negative MCL has been based on microarray analysis, 2,3 a technique which is not available in routine practice.Although IHC for cycD2 and cycD3 has been proposed as a surrogate marker for cycD1 negative MCL, 3 the need to develop a reliable and accessible technique which is useful in the differential diagnosis is of utmost importance.The aim of this study was to investigate means to differentiate 4 cases of cycD2 + MCL with a CCND2 translocation from low-grade Bcell NHL, based on IHC, quantitative RT-PCR and FISH analysis with special interest on CD5 + B-cell NHL, including CLL and a subset of MZL. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Design and Methods",
"section_num": null
},
{
"section_content": "Formalin-fixed and paraffin-embedded biopsies from 35 well-characterized B-cell lymphomas, including 12 CLL, 8 MZL (5 cases CD5 + ), 5 FL and 10 cycD1+ MCL were selected from the files of the Institute of Pathology, Technical University of Munich, Germany.All cases were classified according to the guidelines of the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues. 7Four cases of cycD2 + MCL with a CCND2 translocation were collected from the University Hospital Schleswig-Holstein Campus Kiel, Germany, CHU Sart Tilman, Liege, Belgium, Cleveland Clinic, USA, and Technical University of Munich, Germany.Two of these cases have been the subject of previous publications. 4,6As controls, 9 cases of normal lymph nodes were used. ",
"section_name": "Tissue samples",
"section_num": null
},
{
"section_content": "All cases were previously studied by paraffin section immunohistochemistry (IHC) to assess lymphoid immunophenotype.The expression of cyclin D1 (SP4 clone, LabVision Corporation) and cyclin D2 (rabbit polyclonal, Cell Signaling Technology) was investigated in paraffin-embedded sections.IHC was performed on an automated immunostainer (Ventana Medical Systems, Inc., Tuczon, AZ, USA) according to the com-pany´s protocol. 8 ",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "Real-time quantitative RT-PCR analysis was performed using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA).For the quantification of cycD2 we used the following sequences: 5'-CGCAAGCATGCTCAGACCTT-3', 5'- CLL case with cyclin D2 nuclear expression in a percentage of tumor cells.Note how some paraimmunoblasts reveal stronger cyclin D2 expression (F).Images were recorded using a Hitachi camera HW/C20 installed in a Zeiss Axioplan microscope with Intellicam software.FISH images were acquired with a 63x/1.40oil-immersion objective in a Zeiss Axioskop2 fluorescence microscope (Zeiss, Göttingen, Germany) equipped with the appropriate filter sets (AHF, Tübingen, Germany).Image processing was carried out with Zeiss computer software (AIM 3.2).Maximum projection mode was used to produce extended focus image stacks. TGCGATCATCGACGGTGG-3', 5'-FAM-TGCCACC-GACTTTAAGTTTGCCATGT-TAMRA-3'.The sequences of cycD1, cycD3 and TBP (TATA box-binding protein), as housekeeping gene have already been described. 9,10The assay and analysis were performed as previously described. 11 ",
"section_name": "Real-time quantitative RT-PCR",
"section_num": null
},
{
"section_content": "Locus-specific interphase FISH was performed on paraffin-embedded tissue sections according to the manufacturer's instructions (Abbott/Vysis) with minor modifications.The t(11;14) was investigated using commercially available probes (LSI IGH/CCND1; Vysis, Downers Grove, IL) in all MCL and CD5+MZL.Translocations affecting the CCND2 (12p13) and IGK (2p12) loci were investigated using recently described probes. 3 ",
"section_name": "FISH analysis",
"section_num": null
},
{
"section_content": "The 4 cases of cycD1 negative MCL showed clinical, morphological and phenotypic characteristics of MCL.Cases 1 and 2 are 2 male patients aged 71 and 54 years, who presented with stage IV disease.These cases have been previously reported. 4,6Cases 3 and 4 are 2 novel cases that corresponded to an 82-year old female with involvement of the Waldeyer's ring and cervical lymph nodes (Case 3, Figure 1A-C) and to a 59-year old male with stage IV disease.The lymph nodes in the 4 cases showed a nodular and diffuse growth pattern with a CD20 + , CD5 + , CD10 -, CD23 -(4/4), and p27-(3/3) phenotype, but lack of cycD1 expression.Instead, cycD2 was positive.Interphase FISH demonstrated an IGK-CCND2 fusion indicating the presence of a t(2;12)(p12;p13) translocation in Cases 1 and 3. A cytogenetically cryptic translocation t(12;14)(p13;q32) involving the IGH locus in chromosome 14q32 and leading to IGH-CCND2 juxtaposition was present in Case 2. 6 In Case 4, interphase FISH demonstrated a clear CCND2 break with normal IGK and IGH, indicating the probability of a novel translocation partner, in addition to the already described translocations with IGK and IGH.Unfortunately, hybridization with an IGL probe failed repeatedly.Immunohistochemical analysis was performed in 35 cases of small B-cell lymphomas for cycD1 and cycD2 proteins.Due to the difficulties in the differential diagnosis of CD5 + small Bcell lymphomas, MZL expressing CD5 were preferentially included in the study.CycD1, as expected, was positive only in the 10 MCL cases, all of which had an IGH-CCND1 juxtaposition indicating t (11;14).In contrast, cycD2 was positive in all normal lymph nodes and lymphomas analyzed.This finding is not completely unexpected since cycD2 is the main cyclin expressed in normal B cells.The percentage of positive cells and intensity of positivity varied from case to case; however, CLL cases showed the strongest reactivity among the lymphomas analyzed (Figure 1D-F).This result clearly indicates that immunohistochemical detection of cycD2 is not helpful in the differential diagnosis of cycD1 negative MCL.On the contrary, reliance on cycD2 IHC may well lead to the overdiagnosis of cycD1 negative MCL in phenotypically and morphologically difficult cases, such as CLL and CD5 + Figure 2. Cyclin D2 mRNA expression in mature B-cell NHL.Quantitatitve RT-PCR analysis of cyclin D2 was performed relative to the TBP housekeeping gene and results are depicted as ratio of cyclin D2/TBP transcript numbers.The horizontal line indicates the cut-off value for altered cyclin D2 expression (cycD2/TBP ratio=15.4),which corresponds to the mean value of cyclin D2 in normal lymph nodes (cycD2/TBP ratio=6.8)plus three standard deviations (SD:2.8).The difference in cyclin D2 expression between cyclin D2 + MCL and the remaining cases is statistically significant (p=0.04).MZL.FISH analysis for the IGH-CCND1 fusion indicating t(11;14) and for chromosomal translocations affecting the CCND2 locus in 12p13 was negative in all the CD5 + MZL analyzed.Since the identification of cycD1 negative MCL was based primarily on gene expression profile, 2 it seemed logical to consider that quantitative analysis of cycD mRNA levels could be the appropriate method to diagnose cases of cycD1 negative MCL.Therefore, we investigated the levels of the three D-type cyclins in normal lymph nodes and the selected B-NHL cases.The findings are summarized in Table 1.Normal lymph nodes showed, in every case, preferential expression of cycD2 (cycD2/TBP ratio=6.8)with lower expression levels of cycD1 and cycD3.Interestingly, MCL cases with t(11;14) translocation have very low expression of cycD2mRNA.Accordingly, we recently reported that in MCL the low levels of cycD2 are the consequence of downregulation through the abnormally high levels of cyclin D1. 12 In general, CycD2 mRNA levels were slightly increased in MZL and FL, moderately increased in CLL and strikingly increased in cycD2 + MCL (Figure 2).Although some CLL cases have up to 6 times the amount of cycD2 mRNA found in lymph nodes (mean cycD2/TBP 21 vs. 7, p<0.001), the levels of expression were far below the cycD2 mRNA levels found in cycD2 + MCL with a translocation involving the CCND2 locus (cycD2/TBP 21 vs. 202, p=0.004).These results indicate that quantitative RT-PCR and/or FISH are ideal methods to confirm the diagnosis of cycD2 + MCL.Accordingly, our previous studies concerning cycD1 mRNA expression in MCL 10 and multiple myeloma, 13,14 showed that very high levels of cycD1 mRNA were always associated with translocation involving the CCND1 locus.Importantly, the cases reported of cycD2 + MCL without translocation need to be analyzed carefully concerning the amount of cycD2 mRNA expression, and other possible mechanisms of cycD2 deregulation.It is of note that cycD2 + MCL are extremely rare, and in order to avoid overdiagnosis it is mandatory to perform either quantitative analysis of cycD2 mRNA and/or FISH. ",
"section_name": "Results and Discussion",
"section_num": null
}
] |
[
{
"section_content": "LQ-M was the principal investigator and takes primary responsibility for the paper; LQ-M and FF designed research, coordinated the research, analyzed and interpreted data and drafted the manuscript; JS-H, IK, MK, and SG performed the laboratory work for this study; LdL, EH, RS, and WK, contributed with case material and discussed data. The authors have no conflict of interests to declare. ",
"section_name": "Authorship and Disclosures",
"section_num": null
}
] |
10.1038/s41408-020-0292-7
|
DuoHexaBody-CD37®, a novel biparatopic CD37 antibody with enhanced Fc-mediated hexamerization as a potential therapy for B-cell malignancies
|
<jats:title>Abstract</jats:title><jats:p>Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.</jats:p>
|
[
{
"section_content": "B-cell malignancies comprise a heterogeneous group of lymphoproliferative disorders including non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL).In addition to chemotherapy and small molecule inhibitors, immunotherapy with anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, and obinutuzumab, has significantly improved the outlook for patients with B-NHL and CLL [1][2][3] .However, many patients eventually relapse and become resistant to treatment, creating an unmet need for alternative therapeutic strategies.In recent years, the tetraspanin plasma membrane protein CD37 has gained renewed interest as a promising therapeutic target for B-cell malignancies [4][5][6][7] .CD37 is selectively expressed on mature B cells and has limited or no expression on other hematopoietic cells such as T cells and NK cells, granulocytes, monocytes and dendritic cells [8][9][10] . CD37 is involved in the spatial organization of the B-cell plasma membrane by forming tetraspanin-enriched micro domains (TEMs) through lateral associations with interaction partners, such as other tetraspanins or integrins 11,12 .CD37 is signaling-competent as it contains intracellular functional ITIM-like and ITAM-like motifs that play a role in pro-survival and pro-apoptotic signaling via the PI3K/AKT pathway.In addition, it controls IL-6 receptor signaling through interaction with SOCS3 12,13 . In cancer, CD37 is highly expressed on malignant B cells in a variety of B-cell lymphomas and leukemias, including NHL and CLL 14,15 .To date, multiple CD37targeting agents have shown preclinical or clinical efficacy [5][6][7] , including antibody drug conjugates 16,17 , a small modular immuno-pharmaceutical protein (SMIP) 18 , an antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) capacity 19 , a radiolabeled antibody 20 and chimeric antigen receptor (CAR) T cells 21 .The effector mechanisms of these agents include direct cytotoxicity mediated through conjugated cytotoxic or radioactive payloads, classical FcγR-mediated effector functions such as ADCC, and T-cell mediated cytotoxicity.Interestingly, CD37 antibody-based therapeutics currently in (pre-)clinical development are poor inducers of complement-dependent cytotoxicity (CDC) [5][6][7] , another powerful Fc-mediated effector mechanism for killing hematological cancer cells 22,23 . We have previously reported that activation of the classical complement pathway by IgG antibodies depends on IgG hexamer formation upon binding to membrane bound antigens.IgG hexamers, which form through intermolecular Fc-Fc interactions, provide an optimal docking site for hexavalent C1q [24][25][26] .Activation of C1 triggers the complement cascade involving a series of proteolytic events leading to formation of membrane attack complexes that eventually kill target cells via disruption of their cell membrane.Introduction of a single point mutation, such as E430G, in the IgG Fc domain increases IgG hexamer formation and enhances CDC activity 27,28 .We combined this approach with the bispecific antibody technology DuoBody ® to generate an obligate bispecific antibody for which potency was further increased compared to combinations of the parent molecules.Obligate bispecific antibodies represent a novel and most promising concept in current therapeutic antibody drug development 29,30 . We hereby report the generation of a panel of CD37targeting mAbs with an E430G hexamerizationenhancing mutation and characterized the preclinical mechanism of action and anti-tumor activity of the single mAbs, mAb combinations and CD37 biparatopic (bispecific) antibodies.It was demonstrated that CDC efficacy by single CD37-targeting mAbs was enhanced by combining two non-cross-blocking mAbs, which was most evident in the context of a biparatopic antibody variant, DuoHexaBody-CD37.DuoHexaBody-CD37 also induced potent FcγR-mediated effector functions, including ADCC and antibody-dependent cellular phagocytosis (ADCP).In addition, DuoHexaBody-CD37 showed significant anti-tumor efficacy in vivo in human cell line-and patient-derived xenograft models, indicating that DuoHexaBody-CD37 may serve as a promising novel therapeutic antibody for treatment of human B-cell malignancies. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Anti-CD37 antibodies were generated through immunization (MAB Discovery GmbH, Germany) of rabbits with a mixture of HEK293F cells expressing human (sequence no.NP_001765) or cynomolgus monkey (Macaca fascicularis, sequence no.XP_005589942) CD37 or a mixture of Fc-fusion proteins containing the large extracellular loop of human or cynomolgus monkey CD37.CD37 antibodies were produced recombinantly as chimeric human IgG1s containing the hexamerizationenhancing mutation E430G (HexaBody ® molecules 27 ) and F405L or K409R mutations for bispecific antibody generation by controlled Fab-arm exchange (cFAE; Duo-Body technology 30,31 ) as appropriate.Humanized antibody sequences were generated using CDR-grafting in optimized human germ-line variable region sequences at Abzena (Cambridge, UK).The anti-HIV-1 gp120 mAb IgG1-b12 was used as an negative control antibody (IgG1ctrl) 32 .Rituximab (MabThera ® ), ofatumumab (Arzerra ® ) and obinutuzumab (Gazyva ® ) were commercially obtained. ",
"section_name": "Antibodies",
"section_num": null
},
{
"section_content": "Details on cell lines used in this study are summarized in online Supplementary Table S1.All primary patient cells were obtained after written and informed consent and stored using protocols approved by institutional review boards in accordance with the declaration of Helsinki (see Online Supplementary Information).Blood samples and buffy coats from healthy human donors were obtained from the University Medical Center Utrecht (Utrecht, The Netherlands) and Sanquin (Amsterdam, The Netherlands), respectively.Pooled normal human serum (NHS; AB positive) was obtained from Sanquin.Details on antibodies/reagents used to define cell subsets within samples used for flow cytometry are provided in Online Supplementary Tables S2-S5. ",
"section_name": "Cell lines, patients, donors, and reagents",
"section_num": null
},
{
"section_content": "Antibody binding was assessed using target cells incubated with antibody for 30 min at 4 °C.After washing, cells were incubated with R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (Jackson Immu-noResearch Laboratories, West Grove, PA, USA) for 30 min at 4 °C.Cells were washed and binding was analyzed by determining the geometric mean fluorescensce intensity (gMFI) of the PE signal using flow cytometry. For binding competition assays, target cells were incubated with primary unlabeled antibodies (final concentration 20 µg/ml) for 15 min at room temperature.Next, Alexa Fluor 488 (A488)-labelled antibodies (by reaction with N-hydroxysuccinimidyl-esters following manufacturer's instructions [Molecular Probes, Eugene, OR, USA]) were added to cells at final concentration of 2 µg/ml, followed by incubation for 15 min at room temperature.Cells were washed and gMFI of the A488 signal was determined by flow cytometry. ",
"section_name": "Antibody binding assays",
"section_num": null
},
{
"section_content": "A CD37 single residue alanine library was generated (GeneArt, Regensburg, Germany) in which all amino acid residues in the extracellular domains of human CD37 (UniProt P11049) were individually mutated to alanine, except for cysteines.The library was used to map amino acids in the extracellular loops of human CD37 involved in binding of mAbs Hx-CD37-010 and Hx-CD37-016 (details summarized in Online Supplementary Information). ",
"section_name": "Alanine scanning",
"section_num": null
},
{
"section_content": "CDC assays were performed as described using tumor cells incubated with antibody for 45 minutes at 37 °C in the presence of NHS (20% final concentration) as a complement source 33 . ",
"section_name": "CDC assays",
"section_num": null
},
{
"section_content": "Expression levels of cellular markers were determined as described in ref. 33 using an indirect immunofluorescence assay (QIFIKIT ® , Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. ",
"section_name": "Expression analysis",
"section_num": null
},
{
"section_content": "Activation of FcγRIIa-(H-131) and FcγRIIIa-mediated (V-158) intracellular signaling was quantified using Luminescent Reporter Bioassays (Promega, Madison, WI, USA), according to the manufacturer's recommendations.Chromium-51 (Cr 51 ) release ADCC assays were performed as described 34 and summarized in Online Supplementary Information.ADCP assays were performed using tumor cells labeled with calcein AM (Life Technologies, Carlsbad, CA, USA) or pHRodo TM Red AM Intracellular pH Indicator (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions and opsonized with antibodies for 15 min at 37 °C.Human monocyte-derived macrophages (h-MDM, isolation and culturing detailed in Online Supplementary Information) were added at effector to target (E:T) ratios of 2:1 or 1:1 and incubated for 4 h at 37 °C/5%CO 2 .During incubation, images were captured using an Incu-Cyte S3 Live Cell Analysis System with a ×10 objective lense and acquired/processed using IncuCyte S3 software.Alternatively, tumor cells and h-MDM were stained for surface markers after incubation using fluorochromeconjugated antibodies for 30 min at 4 °C, fixed using 4% paraformaldehyde (ChemCruz, Dallas, TX, USA) and analyzed by flow cytometry.CD11b + /calcein AM + / CD19 -cells were defined as h-MDM that phagocytosed Daudi cells.CD11b -/calcein AM + cells were defined as non-phagocytosed Daudi cells remaining after co-culture, used to determine target cell depletion. ",
"section_name": "ADCC and ADCP assays",
"section_num": null
},
{
"section_content": "Binding and cytotoxicity assays were performed with heparin-and hirudin-treated blood samples from healthy human donors, respectively.For cytotoxicity, blood samples were incubated with antibody for 4 h at 37 °C.Next, red blood cells were lysed and samples were stained for 30 min at 4 °C with fluorochrome-labeled lineage-specific antibodies and TO-PRO-3 to characterize cell subsets and dead or dying cells respectively.For binding, red blood cells were first lysed and subsequently incubated with designated antibody mixtures.Binding was assessed by flow cytometry and expressed as the gMFI of AF488 fluorescence intensity for viable cell subsets.Depletion was determined as: ",
"section_name": "Whole-blood assays",
"section_num": null
},
{
"section_content": "Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) studies were conducted following protocols approved by institutional ethical committees, as provided in Online Supplementary Information and Supplementary Table S6.In vivo pharmacokinetic analysis was performed as described in ref. 27 . ",
"section_name": "Animal studies",
"section_num": null
},
{
"section_content": "Flow cytometry data were analyzed using FlowJo V10 software.Graphs were plotted and analyzed using GraphPad Prism 8.0.Dose-response curves were generated using best-fit values of non-linear dose-response fits using log-transformed concentrations.All data shown are representative of at least two independent replicate experiments.Statistical differences in median animal tumor volumes were compared between treatment groups on the last day all groups were complete.In case of equal variance between groups (Bartlett's test) the parametric One Way ANOVA Uncorrected Fisher's LSD test (Daudi-Luc) was used.In case of unequal variance between groups (Bartlett's test) the non-parametric Mann-Whitney test (JVM-3, DOHH-2, NHL PDX) was used. ",
"section_name": "Data processing",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Rabbits were immunized with CD37 antigen to generate a diverse panel of mAbs recognizing human CD37.After humanization using CDR-grafting, antibodies were expressed in a human IgG1 backbone with and without the E430G hexamerization-enhancing mutation (Hx) and binding to CD37 on human tumor cells was assessed using Daudi cells.Four humanized CD37 mAbs were selected for further studies based on specific and efficient target binding (EC 50 < 0.1 µg/ml), CDR sequence diversity and cross-reactivity with human and cynomolgus monkey CD37.Hx-CD37-004, Hx-CD37-005, Hx-CD37-010 and Hx-CD37-016, and corresponding wild-type (WT) IgG1 variants showed dose-dependent binding to Daudi cells (Fig. 1a) with EC 50 values ranging from 0.42 to 0.92 µg/ml.Comparable binding of WT anti-CD37 mAbs indicated that binding was not affected by the E430G mutation.We next performed cross-block binding experiments on Raji cells to examine cross-competition between the four humanized CD37 mAbs.Hx-CD37-004 competed with CD37-016 for binding, and Hx-CD37-005 competed with Hx-CD37-010 for binding to CD37 (Fig. 1b).The mutually competing antibodies Hx-CD37-004 and Hx-CD37-016 were able to simultaneously bind to CD37 with one of the other mutually competing antibodies Hx-CD37-005 or Hx-CD37-010, thereby indicating that the four antibodies represent two different cross-blocking groups. We selected one mAb candidate from each crossblocking group, Hx-CD37-010 and Hx-CD37-016, and used alanine scanning analysis to map epitopes within the extracellular loops of human CD37.A library with alanine substitutions at all extracellular residues of human CD37, except for cysteines, was generated.Alanine mutants were expressed individually in HEK293F™ cells and binding of Hx-CD37-010 and Hx-CD37-016 was determined by flow cytometry.Loss of Hx-CD37-010 binding to human CD37 was observed with alanine substitutions at position Y182, D189, T191, I192, D194, K195, V196, I197, and P199, while for Hx-CD37-016, loss of antibody binding was observed with alanine substitutions at position E124, F162, Q163, V164, L165 and H175 (Fig. 1c).These results showed that residues identified to be crucial for binding of Hx-CD37-010 are distinct from residues crucial for binding of Hx-CD37-016.Together, the binding analyses demonstrate that Hx-CD37-010 and Hx-CD37-016 bind different residues within the second extracellular domain (EC2) of human CD37 (Fig. 1d). ",
"section_name": "Generation of CD37 mAbs and analysis of their binding characteristics",
"section_num": null
},
{
"section_content": "We previously reported that CDC by CD37 mAbs is potentiated by introducing the hexamerization-enhancing mutation E430G in the IgG Fc domain 33 and therefore investigated the potency of the novel CD37 mAbs to induce CDC in vitro.Whereas WT IgG1-CD37 antibodies were inactive, hexamerization-enhanced variants induced dose-dependent and potent CDC in Daudi cells (EC 50 ranging from 0.15 to 0.75 µg/ml) (Fig. 2a).In addition, introduction of the E430G mutation unlocked CDC activity of the CD37 mAbs in Raji cells (EC 50 ranging from 0.29 to 0.77 µg/ml), which are expected to be less sensitive to CDC due to higher expression of complement regulatory protein CD59 (Fig. 2b) 35 .An alternative way to enhance CDC is by dual epitope targeting using noncross-blocking antibody combinations, as has been previously reported for a number of cell surface antigens, for example EGFR 36 .Also here, the WT IgG1-CD37 mAbs did not induce CDC as single agents, whereas combinations of non-cross-blocking WT IgG1-CD37-010 and IgG1-CD37-016 potentiated CDC to 65% maximum lysis in Raji cells (Fig. 2c).Interestingly, while hexamerizationenhanced variants of these non-cross-blocking CD37 mAbs individually induced 50% maximum lysis in Raji cells, CDC-mediated lysis was strongly enhanced in the combination (87% maximum lysis) (Fig. 2d), thereby clearly outperforming combinations of the WT noncross-blocking CD37 mAbs.These results were confirmed with combinations of the other CD37-specific non-crossblocking mAbs (data not shown).In contrast, combinations of Hx-CD37 mAbs that compete for CD37 binding did not show enhanced CDC activity compared to single antibodies, including the combination of Hx-CD37-005 and Hx-CD37-010 (Fig. 2e) and the mixture of Hx-CD37-004 and Hx-CD37-016 (data not shown).These results indicate that CDC activity of the CD37 mAbs is potentiated by enhanced hexamerization through the E430G mutation and by dual epitope targeting. ",
"section_name": "CDC activity of CD37 mAbs is potentiated by enhanced hexamerization and dual epitope targeting",
"section_num": null
},
{
"section_content": "We next explored the possibility of combining enhanced hexamerization with dual epitope targeting in a bispecific antibody.A biparatopic CD37 mAb variant, DuoHexaBody-CD37, was generated through controlled Fab-arm exchange between Hx-CD37-010 and Hx-CD37-016 30,31 .The CDC activity of DuoHexaBody-CD37 was compared to that of single mAbs and mAb combinations in samples from untreated CLL patients and a CLL patient relapsed/refractory to rituximab, ibrutinib and idelalisib.Strikingly, DuoHexaBody-CD37 induced superior CDC activity in all patient-derived CLL samples compared to either the single where the z-score was higher than 1.5 (Hx-CD37-010) or lower than -1.5 (Hx-CD37-016), indicated by the horizontal dotted lines, were considered as \"loss of binding mutants\".The number above the x-axis refers to amino acid positions in full length human CD37.EC1 = small extracellular loop, EC2 = large extracellular loop of CD37, respectively.d Amino acid residues involved in Hx-CD37-010 (blue) and Hx-CD37-016 (purple) binding to CD37 are depicted in a graphical representation based on UniProtKB P11049.The extracellular domain of CD37 contains six cysteines (Cys, C), which are indicated in red; these form intramolecular disulphide bonds and contribute to the conformation of the tetraspanin.The asparagine residues (Asn, N) in the N-glycosylation site consensus sequence Asn-Xxx-Ser (NXS) are indicated in red. mAbs or the combination, which was most apparent in the refractory CLL sample (Fig. 3a-d).Consistent with results in tumor cell lines, the mAb combination showed enhanced CDC compared to the single mAbs.Comparison of DuoHexaBody-CD37 with the approved CD20 antibodies rituximab, ofatumumab and obinutuzumab demonstrated superior CDC in all patient samples tested.None of the approved CD20 antibodies induced CDC in the refractory CLL sample (Fig. 3a) while in untreated CLL samples, only ofatumumab induced CDC at concentrations of 10 (Fig. 3c,d) and 100 μg/ml (Fig. 3b). The capacity of DuoHexaBody-CD37 to induce CDC in malignant B cells was further confirmed in CDC assays in vitro using 16 tumor cell lines with varying CD37 expression levels, derived from different B-cell lymphoma subtypes (Fig. 3e).DuoHexaBody-CD37 induced potent CDC in 8 of the 16 cell lines tested, with generally higher levels of tumor cell lysis observed in cell lines with CD37 expression levels above 100,000 copies/cell (Fig. 3e,f). ",
"section_name": "A biparatopic hexamerization-enhanced CD37 antibody variant, DuoHexaBody-CD37, induces superior CDC activity in vitro and ex vivo",
"section_num": null
},
{
"section_content": "While the primary rationale behind the development of DuoHexaBody-CD37 was focused on maximizing its capacity to induce CDC, other Fc-mediated effector functions such as ADCC and ADCP, known to contribute to tumor cell kill, were also tested.The potential of DuoHexaBody-CD37 to induce ADCC and ADCP was first evaluated in FcγRIIIa (V-158) and FcγRIIa (H-131) reporter assays, respectively.When bound to Daudi target cells, DuoHexaBody-CD37 induced efficient, dosedependent activation of FcγRIIIa and FcγRIIa signaling in transfected Jurkat effector T cells (Fig. 4a,b).FcγRIIIa and FcγRIIa signaling was at least comparable to that induced by the CD20 antibody rituximab.ADCC induction by DuoHexaBody-CD37 was further evaluated in a 51 Cr release assay using Daudi cells as target cells and human PBMCs from 12 healthy human donors as effector cells (E:T ratio 100:1).DuoHexaBody-CD37 induced efficient, dose-dependent ADCC of Daudi cells (EC 50 = 9.9 ± 10.0 ng/ml), which was comparable to the WT IgG1-CD37 mAbs without the E430G and F405L/ K409R mutations (Fig. 4c,d). Next, ADCP was assessed in a flow cytometry-based assay using calcein AM-labeled Daudi target cells and h-MDM as effector cells.DuoHexaBody-CD37 induced efficient phagocytosis of calcein AM-labeled Daudi target cells by h-MDM, as illustrated by a dose-dependent increase in CD11b + /calcein AM + double positive h-MDM (EC 50 = 13.5 ± 13.1 ng/ml), resulting in almost complete depletion of Daudi target cells (EC 50 = 5.8 ± 2.3 ng/ml) (Fig. 4e).DuoHexaBody-CD37-mediated ADCP was confirmed in an image-based assay using (c,d).e 17 B-lymphoma cell lines and the Epstein-Barr virus (EBV)-positive B-lymphoblastic cell line WIL-2S were evaluated for CD37 molecule expression on the cell surface using quantitative flow cytometry.The dotted line indicates 100,000 CD37 surface molecules/cell.f The capacity of 10 µg/ml DuoHexaBody-CD37 to induce CDC in the presence of 20% NHS was tested in 16 cell lines also described in e and expressed as the percentage lysis determined by the fraction of PI-positive cells.The cell lines are grouped per B-lymphoma subtype: mantle cell lymphoma (MCL), Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL) with activated B-cell (ABC) or germinal center B-cell (GCB) subtypes and an EBV-positive Blymphoblastic cell line.Cell lines with >100,000 CD37 molecules per cell are indicated by black bars, and those with <100,000 CD37 molecules per cell by white bars.Either four (e) or two (f) replicates were used per cell line (mean ± SD).Daudi cells labeled with the pH-sensitive pHRodo dye that becomes increasingly fluorescent in the acidic lysosomal environment.Also here, DuoHexaBody-CD37 induced efficient engulfment and lysosomal degradation of Daudi cells by h-MDM (Fig. 4f,g).Together, these data demonstrate that DuoHexaBody-CD37 induces efficient FcγR-mediated immune effector functions to kill CD37positive tumor cells. ",
"section_name": "DuoHexaBody-CD37 induces efficient ADCC and ADCP in vitro",
"section_num": null
},
{
"section_content": "CD37 is reported to be highly expressed on mature B cells, with low expression levels on other leukocyte subsets [8][9][10] .Since non-malignant B-cell depletion may be used as a safety-and pharmacodynamic biomarker when exploring B-cell targeting therapies, the ability of DuoHexaBody-CD37 to bind and deplete B cells versus other leukocyte subsets was evaluated in whole-blood derived from six healthy human donors.The blood was hirudin anticoagulated to preserve complement activity.DuoHexaBody-CD37 showed efficient binding to CD19 + B cells, while low binding was observed to T cells, NK cells, and neutrophils for all six healthy human donors tested (Fig. 5a).DuoHexaBody-CD37 showed potent depletion of the CD19 + B-cell population compared to the negative control, with 98% ± 1.3% depletion at 10 µg/ ml and an average EC 50 of 0.077 ± 0.039 µg/ml (Fig. 5b).For the T cells, NK cells, and neutrophils that showed low DuoHexaBody-CD37 binding, no depletion was observed at saturating mAb concentrations of 10 µg/ml (Fig. 5c). (see figure on previous page) Fig. 4 DuoHexaBody-CD37 induces efficient ADCC and ADCP in vitro.a, b FcγRIIIa (a) and FcγRIIa (b) crosslinking by DuoHexaBody-CD37 was analyzed in a bioluminescent Reporter Bioassay using Daudi target cells and engineered FcγRIIIa-or FcγRIIa-expressing Jurkat effector T cells that express luciferase upon FcγR crosslinking.Luciferase production is presented as relative luminescence units (RLU).Error bars represent the mean ± SD of duplicate measurements.c ADCC by 2 µg/ml DuoHexaBody-CD37 was evaluated in a classical 51 Cr release assay using Daudi target cells and PBMCs from 12 healthy human donors as a source of effector cells (E:T of 100:1).The percentage lysis was calculated relative to a Triton X-100 control (100% lysis) and no antibody control (0% lysis).****P < 0.00001, paired T-test with two-tailed 95% confidence intervals.d Dose-response ADCC (mean percentage lysis ± SD of three replicate samples) induced by DuoHexaBody-CD37, WT IgG1-CD37-010 and WT IgG1-CD37-016 shown for one representative responsive donor as described in c.Error bars represent the mean ± SD of triplicate measurements.e ADCP induced by DuoHexaBody-CD37 using Daudi target cells and monocyte-derived h-MDM from healthy human donors as a source of effector cells.Calcein AM-labeled Daudi cells opsonized with DuoHexaBody-CD37 were incubated with CD11b + h-MDM at an E:T ratio of 2:1 and ADCP was analyzed by flow cytometry after a 4 h co-culture.The amount of h-MDMs that phagocytosed Daudi cells is presented as percent CD11b+/calcein AM+/CD19 -double positive cells.CD19 was used to exclude macrophages with bound instead of phagocytosed tumor cells.The percentage CD11b -/calcein AM+ cells was determined as an indicator of the amount of non-phagocytosed Daudi cells; presented here as a depleted cell fraction relative to a no antibody control sample.Data from one representative donor out of three is shown.f ADCP of pHRodo-labeled Daudi target cells by h-MDM induced by DuoHexaBody-CD37 over time at an E:T ratio of 1:1, shown for one out of three representative donors.Red fluoresence indicates phagocytosed Daudi target cells by h-MDM.ADCP was quantified by the total sum of the red fluorescent intensity in the image (RCUxµm 2 /image) and presented as the mean ± SD of duplicate measurements.g Phase contrast-and red fluorescent images of DuoHexaBody-CD37-opsonized (1 µg/ml) pHRodo-labeled Daudi target cells co-cultured with h-MDM effector cells at 0 and 4 h incubation as described in f. ",
"section_name": "DuoHexaBody-CD37 depletes B cells, but not other leukocyte populations in human whole blood",
"section_num": null
},
{
"section_content": "Fig. 5 DuoHexaBody-CD37 depletes B cells, but not other leukocyte populations in human whole blood.Binding to-and depletion of different leukocyte cell subsets in human whole blood by DuoHexaBody-CD37 was assessed by flow cytometry using six healthy donors.a Binding of A488-labeled DuoHexaBody-CD37 (30 µg/ml) is expressed as the gMFI ± SD of the A488 signal.b, c Cell depletion induced by a concentration series (b, one representative donor) or 10 µg/ml (c, six donors) DuoHexaBody-CD37 after a four hour incubation period presented as mean percentage depletion ± SD relative to a no antibody control sample.Leukocyte subsets were characterized as: B cells (CD19 + ), T cells (CD3 + ), NK cells (CD56+) and neutrophils (CD66 + /CD16 + ). ",
"section_name": "C",
"section_num": null
},
{
"section_content": "The anti-tumor activity of DuoHexaBody-CD37 in vivo was evaluated in CDX models obtained by intravenous or subcutaneous injection of B-cell lymphoma-derived Daudi-Luc and DOHH-2 cells and CLL-derived JVM-3 cells, that express moderate to high levels of CD37 (Fig. 3e).First, we confirmed that DuoHexaBody-CD37 (which lacks cross-reactivity to murine CD37) has a normal clearance rate comparable to WT IgG1-ctrl in tumor-free mice, i.e. in the absence of target binding (data not shown).Next, SCID mice were injected with Daudiluc, DOHH-2 or JVM-3 cells and treated with 0.1, 1, or 10 mg/kg DuoHexaBody-CD37 after tumors had established.Three weekly doses of DuoHexaBody-CD37 resulted in significantly reduced tumor growth in the Daudi-Luc model at all tested dose levels as compared to the IgG1-ctrl, and at 1 and 10 mg/kg in the JVM-3 and DOHH-2 models (Fig. 6a). The anti-tumor activity of DuoHexaBody-CD37 was also assessed in PDX models, which offer more reliable results for clinical outcomes, because of their more conserved characteristics of the original tumor including heterogeneity, genetic and biological complexity and molecular diversity 37 .In a screening approach, we evaluated the antitumor efficacy induced by DuoHexaBody-CD37 using nine NHL PDX models in an experimental set up using a one mouse per group design.SCID mice were treated with two weekly doses of 5 mg/kg DuoHexaBody-CD37 or PBS after tumors had established, and the ratio between the relative tumor growth in the DuoHexaBody-CD37-treated mouse (ΔT) and the PBS control mouse (ΔC) was used to classify responders (ΔT/ΔC < 10%) versus non-responders (ΔT/ΔC > 70%) and intermediates (10% ≤ ΔT/ΔC ≤ 70%).Three models were classified as responders, three as intermediates and three as non-responders (Fig. 6b).From the three models that showed <10% relative tumor growth (i.e.tumor stasis or tumor regression), two models achieved complete tumor regression and did not grow out during the complete observation period (~60 days).Although CD37 mRNA expression was confirmed for all PDX models, variability between models was limited and could not be associated with response (data not shown).Follow-up cohort studies in two responding PDX models (Ly12638 and Ly13005) b PDX screening in which tumor fragments from 9 NHL patients were subcutaneously implanted in SCID mice, using a one mouse per group design.Mice were dosed with either two weekly doses (QWx2) of 5 mg/kg DuoHexaBody-CD37 or PBS as a negative control.The relative tumor growth is presented as the difference in tumor volume between day of first treatment and day of analysis (7-25 days after initiation of treatment) in the DuoHexaBody-CD37-treated mouse (treatment, ΔT) and the control mouse (control, ΔC).The ratio between the relative tumor growth in the treatment and control mouse, specifically ΔT/ΔC, was used to categorize models as responders (ΔT/ΔC < 10%), intermediates (10% ≤ ΔT/ΔC ≤ 70%), or non-responders (ΔT/ΔC > 70%).c PDX dose response using two responding NHL models as described in b with indicated doses of DuoHexaBody-CD37 or IgG1-ctrl (n = 8 mice/group).Average tumor size is shown ±SEM.*P < 0.01, **P < 0.001, ***P < 0.0001.confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg (Fig. 6c).Collectively, these data indicate that DuoHexaBody-CD37 can mediate significant anti-tumor activity in both CDX and PDX in vivo models derived from different B-cell malignancy subtypes. ",
"section_name": "DuoHexaBody-CD37 shows anti-tumor activity in vivo in xenograft models",
"section_num": null
},
{
"section_content": "The treatment landscape for B-cell malignancies has rapidly evolved since initial approval of the CD20-targeting mAbs.With resistance to CD20-targeted therapies arising, a number of alternative B-cell surface antigens have been evaluated for targeted mAb therapy including CD19, CD22, CD30, CD79b, and CD37 38 .Similar to CD20, CD37 is highly expressed on tumor B cells across all major NHL and CLL subtypes.Although CD37 has long been recognized as a potential target for treatment of B-cell malignancies, it has recently received renewed interest with various agents currently in (pre)-clinical development.While multiple effector mechanisms have been reported for these agents, they are generally poor in inducing CDC [5][6][7] . Here, we introduce DuoHexaBody-CD37, a novel CD37targeted agent generated using an innovative approach that combines DuoBody and HexaBody antibody platform technologies.DuoHexaBody-CD37 is a biparatopic bispecific IgG1 antibody with a hexamerization-enhancing mutation that induces strong anti-tumor activity in preclinical models in vitro and in vivo through potent CDC, ADCC and ADCP (Fig. 7).DuoHexaBody-CD37 exhibits highly potent CDC activity in vitro and ex vivo, which was superior to the parental WT or hexamerization-enhanced CD37 mAbs and to the combinations thereof.DuoHexaBody-CD37 not only outperformed all other CD37 antibody variants evaluated in ex vivo CDC assays using primary CLL patient samples, but also outperformed approved CD20 mAbs rituximab, ofatumumab and obinutuzumab.We demonstrated that the superior CDC efficacy of DuoHexaBody-CD37 is caused by enhanced antibody hexamerization upon target binding and by dual epitope targeting inherent to binding two nonoverlapping epitopes on CD37.Locally increasing the density of Fc-domains through dual epitope targeting can potentiate CDC 36 and may also favorably affect Fc-mediated antibody hexamerization, specifically in the context of the hexamerization-enhancing mutation.Consequently, the threshold for complement activation by DuoHexaBody-CD37 might be lower, which could result in enhanced anti-tumor efficacy in clinical settings.This could be of specific interest for patients who no longer respond to CD20 mAb treatment regimens, where antigen expression, cell surface antigen distribution or expression of complement inhibitors might be limiting factors 22 . While DuoHexaBody-CD37 was shown to bind two non-overlapping epitopes on CD37, the fine mechanism of target binding, either bivalent or monovalent, and subsequent oligomerization into hexameric complexes remains to be elucidated.IgG oligomerization on target surfaces was recently reported to occur via Fc-Fc interaction-mediated recruitment of IgG molecules directly from solution, but also through lateral diffusion on the cell in case of preferentially monovalent binding IgG molecules 25 .CD37 is thought to be highly mobile due to its role in protein trafficking and organization in the plasma membrane and the formation of TEMs 39 .One could speculate that for DuoHexaBody-CD37 specifically, dual epitope targeting may optimize Fc-tail configuration in the process of IgG hexamerization, which may be augmented by lateral diffusion. Besides inducing highly potent CDC, DuoHexaBody-CD37 was shown to efficiently engage with FcγRs in mediating ADCC and ADCP, indicating the mechanism of action of DuoHexaBody-CD37 is multifaceted.DuoHexaBody-CD37 compared favorably to rituximab in both FcγRIIIa and FcγRIIa crosslinking, in cell line models that showed comparable levels of CD20 and CD37 expression.Importantly, DuoHexaBody-CD37 efficiently depleted peripheral blood B cells, but not other leukocyte populations from healthy human whole blood.Furthermore, in xenograft models in vivo, DuoHexaBody-CD37 induced significant inhibition of tumor growth in a CLL CDX model, two NHL CDX models and six out of nine NHL PDX models.Notably, mice are not considered a suitable species to assess CDC-dependent tumor cell kill in vivo, suggesting FcγR-mediated effector functions may largely determine the observed anti-tumor activity 40 .Further studies are required to understand the contribution of individual effector mechanisms in vivo. The current landscape of drug development in B-cell malignancies includes CD20-targeting antibodies such as ofatumumab and the glycoengineered obinutuzumab, and small molecule inhibitors targeting Bruton's tyrosine kinase, Bcl-2 and PI3K-δ, such as ibrutinib, venetoclax, and idelalisib, respectively 3 .These growing numbers of therapeutic agents might also provide opportunities for combination therapy with CD37-targeting antibodies.Combinations of DuoHexaBody-CD37 with anti-CD20 antibodies could be of particular interest, as we have previously shown enhanced CDC in CLL and B-NHL primary patient cells with combinations of CD20 and CD37 mAbs containing hexamerization-enhancing mutations 33 .Furthermore, CD37 has been reported to contain ITIM-like and ITAM-like regulatory motifs that regulate pro-survival and pro-apoptotic signaling processes via the PI3K/AKT pathway 13 .We speculate that combinations of DuoHexaBody-CD37 with pro-apoptotic PI3K-δ-inhibitors such as idelalisib may work synergistically, however the role of DuoHexaBody-CD37 in apoptotic signaling via CD37 remains to be elucidated. In summary, we present here a novel therapeutic antibody that, for the first time, combines the proprietary DuoBody and HexaBody antibody platforms to create a biparatopic CD37 IgG1 antibody with enhanced Fc-mediated hexamerization upon target binding.The potent anti-tumor activity exhibited by DuoHexaBody-CD37 in preclinical Bcell malignancy models highlights its therapeutic potential.Preparations to evaluate the clinical safety and preliminary efficacy of DuoHexaBody-CD37 in a first-in-human clinical trial are currently ongoing. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Supplementary Information accompanies this paper at (https://doi.org/10.1038/s41408-020-0292-7). ",
"section_name": "",
"section_num": ""
}
] |
10.3390/diagnostics13203201
|
Somatic Mutation of the Non-Muscle-Invasive Bladder Cancer Associated with Early Recurrence
|
<jats:p>Next-generation sequencing (NGS) is widely used in muscle-invasive bladder cancer but has limited use in non-muscle-invasive bladder cancer (NMIBC) due to significant heterogeneity and high cancer-specific survival. Therefore, we evaluated the genomic information of NMIBC and identified molecular alterations associated with tumour recurrence. A total of 43 patients with NMIBC who underwent transurethral resection of the bladder were enrolled. We performed NGS using an Oncomine panel of tumour specimens and blood samples corresponding to each specimen. The somatic mutation results were analysed by pairwise comparison and logistic regression according to the recurrence of bladder tumours within 1 year. The median incidence of genetic variations in 43 tumour samples was 56 variations per sample, and a high tumour mutation burden (TMB) was associated with tumour recurrence (median variation 33 vs. 64, p = 0.023). The most mutated gene was adipose tissue macrophages (ATM) (79%), followed by neurofibromatosis-1 (NF1) (79%), and neurogenic locus notch homolog protein 1 (NOTCH1) (79%). In multivariable analysis, mutation of epidermal growth factor receptor (EGFR) (odds ratio [OR], 9.95; 95% confidence interval [CI], 1.40–70.96; p = 0.022) and telomerase reverse transcriptase (TERT) (OR, 7.92; 95% CI, 1.22–51.51; p = 0.030) were significant factors associated with the recurrence of bladder tumour within 1 year. Our results revealed that high TMB, EGFR mutation, and TERT mutation had a significant association with tumour recurrence in NMIBC. In addition, somatic mutations in EGFR and TERT could be useful prognostic biomarkers in NMIBC.</jats:p>
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[
{
"section_content": "Bladder cancer is one of the most common cancers worldwide, and its prevalence has increased by 30% in the last decade [1].The majority of bladder cancers are transitional cell (urothelial) carcinomas, and approximately 75% of patients with bladder cancer present with non-muscle-invasive bladder cancer (NMIBC) [2].Compared to muscle-invasive bladder cancer (MIBC), NMIBC has significant heterogeneity of different stages and grades and a high cancer-specific survival of 98% for low-grade, non-muscle-invasive cancers [2][3][4].However, the prognosis of NMIBC is not homogenous despite standard treatment with transurethral resection of bladder tumour (TURBT) followed by adjuvant intravesical therapy.In addition, patients with NMIBC experience a frequent disease recurrence (50-70%) and progression to MIBC (up to 20%) after initial treatment; most patients must undergo long-term cystoscopic surveillance and multiple invasive treatment procedures with TURBT, which make NMIBC one of the most challenging and expensive cancers to diagnose and treat [2,5].In order to reduce patient and socioeconomic burden and improve post-treatment monitoring in NMIBC, international urological guidelines have stratified disease recurrence and progression according to several clinical and histopathological parameters, but the precise prediction of disease prognosis remains difficult.[2,6] Therefore, more advanced strategies to reveal the risk and predict the prognosis of patients with NMIBC are still needed, leading to growing interest in the genomic evaluation of bladder cancer and identifying accurate non-invasive biomarkers. Next-generation sequencing (NGS) is a high-throughput method allowing simultaneous sequencing of millions of DNA fragments without previous sequence knowledge.This advanced technology has brought a true revolution by enabling sequencing even with a small amount of DNA fragments using clonal amplification, massive parallel, and cyclic sequencing [7].In addition, NGS enables the comprehensive assessment of the cancer landscape by sequencing the tumour genome, which helps with disease risk assessment, informative biomarker identification, biomarker-guided genomic-targeted therapy, and the prediction of treatment response and prognosis.Therefore, the use of NGS is rapidly gaining popularity in the clinical practice of other malignancies, such as non-small cell lung, breast, and colorectal cancers [7]. Among urologic malignancies, bladder cancer is a carcinoma with the highest frequency of somatic mutations after skin melanoma and lung cancer [8].Hence, to date, several studies have evaluated the genomic information of bladder cancer for enabling NGS to be feasible in clinical practice as in other malignancies; however, most have studied MIBC with a less favourable prognosis, and only a few studies have focused on NMIBC due to its significant heterogeneity and high cancer-specific survival [9][10][11][12].Although largescale studies have identified recurrent DNA alterations in NMIBC, such as early, abundant mutations in the TERT promoter and chromatin-modifying genes, as well as activating alterations to oncogenes such as FGFR3 [13,14], there is currently a lack of information on the genetic risk factors for disease recurrence and progression of NMIBC.Therefore, in the present study, we evaluated the genomic information of NMIBC and identified somatic molecular alterations associated with tumour recurrence. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "All patients who underwent TURBT from January 2017 to December 2020 were selected.Bladder tumours were staged according to the 2017 TNM classification of urinary bladder cancer [15].Histologic grading of bladder tumours was performed according to the 2004/2016 World Health Organisation (WHO) grading classification system [16].After confirmation by pathologic examination, patients diagnosed with NMIBC were enrolled in the study.In addition, patients diagnosed with NMIBC were stratified into 4 groups according to the European Association of Urology prognostic-factor risk groups, and patients at intermediate or greater risk of recurrence received adjuvant intravesical Bacillus Calmette-Guerin (BCG) instillation treatment with informed consent.The BCG instillation schedule followed the Southwest Oncology Group method; the 6-week induction course and maintenance course consisted of each week for 3 weeks given at 3, 6, 12, 18, 24, 30, and 36 months from the initiation of induction therapy [17].Bladder tumour specimens for NGS analysis were obtained at the initial TURBT, and blood samples corresponding to each specimen were collected when patients visited the outpatient department according to their schedule.Patients were excluded if they exhibited any of the following conditions: classification as carcinoma in situ (Tis), bladder tumour specimens not suitable for NGS analysis, histologic diagnosis other than pure transitional cell carcinoma, did not collect blood samples or obtain informed consent, or were lost at follow-up.A total of 43 patients were included in the final analysis. Detailed medical histories, including age, body mass index (BMI), sex, diabetes mellitus, hypertension, smoking history, pathologic stage and grade, prognostic-factor risk, adjuvant BCG instillation, and disease recurrence within 1 year, were obtained from the patients' medical records.Patients were examined by cystoscopy regularly every 3 to 6 months during the first 2 years and biannually up to 5 years.Disease recurrence was defined as a new cystoscopically detected bladder tumour with histologic confirmation after the initial TURBT. ",
"section_name": "Study Population and Data Collection",
"section_num": "2.1."
},
{
"section_content": "NGS analysis to evaluate the genomic information of NMIBC was performed on the obtained bladder cancer specimens (formalin-fixed paraffin-embedded tissues) from the initial TURBT and blood samples corresponding to each bladder specimen collected from patients.Slides of all bladder cancer specimens were reviewed by one histopathologic specialist.Genomic DNA was extracted from 43 tumour samples and their corresponding blood samples. ",
"section_name": "Patients and Sample Handling",
"section_num": "2.2."
},
{
"section_content": "Genomic DNA was extracted using the QIAGEN GeneRead DNA FFPE Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions.DNA concentration was quantified using the Qubit™ ds DNA High-Sensitive Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the Qubit fluorometer. ",
"section_name": "DNA Extraction",
"section_num": "2.3."
},
{
"section_content": "Sequencing was performed using the Oncomine Comprehensive Assay v3 (OCAv3) panel (Thermo Fisher Scientific, Waltham, MA, USA).The OCAv3 panel covers 161 cancerassociated genes, including 87 hotspot genes, 43 focal CNV gains, 48 full CDS for DEL mutations, and 51 fusion drivers, in addition to allowing the detection of single nucleotide variants (SNV), multiple-nucleotide variants (MNV), and small insertions/deletions (indel) [18].A library was prepared using the OCAv3 kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's manual.Multiplex PCR was performed using 20 ng of DNA. The completed library was quantified with a High Sensitivity DNA Kit (Cat.5067-4626) on a 4200 TapeStation system (Agilent, Santa Clara, CA, USA) and diluted to a final concentration of 14 pM.Diluted samples were subjected to template prep with Ion Chef XL equipment.Template-prepped samples were loaded into the Ion 530™ Chip Kit (Cat.A45850) and analysed on the Ion S5 XL sequencing equipment.Alignment was performed using t-map (v5.10.1).As the reference genome, Hg19 was used. ",
"section_name": "Library Preparation and Sequencing",
"section_num": "2.4."
},
{
"section_content": "The sequenced data were processed using a series of steps.We aligned the sequenced files (FASTQ file) to the reference genome (human reference genome g1k v37) using the BBmap (38.96) and sorting and indexing were performed using Samtools (samtools-1.3.1).Next, filtered alignments are further processed to improve the alignment quality, including local realignment around indels and base quality score recalibration using the Genome Analysis Toolkit (gatk-4.2.6.1).Base quality score recalibration is carried out to recalculate base quality scores for all sequenced reads based on known polymorphisms.The base and mapping quality scores are used to filter reads during variant calling, and the fine-tuning that occurs in this step is important to ensure only high-confidence variants are called. Variant calling was performed in GATK-Mutect2.Mutect is a method developed for detecting the most likely somatic point mutations in NGS data using a Bayesian classifier approach.We used FilterMutectCalls of the Mutect2 pipeline for a variant filter that filters based on the probability of a somatic variant and optimises the threshold of the \"F score\" by considering the average of sensitivity and precision.For the detection of somatic single-nucleotide polymorphism (SNP) and insertion and deletion (indel), we used Mutect2 (GATK v4) tumour (bladder tissue) and normal (whole blood) samples (\"tumour with matched normal\" mode) on each sample.By performing this variant calling, germ-line mutations were excluded, and we analysed oncogene mutations among pure somatic mutations. The somatic mutation data analyses were carried out using the R4.3.1 package (\"maftools\").The mutation results were analysed by pairwise comparison according to the recurrence of bladder tumours within 1 year. ",
"section_name": "Data Analysis",
"section_num": "2.5."
},
{
"section_content": "Statistical analysis was performed using the R4.3.1 package (\"maftools\").A descriptive analysis was performed to assess patient demographics.Continuous variables are presented as means and standard deviations, and categorical variables are presented as frequencies (%).Clinical and histopathological factors were compared according to the recurrence of bladder cancer within 1 year using Pearson's χ 2 -test and Fisher's exact test for categorical variables and a t-test for continuous variables.A logistic regression test was performed to identify molecular alterations associated with tumour recurrence.p < 0.05 indicated statistical significance. ",
"section_name": "Statistical Analysis",
"section_num": "2.6."
},
{
"section_content": "The study protocol was reviewed and approved by the institutional review board of the Chonnam National University Hospital (IRB-approved protocol: No. CNUH-2021-205).The study was performed in accordance with the principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Studies.Informed consent was obtained from all patients. ",
"section_name": "Ethics Statement",
"section_num": "2.7."
},
{
"section_content": "The characteristics of the patients who received transurethral resection for NMIBC are summarised in Table 1.The mean age and BMI of the enrolled patients were 70.6 ± 9.6 years and 25.2 ± 3.3 kg/m 2 , respectively.Thirty-seven (86.0%) patients were male, 25 (58.1%)patients had hypertension, 12 (27.9%)patients had diabetes mellitus, and 25 (58.1%)patients had a smoking history.Regarding histopathologic results, the numbers of patients with Ta-low grade, Ta-high grade, or T1-high grade were 31 (72.1%),11 (25.6%), and 1 (2.3%), respectively.According to the European Association of Urology prognostic-factor risk groups, 14 (32.6%)patients were low-risk, 21 (48.8%)patients were intermediate-risk, and 8 (18.6%) patients were high-risk.Twenty-four (55.8%) patients were administered adjuvant intravesical BCG treatment as part of a planned schedule.During the follow-up period, 22 patients (51.2%) had disease recurrence within 1 year regardless of adjuvant treatment and additionally received treatment with TURBT.Of 22 patients with disease recurrence within 1 year, 17 (77.3%)patients had Ta-low-grade recurrence, and there was no disease progression to T2.Regarding comparisons of clinical and histopathological features according to the recurrence of bladder cancer within 1 year, there were no significant differences between the two groups except smoking history (Table 2).The results of analysing somatic mutations in tumour tissues and blood samples corresponding to each bladder specimen are presented in Figure 1.The median incidence of genetic variations in 43 tumour samples was 56 variations per sample, and a frameshift deletion was the most common variant classification.Among the six single nucleotide variation (SNV) classes, C > T was the most common SNV pattern.The most mutated gene was ATM (79%), followed by NF1 (79%) and NOTCH1 (79%).High TMB was associated with tumour recurrence (median variation 33 vs. 64, p = 0.023) (Supplementary Figure S1).However, none of the top 10 mutated genes had a significant difference in a pairwise comparison according to the recurrence of bladder tumours within 1 year, as shown in Figure 2.However, EGFR mutation (3 vs. 10, p = 0.026) and TERT mutation (4 vs. 11, p = 0.033) were significantly different in further pairwise comparison analyses (Figure 3).The most mutated gene was ATM (79%), followed by NF1 (79%) and NOTCH1 (79%).High TMB was associated with tumour recurrence (median variation 33 vs. 64, p = 0.023) (Supplementary Figure S1).However, none of the top 10 mutated genes had a significant difference in a pairwise comparison according to the recurrence of bladder tumours within 1 year, as shown in Figure 2.However, EGFR mutation (3 vs. 10, p = 0.026) and TERT mutation (4 vs. 11, p = 0.033) were significantly different in further pairwise comparison analyses (Figure 3).The predictive factors associated with tumour recurrence are shown in Table 3. Univariate analyses revealed a significant association of TMB, EGFR mutation, and TERT mutation with tumour recurrence within 1 year (p = 0.030, p = 0.033, and p = 0.039, respectively).In addition, multivariable analyses showed somatic mutations in EGFR (odds ratio ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Although NMIBC has a significantly higher cancer-specific survival rate compared MIBC, patients with NMIBC have a highly diverse clinical course according to histopathologic and patient-related factors [2][3][4].In addition, some patients with NMIBC experience frequent tumour recurrence despite receiving standard treatment with TURBT followed by adjuvant intravesical therapy, which leads to a significant patient and socioeconomic burden [5].Therefore, there is a growing interest in more advanced strategies to reveal risk and predict prognosis, and several physicians have focused on the genomic evaluation of bladder cancer.Thus, we evaluated the genomic information of NMIBC and identified molecular alterations associated with tumour recurrence.We found that EGFR mutations or TERT mutations might have prognostic value for the tumour recurrence of NMIBC. Recently, the rapid development of NGS technology has enabled us to identify comprehensive genetic information related to cancer, and leveraging genomic data based on NGS analysis has improved disease risk assessment, genomic-targeted therapy, and the prediction of treatment responses and prognosis [7].Bladder cancer, especially NIMBC, has significant heterogeneity with different stages, grades, and genomic alterations [2][3][4]8].Therefore, many physicians have made an effort to evaluate the relationship between genomic alterations and NMIBC.Indeed, recently, the tumour mutation burden (TMB) was reported to be an emerging biomarker for the prediction of tumour behaviour.Jia Lv et al. analysed the clinical significance of TMB in 443 bladder cancer samples obtained from The Cancer Genome Atlas (TCGA) and found that TMB was related to high-grade bladder cancer [19].In the present study, the median incidence of genetic variations in 43 tumour samples was 56 variations per sample, and a high TMB was associated with tumour recurrence, which is consistent with previous reports.In addition, previous studies reported that patients with a high TMB had better overall survival and response to immunotherapy compared with those with a low TMB [19,20].However, in the present study, overall survival was not analysed because NMIBC has a high cancer-specific survival rate. The most frequently mutated genes in bladder cancer samples in the present study were ATM, NF1, and NOTCH1.ATM is a protein-coding gene that encodes a PI3K-related serine/threonine protein kinase that helps maintain genomic integrity.ATM gene mutations are commonly associated with increased predisposition to various cancers (more often in hematologic malignancies) and poor prognosis because ATM has a crucial role in the repair of DNA double-strand breaks [21].The NF1 gene, highly related to neurofibromatosis, encodes neurofibromin, a tumour suppressor protein that acts as a negative regulator of the Ras/MAPK and PI3K/mTOR signalling pathways [22].The Notch signalling pathway relies on cell-cell contact to influence cellular behaviour, and it has been implicated in human cancer as an oncogene and tumour suppressor [23].Based on previous reports, mutations of ATM, NF1, and NOTCH1 in bladder cancer are found in approximately 15%, 10%, and 50% of cases, respectively [21,23,24].These results are inconsistent with those of the present study.In addition, previous large-scale studies of NMIBC have identified early, general mutations in the TERT promotor and chromatin-modifying genes such as KDM6A and ARID1A, as well as activating alterations to oncogenes such as FGFR3 and ERBB2 [13,14,20].In the present study, these gene mutations were not included in the top 10 mutated genes, except for ARID1A.(ARID1A 67%, FGFR3 47%, TERT 35%, and ERBB2 23%) This may be due to the number of targeted genes included in the gene panel and the small size of our study population.Therefore, further research on the relationship between these gene mutations and NMIBC is needed in the future. In the present study, the mutation results were analysed by pairwise comparison according to the recurrence of bladder tumours within 1 year.The results revealed that the EGFR mutation and the TERT mutation had a significant association with tumour recurrence.In addition, multivariable analyses showed that EGFR and TERT mutations were significant factors associated with the recurrence of NMIBC.EGFR is the product of the c-erbB1 proto-oncogene, which belongs to the tyrosine kinase receptor family.It is a receptor for several growth factors, such as epidermal growth factor, transforming growth factor alpha, amphiregulin, heparin-binding EGF-like factor, betacellulin, and epiregulin [25].EGFR causes transcriptional activation and cell proliferation via intracellular cascade events.Indeed, several studies reported an association between EGFR overexpression and malignancies, such as lung, breast, and colon cancers [26,27].Regarding bladder cancer, the results of previous studies have revealed that more than half of cases of bladder cancer had EGFR overexpression, which was associated with high tumour grade, stage, tumour progression, and poor prognosis [25,28].These results are consistent with those of the present study, which strongly suggest that EGFR might be a useful prognostic biomarker in bladder cancer, even in NMIBC. Activating mutations in the TERT gene increase telomerase expression, allowing some neoplasms to overcome the end-replication problem and avoid senescence [29].TERT mutations have the highest prevalence in glioblastoma, liposarcoma, oligodendroglioma, urothelial carcinoma, melanoma, and hepatocellular carcinoma [30].Regarding bladder cancer, TERT mutations are an early event in bladder tumourigenesis, and a recent study reported the potential of TERT mutations as a promising non-invasive biomarker for the early detection of bladder cancer [31].In the present study, our results showed that TERT might be a potential biomarker for tumour recurrence.In addition, our results may support the importance of TERT mutations in NMIBC recurrence, and further studies will be needed to validate these findings and evaluate their clinical application. The present study had some limitations.First, this was a small-scale study and only included patients from a single institution.Further studies using more bladder cancer samples from multiple institutions are needed to identify the precise role of gene mutations in bladder cancer recurrence.Second, our targeted sequencing approach for cancer sample tissues can be limited by biopsy bias, and it is difficult to evaluate precise evolutionary relationships over time.Third, we did not consider the effect of adjuvant intravesical BCG treatment.As BCG can provide temporal changes in the genomic environment of early bladder cancer [20], further studies on recurrent bladder cancer samples are needed to account for this aspect.However, our results indicate that ancestor gene mutations are associated with NMIBC recurrence.In addition, considering the results of this study, current strategies for the surveillance of bladder cancer via macroscopic cystoscopy and urine cytology can miss the possibility of residual disease, so including a genetic evaluation should be considered for the stratification of recurrent risk. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The clinical application of NGS-based analysis is already accepted as an essential step towards precision oncology.NGS-based analysis and leveraging genomic data are anticipated to improve the classification of bladder cancer patients with recurrent risk.Considering this trend, our results may support the importance of somatic mutations in NMIBC recurrence and the fact that somatic mutations in EGFR and TERT have a prognostic value for tumour recurrence in NMIBC.However, additional research to validate these findings and evaluate their clinical application is needed. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "The data presented in this study are available in this article. The authors declare no conflict of interest. ",
"section_name": "Data Availability",
"section_num": null
},
{
"section_content": "The data presented in this study are available in this article. ",
"section_name": "Data Availability",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/diagnostics13203201/s1, Figure S1: Results of tumour mutation burden according to tumour recurrence. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
10.3390/cancers12030652
|
Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA
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<jats:p>Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.</jats:p>
|
[
{
"section_content": "About 95% of coding genes in humans are subjected to alternative splicing, a highly regulated and complex mechanism that diversifies the proteome by creating multiple proteins from the same gene.Deregulation of splicing is observed in many cancers and hematological diseases [1,2].Recent massive sequencing of many cancer genomes allowed the identification of recurrent mutations in genes encoding splicing factors, including SF3B1 (Splicing Factor 3B subunit 1), SRFS2 (Serine and arginine rich splicing factor 2), U2AF1 (U2 small nuclear RNA auxiliary factor 1), and ZRSR2 (Zinc finger CCCH-type, RNA binding motif and Serine/Arginine Rich 2), suggesting that somatic alterations of genes involved in splicing are common in cancer [2,3].Strikingly, in myelodysplastic syndromes (MDS), over 50% of patients have acquired mutations that affect the splicing machinery [4,5].MDS are complex and heterogeneous acquired pathologies of the bone marrow, characterized by a clonal and ineffective hematopoiesis, resulting in a deficit of mature myeloid blood cells and a risk of clonal progression including evolution to acute myeloid leukemia.Mutations in genes involved in epigenetic regulation are also frequently present in MDS.While many mutations may coexist in MDS and other Cancers 2020, 12, 652 2 of 16 neoplasms, splice factor mutations occur in a mutually exclusive manner [4,6] Remarkably, mutations in SF3B1 occur in up to 85% of sideroblastic MDS (MDS-RS) [4,5,7], a subtype of MDS characterized by the presence of ring sideroblasts (RS) in the bone marrow, and associated with good prognosis.SF3B1 mutations are also found in chronic lymphoid leukemia (CLL) [8,9], where they are associated with poor outcome, in uveal melanoma (20%) [10] and to a lesser extent in breast [11] and pancreatic cancers [12]. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle (snRNP) that interacts with branch point sequences close to the 3' splice site during pre-mRNA splicing [13].SF3B1 is the largest subunit of SF3b, a heptameric protein complex of U2 snRNP.SF3B1 consists of an unstructured N-terminal domain, and a C-terminal HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) domain composed of 20 tandem repeats structured as a superhelix [14], providing a scaffold together with other SF3b subunits within the U2 snRNP.Most of SF3B1 mutations are heterozygous change-of-function mutations leading to amino acid substitutions at restricted sites in H4-H8 repeats of the highly conserved HEAT domain.While most of these key residues contribute to SF3B1 structure, others seem rather to be directly involved in the interactions with the intron at the branch point site and/or with spliceosomal proteins, including the K700 residue [14].Recent RNA-sequencing (RNA-seq) analyses in various malignancies, including CLL [15] and breast cancer [16], uveal melanoma [16][17][18] and MDS [19][20][21], indicate that the most common mutations of SF3B1 lead to a global splicing defect characterized by the production of aberrant transcripts through the use of cryptic 3' splice sites and alternative branch points.However, the number of altered transcripts remains limited (approximately 1% of the transcriptome) likely because only introns that possess peculiar sequences appear to be sensitive to the altered form of SF3B1 [16].Approximately half of aberrant transcripts are predicted to be degraded by the non-sense mediated mRNA decay (NMD), while the other half would be translated into proteins with potentially altered function [16].Importantly, the differentially expressed genes and aberrant splicing events observed in cells with disease-related SF3B1 point mutations appear to be different from those observed upon depletion of SF3B1 [22,23], stressing the fact that SF3B1 mutants bear specific functions.Interestingly, Zhang et al. recently reported that disease-causing mutations in SF3B1 alter splicing by disrupting interaction with the spliceosomal protein SUGP1 [24], providing a possible mechanistic explanation for the phenotype of SF3B1 point mutants.Moreover, silencing of SF3B1 by shRNA alters the proliferation of erythroid progenitors in vitro [23] and in vivo in a mouse model [25], highlighting its essential role during erythropoiesis.Notably, a dynamic intron retention program enriched in RNA processing genes, including SF3B1, regulates gene expression during terminal erythropoiesis [26,27].However, studies on SF3B1 splice variants remain scarce. Upon analyzing published RNA-seq raw data, we found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, including in MDS-RS.This aberrant transcript is expected to encode a novel SF3B1 protein isoform harboring an insertion of eight amino acids in the H3 repeat of the HEAT domain.As the functional consequences of disease-causing SF3B1 mutations are only partially understood, we aimed to investigate whether this aberrant SF3B1 splice variant, which is a minor form, would interfere with the overall function of the spliceosome.Here, we show that cancer-associated SF3B1 point mutations drive the formation of this SF3B1 aberrant transcript in MDS-RS patients and in human myeloid cell lines.Using complementary approaches in both human and yeast cells, we show that insertion of these eight amino acids in wild-type or mutant SF3B1 protein abolished SF3B1 essential splicing activity, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.Finally, splice-switching oligonucleotides were used to favor the formation of this peculiar SF3B1 transcript, which might represent a novel way to sensitize SF3B1 mutated cells to splicing modulators. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results and Discussion",
"section_num": "2."
},
{
"section_content": "By exploring publicly available RNA-seq raw data obtained from patients with SF3B1 mutations, we identified a transcript of SF3B1 that was solely detected in SF3B1 mutated samples in various neoplasms, including breast cancer [16], uveal melanoma [19] and recently in MDS [28].This transcript, which we named SF3B1ins, results from the retention of 24 nucleotides from intron 12 into exon 13, through the recognition of a cryptic AG' 3' splice site located 24 nucleotides upstream of the canonical AG 3' splice site (Figure 1A).As no stop codon is generated, SF3B1ins transcript is predicted to produce a protein with an insertion of eight amino acids (LLLFSLFQ) in the H3 repeat of the highly conserved HEAT domain of SF3B1 (Figure 1B).We first examined whether SF3B1ins transcript was effectively detected in a series of bone marrow mononuclear cell samples derived from 11 MDS-RS patients, in comparison to patients with Idiopathic Cytopenia of Undetermined Significance (n = 4) or from MDS patients without RS (n = 6).Most patients had normal karyotypes (Table S1).The mutational status of a panel of 26 genes known to be frequently mutated in myeloid malignancies, including SF3B1, was determined by targeted NGS for MDS-RS patients (Table S1).SF3B1 was mutated in 9 out of 11 MDS-RS patients, with a variant allele fraction ranging from 12.3% to 48%.The two SF3B1 wt MDS-RS patients had mutations in the other splicing factor encoding genes ZRSR2 or SRSF2, and in TET2. To validate splicing abnormalities in the series of MDS patients, we first analyzed the splicing of TMEM14C and ENOSF1, two genes known to present an alternative cryptic AG' splice site efficiently selected in SF3B1 mutated background [19,20,29].As expected, aberrant transcripts of TMEM14C and ENOSF1 were exclusively detected in SF3B1-mutated MDS-RS patients.Using primers designed to specifically amplify the SF3B1ins transcript, we detected SF3B1ins systematically and exclusively in SF3B1-mutated samples (Figure 1C).The relative abundance of SF3B1ins was less than 10% of total SF3B1 transcripts in mononuclear cells, a proportion similar to that reported in published RNA-seq data [19].However, the proportion of SF3B1ins protein in SF3B1 mutated cells could not be established, as SF3B1ins cannot be distinguished from SF3B1 by Western blot. To investigate whether production of SF3B1ins transcript was indeed driven by SF3B1 hot spot mutations, we first generated human myeloid leukemic cell lines (K562 and UT-7) expressing SF3B1 WT FLAG or SF3B1 K700E FLAG under the control of a doxycycline inducible promoter.We chose to study the K700E substitution, which is the most common in MDS.An optimized version of SF3B1 was used because it was impossible to clone the natural sequence, as already reported [19].Importantly, the amount of recombinant SF3B1 FLAG protein was similar to that of endogenous SF3B1 (Figure S1), mimicking a heterozygous SF3B1 wt/K700E background.We first analyzed the splicing of specific genes in different independent conditions.Aberrant splice variants of ENOSF1, TMEM14C, and DPH5 were exclusively found when SF3B1 K700E FLAG was expressed, as shown in three K562 independent clones (Figure 1D) and two independent UT-7 cell populations (Figure 1E), validating the cell models used in this study.While only traces of SF3B1ins transcript were detected in SF3B1 WT FLAG -transfected cells or in the absence of transgene induction, this alternative transcript was clearly produced when SF3B1 K700E FLAG was expressed.Expression of SF3B1 K700E was thus sufficient to promote the formation of SF3B1ins transcript, even in the presence of endogenous SF3B1 WT , as observed in patient cells.To check whether the SF3B1ins transcript was also produced when other SF3B1 hot spot mutations were expressed, we transfected K562 cells with plasmids expressing SF3B1 WT , SF3B1 K700E , SF3B1 E622D , SF3B1 H662Q , or SF3B1 K666E .Here again, SF3B1ins transcript was exclusively detected upon expression of the mutated versions of SF3B1 (Figure 1F).Consequently, expression of the main disease-related SF3B1 mutations is sufficient to generate SF3B1ins transcripts, even in the presence of a functional SF3B1 wt protein. ",
"section_name": "Expression of the Main SF3B1 Mutations in MDS Patients and in Myeloid Cell Lines Drives the Formation of SF3B1ins Transcript",
"section_num": "2.1."
},
{
"section_content": "",
"section_name": "GTTTGT TCTCCTTA G AT G T G A A A G T G T A G ' C T T C T T C T C T T T T C T C T T T T T C",
"section_num": null
},
{
"section_content": "To study the functionality of the SF3B1ins isoforms, we inserted the sequence encoding the LLLFSLFQ peptide into the plasmids expressing wild type SF3B1 FLAG or SF3B1 K700E FLAG .The resulting constructs were first introduced into K562 cells by transient transfection.SF3B1 wt INS and SF3B1 K700E ",
"section_name": "SF3B1ins and SF3B1 K700E ins Proteins Are Defective for Splicing",
"section_num": "2.2."
},
{
"section_content": "proteins were both correctly produced, although in somewhat lower abundance than SF3B1 wt and SF3B1 K700E proteins (Figure 2A), and were both properly addressed to the nucleus, as shown by immunofluorescence analysis (Figure S2).Remarkably, transient expression of SF3B1 K700E INS did not lead to the production of aberrant ENOSF1 or TMEM14C transcripts, in contrast to transient expression of SF3B1 K700E (Figure 2B).Thus, inserting the LLLFSLFQ sequence into the H3 repeat impeded the phenotypic expression of the K700E substitution.This may have occurred either by a mechanism whereby the LLLFSLFQ insertion in cis would suppress SF3B1 K700E -dependent splicing anomalies (potentially through conformational modifications), or by inactivation of SF3B1 protein function, leading to a loss-of-function phenotype.In order to distinguish between these two hypotheses, we needed a model in which most of SF3B1 function would be assumed by the transfected constructs.This necessitated reducing the production of endogenous SF3B1, which was done by using two siRNAs directed against the 3'UTR of SF3B1.While the endogenous SF3B1 protein was efficiently depleted (Figure 2C), the FLAG-tagged SF3B1 protein was depleted in a similar way, for unknown reasons, even though there was no sequence overlap between the SF3B1 3' UTR and the engineered SF3B1 construct.Despite this general SF3B1 decreased expression, we carried out RT-PCR experiments to study splicing events specifically altered upon downregulation of SF3B1, in order to evaluate the capacity of SF3B1 wt INS and SF3B1 K700E INS to compensate for the splicing defects caused by SF3B1 depletion.We decided to study specific exon skipping events in RBM5, DUSP11, CCNA2, and STK6, the splicing of which was known to be modified upon either SF3B1 silencing [22,30] or treatment by the spliceosome inhibitor spliceotastin A [31].Depletion of SF3B1 in siRNA-only transfected cells or in K562 cells co-transfected with the empty vector favored skipping of exon 6 in RBM5, of exon 6 in DUSP11, of exon 5 in CCNA2 and of exons 4-5-6 in STK6 (Figure 2D,E), as previously described.While expression of both SF3B1 WT and SF3B1 K700E partially prevented exon skipping, expression of both SF3B1 wt ins and SF3B1 K700E ins did not.The inability of SF3B1 wt ins and SF3B1 K700E ins to restore the normal splicing of specific transcripts known to be altered upon silencing of SF3B1 implies that both SF3B1 wt ins and SF3B1 K700E ins are inactive for splicing. To further address this question, we generated K562 cell lines that stably expressed SF3B1 WT ins or SF3B1 K700E ins in combination with shSF3B1, both under the control of a doxycycline-inducible promoter.Here again, the splicing pattern of RBM5, DUSP11, CCNA2, and STK6 was not rescued by expression of SF3B1 wt ins or SF3B1 K700E ins (Figure S3).Silencing of SF3B1 by shSF3B1 affected cell growth, as previously reported in other cell lines [32] or in CD34+ cells [23].Notably, induction of SF3B1 wt INS or SF3B1 K700E INS did not complement the growth phenotype due to SF3B1 silencing (Figure 2F).Taken together, our results indicate that insertion of the LLLFSLFQ sequence into the H3 repeat of SF3B1 abolishes the splicing and essential function of SF3B1, underlining the crucial role of the H3 repeat in the splicing function of SF3B1.Moreover, the fact that splicing of RBM5 and DUSP11 was not affected when SF3B1 wt ins or SF3B1 K700E ins was expressed in regular conditions (i.e., without siSF3B1) suggests that SF3B1ins proteins do not seem to exert a dominant negative effect (Figure 2B).The effect of SF3B1ins on the pathophysiology of MDS and other neoplasms may be limited due to its low abundance.Nevertheless, while cancer-associated SF3B1 substitutions lead to an improper recognition of the branch site and to the use of cryptic splice sites, it remains possible that a fraction of the SF3B1 pool, i.e., SF3B1 wt ins and SF3B1 K700E ins, may drive improper U2 snRNP assembly in SF3B1 +/K700E cells.A-E) K562 cells were transfected with plasmids encoding different versions of SF3B1 (SF3B1 WT , SF3B1 K700E , SF3B1 WT ins, and SF3B1 K700E ins).(A) Total SF3B1 proteins (endogenous and exogenous) were detected by Western blot analysis using anti-SF3B1 antibody.Plasmid-encoded SF3B1 was detected using anti-FLAG antibody.(B) Analysis of splicing events known to be specifically altered in SF3B1-mutated background (ENOSF1, TMEM14C, DPH5) or upon SF3B1 loss of function (RBM5, DUSP11).RT-PCR was performed using primers allowing specific detection of distinct splicing events in SF3B1, ENOSF1, TMEM14C, DPH5, RBM5, and DUSP11.(C-E) K562 cells were co-transfected with an siRNA specific to endogenous SF3B1 and with plasmids encoding different versions of SF3B1.(C) Western blot analysis of SF3B1 proteins as A-E) K562 cells were transfected with plasmids encoding different versions of SF3B1 (SF3B1 WT , SF3B1 K700E , SF3B1 WT ins, and SF3B1 K700E ins).(A) Total SF3B1 proteins (endogenous and exogenous) were detected by Western blot analysis using anti-SF3B1 antibody.Plasmid-encoded SF3B1 was detected using anti-FLAG antibody.(B) Analysis of splicing events known to be specifically altered in SF3B1-mutated background (ENOSF1, TMEM14C, DPH5) or upon SF3B1 loss of function (RBM5, DUSP11).RT-PCR was performed using primers allowing specific detection of distinct splicing events in SF3B1, ENOSF1, TMEM14C, DPH5, RBM5, and DUSP11.(C-E) K562 cells were co-transfected with an siRNA specific to endogenous SF3B1 and with plasmids encoding different versions of SF3B1.(C) Western blot analysis of SF3B1 proteins as described in (A). Average quantification of endogenous and exogenous levels of SF3B1 protein from three independent experiments is indicated.(D) Analysis of specific splicing events as described in (B).The proportion of exon skipping in RBM5 and DUSP11 (average from three independent experiments) is indicated below the corresponding gels.(E) RT-qPCR was performed using primers allowing specific quantification of exon skipping events in CCNA2 (exon 5) and STK6 (exons 4, 5, and 6) transcripts, normalized to GAPDH.Relative quantification is indicated, and error bars represent ± SEM from three independent experiments.(F) Growth of K562 cells expressing inducible SF3B1 WT versus SF3B1 WT ins (top) and SF3B1 K700E versus SF3B1 K700E ins (bottom) upon SF3B1 silencing (shSF3B1), following doxycycline induction.Error bars represent ± SEM from four independent experiments.A Mann-Whitney test was applied (p value < 0.05).Data information: In (A-D), representative results of at least three independent experiments are presented.In (C and E), error bars represent ± SEM from three independent experiments.L: ladder Interestingly, elegant structural studies have indicated that HEAT repeat H3, as well as H5-H6 repeats, contact the β-propeller A domain of SF3B130, another core component of SF3b complex [14].The H3 repeat seems also to be directly implicated in the interaction of the concave surface of SF3B1 with SF3b14b, a highly conserved protein required for proper assembly of yeast U2 snRNP and stability of the spliceosome [33].SF3b14b appears to shape the structure of SF3B1 through direct contact with H2-H3 at the N-terminus of the superhelix and H15, H17, and H18 at the C-terminus of the superhelix of SF3B1.These structural features suggest that insertion of the LLLFSLFQ loop into the H3 repeat might modify the overall structure of the SF3b complex through modification of protein-protein interactions. ",
"section_name": "INS",
"section_num": null
},
{
"section_content": "To further address the question of the functionality of SF3B1ins, we performed functional complementation experiments in Schizosaccharomyces pombe, an alternative model of choice for the study of RNA splicing [34].The splicing machinery is relatively well conserved between S. pombe and H. sapiens, including the presence of orthologues of SR proteins, a class of splice regulatory proteins.The S. pombe SF3B1 orthologue, SpSAP155 [35], which is essential for viability, shares 52% sequence identity (71% sequence identity within the HEAT domain), and 67% sequence similarity with HsSF3B1.Most substitutions found in MDS patients affect amino acids conserved between the two species, in the HEAT domain (Figure S4), including the K700 residue. We took advantage of the thermosensitive growth phenotype of the yeast prp10-1 mutant strain [35], which harbors two point mutations in SAP155, initially selected in a genetic screen, to study the ability of various Sap155 alleles to restore growth at restrictive temperature (37 • C).We first introduced the counterparts of E622N, H662Q, K666N (R666N), and K700E substitutions into the S. pombe SAP155 sequence by site-directed mutagenesis.The growth deficiency of the prp10-1 strain at 37 • C was restored upon expression of wild-type SAP155 or the different mutated versions of SAP155 (Figure S4), indicating that the main counterparts of Sap155 pathogenic mutations do not affect the growth of S. pombe, as also observed in S. cerevisiae [36].Thus, the various cancer-related SAP155 point mutations do not alter splicing of genes essential for yeast viability, in both S. pombe and S. cerevisiae.We next investigated the effect of these mutations on the ability to splice specific pre-mRNAs following a switch to the restrictive 37 • C temperature for two hours.This led to the defective splicing of TFIID, NDA3, and RPL7 in the prp10-1 strain, due to the loss of function of the endogenous sap155 (prp10-1) allele (Figure 4), as previously reported [35].Expression of SAP155 wt , SAP155 K700E , or the selected main variants in the prp10-1 strain restored the correct splicing of TFIID, NDA3, and RPL7 (Figure 3 and Figure S4).Hence, yeast counterparts of disease-related SF3B1 mutants do not alter the splicing of these specific transcripts in S. pombe.Nevertheless, given that disease-related mutations appear to alter the fidelity of branch site selection of a mini-gene construct in S. cerevisiae [36], it is likely that the K700E counterpart impacts other transcripts in S. pombe upon selection of cryptic splice sites.The latter could be predicted by in silico analyses or identified by RNA-seq analysis, as previously done in mice.While Cancers 2020, 12, 652 8 of 16 the nature of splicing defects observed in a conditional knock-in Sf3b1 K700E/+ mouse model mimicked that described in human MDS, with numerous aberrant 3' splice-site selections, only a few mis-spliced transcripts were found in common between mouse and human.This resulted from the relatively poor interspecies conservation of intronic sequences able to function as aberrant splice sites [37].Similar discrepancies are expected to be observed in S. pombe due to even higher divergency between yeast and human intronic sequences. Cancers 2020, 12, x 8 of 15 conditional knock-in Sf3b1 K700E/+ mouse model mimicked that described in human MDS, with numerous aberrant 3' splice-site selections, only a few mis-spliced transcripts were found in common between mouse and human.This resulted from the relatively poor interspecies conservation of intronic sequences able to function as aberrant splice sites [37].Similar discrepancies are expected to be observed in S. pombe due to even higher divergency between yeast and human intronic sequences.As the insertion site of the LLLFSLFQ loop maps to an extremely conserved region of SF3B1 in the H3 repeat [14], we next introduced this loop at the exact same position in the wild-type or mutated SpSAP155 sequence to investigate how this could interfere with the function of SpSAP155 in S. pombe.Wild-type and prp10-1 mutant strains were transformed with plasmids expressing SAP155 WT , SAP155 K700E , SAP155 WT ins, or SAP155 K700E ins.Growth of prp10-1 cells expressing SAP155 WT ins or SAP155 K700E ins was affected at 25 °C.The growth phenotype was even stronger when K700E was associated to the eight-amino-acid insertion.Moreover, expression of SAP155 WT ins and SAP155 K700E ins in prp10-1 mutant strain did not complement at all the defective growth of prp10-1 As the insertion site of the LLLFSLFQ loop maps to an extremely conserved region of SF3B1 in the H3 repeat [14], we next introduced this loop at the exact same position in the wild-type or mutated SpSAP155 sequence to investigate how this could interfere with the function of SpSAP155 in S. pombe.Wild-type and prp10-1 mutant strains were transformed with plasmids expressing SAP155 WT , SAP155 K700E , SAP155 WT ins, or SAP155 K700E ins.Growth of prp10-1 cells expressing SAP155 WT ins or SAP155 K700E ins was affected at 25 • C. The growth phenotype was even stronger when K700E was associated to the eight-amino-acid insertion.Moreover, expression of SAP155 WT ins and SAP155 K700E ins Cancers 2020, 12, 652 9 of 16 in prp10-1 mutant strain did not complement at all the defective growth of prp10-1 strain at restrictive temperature (Figure 3A), in contrast to insertion-less constructions.The splicing of TFIID, NDA3, and RPL7 was not restored upon expression of SAP155 WT ins or in prp10-1 strain (Figure 3B).Due to the major growth defect of the prp10-1 mutant strain expressing SAP155 K700E ins, we could not study the splicing in such a background.Altogether, these results indicate that insertion of LLLFSLFQ in the H3 repeat of S. pombe SAP155 alters its essential function, as observed in human cell lines. ",
"section_name": "Study of Splicing Defects due to Expression of SF3B1ins or Disease-Related SF3B1 Mutations in Schizosaccharomyces pombe",
"section_num": "2.3."
},
{
"section_content": "Modulation of splicing catalysis by small molecules was proposed for therapeutic targeting of cancer cells with mutations in genes encoding spliceosomal proteins [38].Importantly, several splicing inhibitors identified in screens for cell growth inhibitors target SF3B1, including pladienolide B, spliceostatin A, and herboxidien [39].The pladienolide B E7107 derivative was tested in a phase 1 clinical trial in solid tumors [40] but tests were suspended due to adverse events.Recently, another pladienolide B derivative H3B-8800, which modulates both wild-type and mutant spliceosome activity, was shown to preferentially kill SF3B1-mutant cells.Thus, cells with altered splicing appear to be more sensitive to splicing inhibitors than normal cells, mainly by further alteration of genes involved in splicing regulation [41,42].We speculated that increasing the splicing defects in SF3B1 K700E/+ cells may make them more sensitive to splicing modulators.To test whether it was actually possible to increase SF3B1ins formation, thereby increasing splicing dysfunction in target cells, we designed splice switching oligonucleotides (SSO) with the intention to favor use of the AG' cryptic site at the intron 12/exon 13 junction.Four different Locked Nucleic Acid SSO were chosen to mask the canonical AG splice site in different ways at this specific junction (Figure 4A).We introduced the SSO in readily transfectable HEK293T cells that were co-transfected with plasmids expressing either SF3B1 WT or SF3B1 K700E , in comparison to cells transfected with a control fluorescent SSO.SSO #1, the center of which masks the 3' AG canonical site, was able to increase SF3B1ins formation in cells expressing SF3B1 K700E , but also in SF3B1 WT cells (Figure 4B,C).Nevertheless, the level of SF3B1ins in SF3B1 WT upon SSO #1 treatment was still lower than that observed in untreated SF3B1 K700E cells.We thus provide the proof of concept that splicing can be specifically oriented to favor the use of a cryptic AG' splice site.Further studies are required to determine if favoring such an event could increase the sensitivity to splicing modulators, including the promising H3B-8800 compound.A similar approach that would specifically orient the spliceosome to a specific cryptic splice site in other splicing factor encoding genes might be exploited in the future to make SF3B1 K700E/+ cells more sensitive to splicing modulators.untreated SF3B1 K700E cells.We thus provide the proof of concept that splicing can be specifically oriented to favor the use of a cryptic AG' splice site.Further studies are required to determine if favoring such an event could increase the sensitivity to splicing modulators, including the promising H3B-8800 compound.A similar approach that would specifically orient the spliceosome to a specific cryptic splice site in other splicing factor encoding genes might be exploited in the future to make SF3B1 K700E/+ cells more sensitive to splicing modulators.g t g a a a g t g t a g c t t c t t c t c t t t t c t c t t t t t c a g ATCCTCGTGGTCATTGAACCGCTAT ",
"section_name": "Use of Splice-Switching Oligonucleotides to Modulate the Production of SF3B1ins Transcript",
"section_num": "2.4."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "3."
},
{
"section_content": "Bone marrow samples were obtained from patients followed up in Brest University Hospital and in St. Brieuc Hospital, and collected in the Centre de Ressources Biologiques (CRB) of Brest Hospital, which is referenced by the BioBank structure under a unique identifier number BB-0033-00037.The CRB is certified according to the French norm NF S 96-900: \"CRB management system and quality of biological resources.\"This was a non-interventional retrospective study approved by the ethics committee of Brest Hospital (ethic code 29BRC20.0029).Mononuclear cells were isolated by Ficoll gradient from bone marrow aspirates of patients diagnosed with MDS according to the World Health Organization classification, or from individuals with normal bone marrow.Written informed consent was obtained from all subjects, and the experiments conformed to the principles set out in the Declaration of Helsinki. ",
"section_name": "Patient Samples",
"section_num": "3.1."
},
{
"section_content": "The next-generation sequencing panel included 26 genes (ASXL1, BCOR, CBL, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KRAS, MPL, NRAS, PDGFRA, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, STAG2, TET2, TP53, U2AF1, ULK1, and ZRSR2) and the sequencing was performed using Ampliseq TM (Thermo Fisher Scientific, Foster City, CA, USA) custom design.Library preparation and sequencing using Ion PGM TM (Thermo Fisher Scientific) were performed according to the manufacturer's instructions.Mutations were detected using the Variant Caller v4.2 plugin from Torrent Suite Software and IonReporter v5.2 (Life Technologies, Carlsbad, CA, USA).For mutation calling, arbitrary filters were fixed with variant allele frequencies >2% and depth >50X.False positive variants were dropped after BAM analysis on Alamut (Interactive Biosoftware, Rouen, France).Only exonic non-synonymous mutations were analyzed. ",
"section_name": "Next-Generation Sequencing",
"section_num": "3.2."
},
{
"section_content": "Due to the impossibility of cloning the natural sequence of SF3B1 cDNA, a codon-optimized version of SF3B1 (from SF3B1-201 ENST00000335508.10),originally cloned in pCMV-3tag-1A vector [19], was used for all experiments.pCMV-3tag-1A-SF3B1 WT and pCMV-3tag-1A-SF3B1 K700E were generously given by Angelos Constantinou.Plasmids encoding E622D, H662Q, and K666E variants of SF3B1 were obtained by site-directed mutagenesis from pCMV-3tag-SF3B1 WT plasmid using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).The same strategy was used to produce the counterparts of E622N, H662Q, K666N (R666N), and K700E substitutions into the S. pombe SAP155.SF3B1ins coding sequence was created by pseudo-inverse PCR using specific primers to incorporate the 24 nucleotides corresponding to the retained part of the intron 12 in the optimized sequence of SF3B1 (SF3B1 WT and SF3B1 K700E ), in both pCMV-3tag-1A and pCW57.1 plasmids.Primers used for cloning and directed mutagenesis are listed in Supplementary Table S2. ",
"section_name": "Cloning and Site-Directed Mutagenesis",
"section_num": "3.3."
},
{
"section_content": "The K562 and UT-7 cell lines were obtained from the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), respectively.K562 and K562-derived cell lines were cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM of L-glutamine (Gibco), at 37 • C and 5% of CO 2 .UT-7 cells were cultured in IMDM medium (Gibco), 10% fetal bovine serum (Gibco), 4 mM of L-glutamine (Gibco), 100 UI/mL of penicillin and streptomycin (Gibco), and 1 U/mL of erythropoietin (EPO).K562 cells were transfected by electroporation using Cell Line Nucleofector TM Kit V (Amaxa, Lonza) according to the manufacturer's instructions.Transfections were done with 2 × 10 6 cells and 4 µg of plasmid and/or 30 pmol of SF3B1 siRNAs designed to specifically target the 3'UTR sequence (siSF3B1-1: 5'-GUGUAGAACUGGUCAUAGA-3', siSF3B1-2: 5'-CUCAUUCCUUGUGUUUAAA-3').HEK293T cells were cultured in EMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and were transfected with lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.Transfections were done with 400,000 cells, 1 µg of plasmid and 500 pmol of SSO.SSO were synthetized and modified by the locked nucleic acid technology (Qiagen).SSO control was labeled with a 5'FAM to estimate the transfection efficacy.(SSO control: 5'-TAACACGTCTATACGCCCA-3'; SSO #1: 5'-CCACGAGGATCTGAAAAA-3'; SSO #2: 5'-ATGACCAGGATCTGA-3'; SSO #3 : 5'-TTCAATGACCACGAGGAT-3'; SSO #4 : 5'-CTGAAAAAGAGAAAGAG-3').All analyses were performed at 48 h post-transfection. ",
"section_name": "Cell Culture and Transfection",
"section_num": "3.4."
},
{
"section_content": "The optimized coding sequences of SF3B1 WT and SF3B1 K700E , amplified from pCMV-3tag-1A-SF3B1 WT and pCMV-3tag-1A-SF3B1 K700E plasmids, respectively, were introduced in the lentiviral plasmid pCW57.1 using In-Fusion®HD Cloning Plus kit (Takara).shSF3B1 targeting endogenous SF3B1 mRNA was cloned into the pLKO-Tet-on plasmid, an all-in-one inducible system.Sequences of the oligonucleotides used for shSF3B1 (#5) were as described in [32], and did not overlap with the optimized coding sequence of SF3B1.Forward: CCGGCCTCGATTCTACAGGTTATTACTCGAGTAATAACCTGTAGAATCGAGGTTTTTG, Reverse: AATTCAAAAACCTCGATTCTACAGGTTATTACTCGAGTAATAACCTGTAGAATCGAGG Lentival particles were produced by the Vect'UB vectorology core facility (Bordeaux, France).Cells were transduced by lentivirus particles with a multiplicity of infection of 5. Selection of stable positive cells was done with puromycine (Sigma) for SF3B1 inducible cell lines or G418 (Sigma) for shSF3B1 inducible cell lines.Transduction with the shSF3B1 construct was done on both K562 and K562 stable cell lines expressing SF3B1 wt , SF3B1 K700E , SF3B1 wt ins, and SF3B1 K700E ins to obtain co-transduced stable cell lines.SF3B1 expression was induced by treating cells with 2 µg/mL or 3 µg/mL (shSF3B1) of doxycycline (Sigma).All analyses were performed 48 h after induction with doxycycline.To determine cell growth, cells were counted every 24 h following doxycycline induction, using a hemocytometer. ",
"section_name": "Generation of SF3B1 and shSF3B1 Inducible Cell Lines",
"section_num": "3.5."
},
{
"section_content": "RNA was extracted from bone marrow mononuclear cells of MDS patients using the Nucleospin RNA kit (Macherey-Nagel) or TRIzol (Invitrogen)-phenol/chloroform extraction.RNA was extracted from K562 and HEK293T cells using nucleospin RNA kit (Macherey-Nagel) or nucleospin RNA XS (Macherey-Nagel).RNA integrity was checked using Bioanalyser or by agarose gel electrophoresis.Reverse transcription was performed with High Capacity cDNA reverse transcription kit (Applied Biosystems).PCR was performed with the GoTaq®G2 DNA Polymerase kit (Promega).Primers used are listed in Supplementary Table S2.PCR products were analyzed on 2% or 3% agarose gels (Molecular Biology Grade Agarose, Eurobio).Quantification of DNA bands was performed with Image studio lite software.Sequencing of extracted gel bands was achieved to confirm the partial intron retention observed in SF3B1ins transcript.Quantitative real-time PCR was performed with Power SYBR™ Green PCR Master Mix with a StepOnePlus Real-Time PCR System.The 2 ∆∆-Ct method was used to analyze the results using GAPDH as a reference gene. ",
"section_name": "RNA Extraction and RT-PCR",
"section_num": "3.6."
},
{
"section_content": "Proteins were extracted from cells with a lysis buffer (NP40 1%, SDS 0.1%) and quantified with the Dc protein kit (Biorad).Forty µg of total proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes.We used the following primary antibodies: anti-SF3B1 (1:5000, #170854 Abcam), anti-FLAG (1:5000, #F3165, Sigma), anti-GAPDH (1:5000), and anti-b-actin (1:1000, #ab8226, Abcam).Donkey anti-mouse (1:5000, #926-32222, LI-COR) and goat anti-rabbit (1:5000, #926-32211, LI-COR) secondary antibodies were used.Immunofluorescence was detected using Odyssey Infrared Imaging system (LI-COR).Quantification was performed with Image Studio Lite software.β-actin or GAPDH were used as references to normalize quantification. ",
"section_name": "Western Blot Analysis",
"section_num": "3.7."
},
{
"section_content": "Wild-type and prp10-1 Schizosaccharomyces pombe strains [35] were transformed with pREP41-HA plasmids using a slightly modified version of the lithium acetate method [43] in which heat shock was replaced by an hour-long incubation at 25 • C due to the thermosensitive phenotype.Transformants were selected through their ability to grow on leucine free EMM 2% glucose medium (Formedium).To analyze splicing defects, cells were cultured in EMM 2% glucose at 25 • C, and subjected to a 2-h shift at 37 • C (restrictive temperature).Cell wall was broken by vortexing cells for 20 min with acid-washed glass beads (Sigma) at 4 • C and RNA was extracted using Nucleo-spin RNA kit (Macherey-Nagel).Reverse transcription and RT-PCR were performed as described in the preceding section, using primers allowing detection of intron retention in TFIID, NDA3, RPL7 (Table S2). ",
"section_name": "Yeast Growth and Molecular Biology",
"section_num": "3.8."
},
{
"section_content": "Statistical analysis and histograms were created with Prism 8 software.When appropriate, a Mann-Whitney test was used with a p-value < 0.05 to be considered significantly different between two groups. ",
"section_name": "Statistical Analysis",
"section_num": "3.9."
},
{
"section_content": "RNA splicing was reported as a process significantly affected (i.e., numerous splicing genes with abnormal expression or splicing) in all MDS harboring mutations in splice factor genes [44].In this study, we identified and characterized a novel SF3B1 transcript that is specifically produced in neoplasms carrying an SF3B1 mutation, including in myelodysplastic syndromes.This transcript encodes an aberrant SF3B1 protein isoform with an insertion of the LLLFSLFQ sequence into the H3 repeat of the HEAT domain.Using complementary approaches in both human and yeast cells, we showed that this insertion abolished SF3B1 splice function, highlighting the functional importance of the H3 repeat in SF3B1.SF3B1ins transcript level might vary differently in a disease-specific manner, and modulate the phenotypical expression of SF3B1 point mutants.SF3B1 mutant cells are expected to accumulate numerous other aberrant proteins, which could participate in carcinogenesis.So far, to our knowledge, only two studies dealt with the neoproteins specifically produced in SF3B1 mutated cells ( [28] and this work).Significant progress is expected in the characterization of these splicing-derived cancer specific proteins in order to propose novel biomarkers and therapeutic options.Finally, splicing inhibitors were recently proposed for therapeutic targeting of cancer cells with mutations in genes encoding spliceosomal proteins.As the selectivity still remains an important issue, increasing splicing alterations in SF3B1 K700E/+ cells in a specific way, through preferential selection of splice sites by SSO, might represent a novel way to sensitize cells to splicing inhibitor-induced apoptosis. ",
"section_name": "Conclusions",
"section_num": "4."
},
{
"section_content": "The following are available online at http://www.mdpi.com/2072-6694/12/3/652/s1, Figure S1: Complementary results obtained in K562 and UT-7 cells upon inducible expression of SF3B1 K700E or upon transitory expression of other important SF3B1 variants; Figure S2: SF3B1ins FLAG proteins are correctly addressed to the nucleus; Figure S3: Splicing analysis of RBM5, DUSP11, CCNA2 and STK6 in inducible K562 cell lines expressing shSF3B1 alone or in combination with various SF3B1 constructs; Figure S4: Expression of the main disease-related SF3B1 mutations does not affect growth of the Schizosaccharomyces pombe prp10-1 strain; Table S1: Features of the MDS patients included in this study; Table S2: Primers used in this study. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Angelos Constantinou (IGH, Montpellier) for providing the pCMV-FLAG-SF3B1 wt and pCMV-FLAG-SF3B1 K700E plasmids containing an optimized version of HsSF3B1 [19], Tokio Tani (Kumamoto University, Japan) for providing the Schizosaccharomyces pombe wild-type and prp10-1 mutant strains, as well as pREP41-HA-SAP155 plasmid [35], the vectorology core facility Vect'UB in Bordeaux for the production of lentival particles, Aurélie Chauveau, Valoris Le Brun, and Lydie Lecucq in the assistance with NGS experiments, and Marie-Bérengère Troadec for critical reading of the manuscript. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by la Ligue contre le Cancer (Grant to D.B.).T.B. was funded by the Brittany Region and the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.3389/fbioe.2021.623886
|
Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells
|
<jats:p>Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (<jats:italic>KLF4</jats:italic>, <jats:italic>OCT4</jats:italic>, <jats:italic>NOTCH1</jats:italic>, <jats:italic>SOX2</jats:italic>, <jats:italic>NANOG</jats:italic>, <jats:italic>LIN28a</jats:italic>, <jats:italic>CMYC</jats:italic>), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (<jats:italic>NRF1</jats:italic>, <jats:italic>HIF1</jats:italic>α, <jats:italic>PPARGC1A</jats:italic>, <jats:italic>ERR</jats:italic>α, <jats:italic>PKM</jats:italic>, <jats:italic>PDK1</jats:italic>, <jats:italic>LDHA, NFKB1, NFKB2, RELA, RELB, REL</jats:italic>) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (<jats:italic>Nestin</jats:italic>, <jats:italic>MAP2</jats:italic>, <jats:italic>TUBB3</jats:italic>, <jats:italic>ALDH1L1</jats:italic>, <jats:italic>GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2</jats:italic>) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.</jats:p>
|
[
{
"section_content": "Nowadays, alternative sources of potent stem cells are of great interest and human amniotic fluid could be an attractive option.Stem cells isolated from amniotic fluid display several key characteristics that are essential for therapeutic applications.Amniotic fluid stem cells (AFSC) possess the ability to self-renew, differentiate into cell lineages from all three germ layers, they do not form teratomas in vivo and have low immunogenicity (Bossolasco et al., 2006;De Coppi et al., 2007;Roubelakis et al., 2007;Da Sacco et al., 2011).These cells are autogenous to the fetus and semiallogeneic to each parent and are said to be more potent than stem cells from other sources, such as bone marrow, umbilical cord blood, endometrium and other (Yan et al., 2013;Bonaventura et al., 2015;Alessio et al., 2018).Although AFSCs are somewhat similar to pluripotent stem cells, they are still considered as multipotent stem cells, and one approach to improve the plasticity and applicability of AFSCs could be by using small molecules.The use of small molecules is a relatively new technique of cell reprogramming and transdifferentiation (Kim et al., 2020).Nuclear transfer, transcription factor transfection, mRNA based reprogramming methods face many challenges in time and yield efficiency, complexity of delivery and the risk of integrating exogenous genetic material.Meanwhile, small molecules are inexpensive and easy to apply and control time, concentration and combination wise, they usually easy to manufacture and have a long shelf life.In addition, small molecules are cell permeable, nonimmunogenic and can be prescribed to patients as drugs to promote endogenous cell repair and regeneration (Baranek et al., 2017;Ma et al., 2017).Our study was designed to determine how our selected epigenetically active compounds affect stem cell characteristics, such as surface marker and pluripotency associated gene expression, as well as what effect small molecules of interest have on metabolic phenotype and neurogenic differentiation of AFSCs.The aim of this study was to investigate whether uncomplicated and short-term treatments with small molecules improve stem cell characteristics and also provide an improved differentiation efficiency of AFSCs toward neurogenic lineage.We investigated the impact of the following small molecules on primary stem cell lines: HDAC inhibitors trichostatin A (TSA), sodium butyrate (NaBut) and multifunctional molecules of natural origin retinoic acid (RA) and vitamin C (vitC).We demonstrated that the concentrations and combinations of small molecules do not have a cytotoxic effect on AFSCs, but they do affect gene expression patterns with an increased expression of pluripotency markers and neurogenesis associated transcription factors (OCT4, NANOG, LIN28a, CMYC, NOTCH1, SOX2), although at protein level the changes are mild except for significant downregulation of Notch1.Also, small molecules affect the expression of surface markers (SSEA4, CD117, TRA-1-81, CD105).Treated stem cells with combinations of small molecules showed altered metabolic profile as evident by the changes in mitochondrial and glycolytic activity and expression of genes involved in cellular metabolism (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, and LDHA) and NF-κB pathway (NFKB1, NFKB2, RELA, RELB, REL).To test the small molecule combination treatments, we examined the neurogenic differentiation potential of AFSCs and showed that adding a pre-induction step of small molecule treatment improved secretion levels of BDNF and the expression of tested neurogenic genes (Nestin, MAP2, TUBB3, ALDH1L1, GFAP) and genes of several ion channels (CACNA1D, KCNJ12, KCNJ2, KCNH2).In summary, this study demonstrates that short treatments with small molecule combinations could be used as pre-induction steps to improve differentiation efficiency. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Human amniotic fluid samples were obtained by amniocentesis from healthy women (age average-39 years) who needed prenatal diagnostics, but no abnormalities were detected by genetic and karyotype analysis (protocols approved by the Ethics Committee of Biomedical Research of Vilnius District, No 158200-123-428-122).AFSC were isolated using two-step isolation protocol as previously described (Tsai et al., 2004;Savickiene et al., 2015).Isolated cells were maintained in DMEM media, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, Thermo Fisher Scientific).To observe cells in culture, phase contrast microscope (Nicon Eclipse TS100) was used. ",
"section_name": "Isolation and Expansion of Human Amniotic Fluid Stem Cells",
"section_num": null
},
{
"section_content": "AFSCs were characterized by the expression of their surface markers.Cells were collected and washed twice with phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA).A total of 6•10 4 cells/sample were resuspended in 50 µl of PBS with 1% BSA and incubated with the following mouse anti-human antibodies: phycoerythrin (PE) conjugated CD166 (Biolegend) TRA-1-60 (Invitrogen), fluorescein isothiocyanate (FITC) conjugated CD34, CD73, CD90, CD105 (Biolegend), Alexa Fluor R 488 conjugated CD31, HLA-ABC, HLA-DR (Biolegend), allophycocyanin (APC) conjugated CD44, CD117, CD146, SSEA4 (Biolegend), TRA-1-81 (Invitrogen).Appropriate mouse isotype controls were used, such as IgG1-PE (Biolegend), IgM-PE (Invitrogen), IgG1-FITC, IgG2a-FITC (Biolegend), IgG1-Alexa Fluor R 488, IgG2a-Alexa Fluor R 488 (Biolegend), IgG1-APC, IgG3-APC (Biolegend), IgM-APC (Invitrogen).Labeled cells were incubated in the dark at 4 • C for 30 min, then washed twice with PBS with 1% BSA and then analyzed.For intracellular staining cells were washed with PBS and fixed using 2% paraformaldehyde at RT for 10 min.After washing step cells were permeabilized with 0.1% Triton X-100 in PBS/1% BSA solution at RT for 15 min.After centrifugation cells were resuspended in permeabilization solution and incubated for 30 min at 4 • C in the dark with the following mouse anti-human antibodies: Alexa Fluor R 488 conjugated Sox2 (Biolegend), Alexa Fluor R 647 conjugated Nanog, Oct4 (Biolegend), unconjugated Lin28a and c-Myc.Goat anti-mouse IgG (H + L) Highly Cross-Absorbed Alexa Fluor R 488 (Invitrogen) conjugated secondary antibodies were used to label Lin28a and c-Myc samples for another 30 min at 4 • C in the dark.After incubations cells were washed with PBS/1% BSA and analyzed.The measurements were performed using BD FACSCanto TM II flow cytometer with BD FACSDIVA TM software (BD Biosciences).Ten thousand events were collected for each sample. ",
"section_name": "Flow Cytometry Analysis",
"section_num": null
},
{
"section_content": "To determine origin of AFSCs, karyotype analysis was performed and only samples with confirmed male fetus were chosen.AFSCs were treated with 0.6 µg/ml of colchicine for 3 h, then cells were collected by trypsinization and incubated with pre-warmed hypotonic 0.55% KCl solution for 30 min.at 37 • C. The cells were fixed with fixation solution consisting of methanol and glacial acetic acid (3:1) at -20 • C for 30 min., centrifuged and fixation repeated two more times.A few drops of cell suspension were transferred on a microscope slide and stained with 5% Giemsa (Merck) solution in Sorensen's phosphate buffer for 5 min.Slides were analyzed at a magnification of 1,000× (Nikon ECLIPSE E200).Only well-spread metaphases with 42 ± 1 chromosomes were used for the analysis. ",
"section_name": "Karyotyping AFSCs",
"section_num": null
},
{
"section_content": "Cells were seeded into 48-well plates and treated with different concentrations and combinations of epigenetically active compounds [Decitabine, Trichostatin A (TSA), Sodium butyrate (NaBut), Retinoic acid (RA) and Vitamin C (VitC)], three replicates each.After incubation periods the medium was removed from the cells and to measure cell viability 100 µl of 0.2 mg/ml MTT reagent in PBS (Sigma-Aldrich) was added to each well and then incubated for 1.5 h at 37 • C. The precipitate was dissolved in 200 µl 96% ethanol and optical density (OD) of each well was measured using spectrophotometer Infinite M200 Pro (Tecan) at 570 and 630 nm wavelength.Cell viability (in percent) was calculated as the ratio between ODs (570 and 630 nm) of treated samples and untreated control. ",
"section_name": "Treatment With Agents and MTT Assay",
"section_num": null
},
{
"section_content": "To differentiate AFSCs toward neurogenic lineage, several induction protocols were used.Induction medias consisted of DMEM/F12 with GlutaMax TM , 3 µM of RA (Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin and either 1% of N2 supplement, 2% B27 supplement or their combination (Gibco, Thermo Fisher Scientific).Cells were induced to differentiate at 40-60% confluence with media changes every 3 days.To test the effect of epigenetically active molecules, a pre-treatment step was added.AFSCs were treated for 24 h with 25 µg/ml VitC, 20 nM TSA, 0.1 µM RA in DMEM/F12 supplemented with 5% FBS and 100 U/ml penicillin and 100 µg/ml streptomycin.Then the pretreatment media was changed to differentiation medias.I-1% of N2 and 3 µM of RA, II-2% B27 and 3 µM of RA, III-1% of N2, 2% B27 and 3 µM of RA, VI-preinduction, then 1% of N2 and 3 µM of RA, V-preinduction, then 2% B27 and 3 µM of RA, VI-preinduction, then 1% of N2, 2% B27 and 3 µM of RA.Morphological changes were observed with phase contrast microscopy.More information on optimization of neurogenic differentiation media and differentiation induction design is provided in Supplementary Figures 1,2. ",
"section_name": "Neurogenic Differentiation",
"section_num": null
},
{
"section_content": "Total RNA from AFSCs was isolated using TRIzol R reagent (Applied Biosystems) as recommended by the manufacturer.For the gene expression analysis, cDNA synthesis was performed using SensiFAST TM cDNA Synthesis Kit (Bioline).RT-qPCR was performed with SensiFAST TM SYBR R No-ROX Kit (Bioline) on the Rotor-Gene 6000 thermocycler with Rotor-Gene 6000 series software (Corbett Life Science).GAPDH gene was used for normalization of the mRNA amount and the relative gene expression was calculated using Ct method (compared to untreated or undifferentiated control).The list of primers (Metabion International AG, Planegg-Steinkirchen, Germany) is provided in Supplementary Material. ",
"section_name": "Gene Expression Analysis",
"section_num": null
},
{
"section_content": "Control and treated cells were characterized by their energetic profile which was determined using Seahorse XFp Extracellular Flux Analyzer and Cell Energy Phenotype Test Kit (Agilent Technologies, CA, United States).Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured simultaneously firstly without inhibitors (the baseline), and then after the addition of oligomycin and FCCP (inhibitors of the electron transfer chain).After the measurements cell protein lysates were obtained using RIPA buffer (150 mM NaCl, 10 mM EDTA, pH 8.0, 10 mM Tris, pH 7.4, 0.1% SDS, 1% deoxycholate, 1% NP-40 in PBS, pH 7.6) and protein concentrations were measured with spectrophotometer Infinite M200 Pro (Tecan, Switzerland) using DC Protein Assay (Bio-Rad Laboratories, CA, United States).OCR and ECAR values were normalized to the total amount of protein in each well.Cell energy phenotype as the ratio of normalized OCR to normalized ECAR (OCR/ECAR) and the metabolic potential as the percentage increase of stressed OCR over baseline OCR and stressed ECAR over baseline ECAR, were assessed from Cell Energy Phenotype Test data using Seahorse Wave Desktop Software. ",
"section_name": "Extracellular Flux Analysis",
"section_num": null
},
{
"section_content": "To assess neurogenic differentiation, AFSCs were seeded on coverslips and cultivated as undifferentiated control or differentiated toward neurogenic lineage using I-VI protocols for 14 days.Cells were with 4% paraformaldehyde for 15 min at RT and permeabilized using 0.2% Triton X-100 in PBS for 20 min.at RT.After washing with PBS, cells were blocked using 1% BSA/10% goat serum/PBS for 30 min at 37 • C. Detection of NCAM: cells were incubated with primary mouse antibodies against NCAM1 (15 µg/ml) (Abcam) and secondary goat anti-mouse IgG (H + L) Highly Cross-Adsorbed, Alexa Fluor R 594 antibodies (1:400) (Invitrogen) for 1 h each at 37 • C in a humid chamber.Detection of TUBB3 and Vimentin: cells were incubated with FITC conjugated rabbit anti-beta III tubulin antibodies (1:100) (Abcam) or Alexa Fluor R 488 conjugated rabbit anti-Vimentin antibodies (1:150) (Abcam) for 1 h at 37 • C in a humid chamber.F-actin was labeled with Alexa Fluor R 594 Phalloidin (Thermo Fisher Scientific) for 30 min RT.After each incubation coverslips were washed several times with PBS/1% BSA.Nuclei were stained using 300 nM DAPI solution (Invitrogen) for 10 min RT.Coverslips were mounted using Dako Fluorescent Mounting Medium (Agilent Technologies) and analyzed using Zeiss Axio Observer (Zeiss) fluorescent microscope, ×63 magnification with immersion oil and Zen BLUE software. ",
"section_name": "Immunofluorescence Analysis",
"section_num": null
},
{
"section_content": "ELISA method was used to determine the secreted levels of BDNF in conditioned media of control AFSCs and AFSCs differentiated toward neurogenic lineage.Cells were seeded in wells of 6-well plates and were cultivated as control or induced to differentiate toward neurogenic lineage using I-VI protocols.Cells were washed with PBS and NutriStem R hPSC XF medium (Biological Industries) was added for 3 days, after which both the cells and the media were collected separately.BDNF detection kit was purchased from R&D Systems and all procedures were carried out according to the manufacturer instructions.Plates were read with spectrophotometer Infinite M200 Pro (Tecan).AFSC protein lysates were obtained using RIPA buffer (150 mM NaCl, 10 mM EDTA, pH 8.0, 10 mM Tris, pH 7.4, 0.1% SDS, 1% deoxycholate, 1% NP-40 in PBS, pH 7.6) and protein concentrations were measured with Infinite M200 Pro using DC Protein Assay (BioRad Laboratories).BDNF values were normalized to total amount of cell protein. ",
"section_name": "Enzyme-Linked Immunosorbent Assay of BDNF",
"section_num": null
},
{
"section_content": "All experiments were repeated at least 3 times (three independent experiments).Data were expressed as mean values with SDs.For statistical analysis, repeated measures analysis of variance with Tukey's multiple comparison test or two-way analysis of variance with Bonferroni post-test in the GraphPad Prism Software (La Jolla, CA) was used. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "AFSCs were isolated from amniocentesis samples of healthy donors at midsecond (17-20 weeks) trimester of gestation.Stem cells were successfully isolated by a two-step protocol and when grown in culture demonstrated spindle-shaped morphology (Figure 1A).To confirm the fetal origin of the cells karyotype analysis was performed (Figures 1B,C).Only the samples with male fetus were chosen and Y chromosome was present in all instances.Relative expression of stemness markers CMYC, NOTCH1, OCT4, NANOG, LIN28a, KLF4, and SOX2 was also detected by RT-qPCR (Figure 1D).AFSCs were strongly positive (over 90%) for mesenchymal cell surface markers, such as CD44, CD90, and CD105 and immunological marker HLA-ABC, negative (<5%) for hematopoietic marker CD34, endothelial marker CD31 and immunological marker HLA-DR (Figure 1E) as measured by flow cytometry. ",
"section_name": "Characterization of Isolated AFSCs",
"section_num": null
},
{
"section_content": "Small molecule treatments were tested for their toxicity as single molecules (Figure 2A) and in combinations (Figure 2B) using MTT assay.We investigated the effects of HDAC inhibitors TSA and NaBut, and multifunctional molecules RA and vitC on cell viability of AFSCs every 24 h for 4 days.The concentrations for used small molecules were chosen regarding previous studies (Huangfu et al., 2008;Esteban et al., 2010;Han et al., 2010;Hou et al., 2013).The results revealed that these compounds affect cell viability but do not induce cellular cytotoxicity at given concentrations and combinations.When treated with small molecule compounds separately cell viability did not decrease lower than 90% and treatment with NaBut even stimulated cell proliferation since cell viability improved during treatment time.After treating AFSCs with small molecule combinations a gradual decrease in cell viability was observed and after 4 days it reached around 65-75%.We also tested the effects of DNMT inhibitor decitabine (Supplementary Figure 4) as a single compound and in combinations with other small molecules.However, due to insufficient efficacy, we did not use these combinations in further studies. AFSCs were treated with combination A (25 µg/mL vitC, 20 nM TSA, 0.1 µM RA) and B (25 µg/mL vitC, 10 µM NaBut, 0.1 µM RA) for 24 and 96 h and some variations in the expression of genes and corresponding proteins that are involved in maintaining pluripotency was observed (Figure 3).Incubation with small molecule combinations induced changes of gene expression in an adversative manner (Figure 3A).Expression levels of OCT4, NOTCH1, SOX2, and NANOG were higher with combination A after 24 h when compared to 96 h of incubation and combination B shows upregulated expression after 96 h of treatment.Expression of LIN28a increased only with combination B and CMYC showed slight upregulation with A combination after 96 h and with combination B at both time points.This indicates that even though these combinations differ by only one substance with similar function (TSA in combination A and NaBut in combination B), it can influence the cellular response and gene expression activation differently.The results of protein expression changes induced by combination A and B treatments reveal different response to small molecule stimulation.The changes in expression level of Oct4, Nanog and c-Myc are quite mild with only c-Myc displaying significant decrease with combination B after 96 h treatments when comparing to 24 h incubation.Sox2 is upregulated only with B combination after 96 h of treatments and Lin28a is downregulated except for combination A treatment after 96 h, although these expression changes are insignificant.Notch1 is significantly downregulated with both combination treatments at both time points.Differences in gene and protein expression patterns after small molecule treatments show that stimulation of gene transcription and protein translation are regulated differently. The effect of A and B combinations on typical mesenchymal and pluripotent stem cell surface marker and MHC class I and II surface receptor expression were tested by flow cytometry (Figure 4).After 96 h of treatment with small molecule combinations A and B the expression of CD90, CD166, HLA-ABC, TRA-1-61, and TRA-1-81 surface markers remained similar to untreated control.Compared to control cells, treatment with combination A did not have much effect on the expression of CD44, CD73, CD146, SSEA4, and HLA-DR, while with combination B the expression level of these markers decreased 10-15%, except for HLA-DR when a slight increase of approximately 10% was noted.The expression of CD105 decreased when cells were treated with both combinations and the expression of CD117 decreased with combination B, but an increase of 10% was observed with combination A. ",
"section_name": "Evaluation of Small Molecule Effects on AFSCs",
"section_num": null
},
{
"section_content": "After the effects of small molecule treatments were established, the changes in metabolic profile of AFSCSs were determined.AFSCs were treated with combinations A and B for 24 and 96 h and their mitochondrial and glycolytic activity was assessed using Seahorse extracellular flux analyzer and expression of genes associated with mitochondrial respiration, glycolysis and cellular metabolism was examined (Figure 5).The rates of oxygen consumption (OCR) and extracellular acidification (ECAR) were measured simultaneously under basal and induced stressed (after addition of electron transport chain (ETC) inhibitors oligomycin and FCCP) conditions (Figure 5A).The data suggest that 96 h treatments result in more energetically active cells compared to untreated cells or 24 h incubations with small molecule combinations.Analysis of gene expression (Figure 5B) reveal that genes related to glycolysis (ERRα, PKM, PDK1, LDHA) are upregulated more significantly than genes linked to mitochondrial respiration (NRF1, HIF1α, PPARGC1A) and after 96 h treatments.Also, genes encoding transcription factors of NF-κB signaling pathway (NFKB1, NFKB2, RELA, RELB, REL) were examined (Figure 5C) and upregulated expression after treatments with both combinations was registered, especially with combination A after 96 h of treatment.The functional analysis and gene expression results suggest that small molecule combination treatments stimulate AFSCs to enter a more energetically active state. ",
"section_name": "Changes in Metabolic Phenotype",
"section_num": null
},
{
"section_content": "AFSCs were induced to differentiate toward neurogenic lineage and gene expression was analyzed at early (day 7) and late (day 15) differentiation stages (Figure 6).Several differentiation medias were used containing such supplements as 1% N2 with 3 µM RA (I), 2% B27 with 3 µM RA (II) or their combination with 3 µM RA (III).Taking into account that combination C stimulates SOX2 and NOTCH1 expression after 24 h of treatment and other investigated genes are upregulated (except for CMYC) at given time point, we selected A combination and 24 h treatment for the preinduction step, especially since SOX2 and NOTCH1 are associated with neurogenesis.After 24 h the media with preinduction compounds was changed to differentiation medias with 1% N2 with 3 µM RA (IV), 2% B27 with 3 µM RA (V) and N2/B27 combination with 3 µM RA (VI).Morphological (Supplementary Figure 3) and gene expression changes under different conditions were observed.A preinduction step upregulated expression of astrocyte markers ALDH1L1 and GFAP when compared to differentiations without preinduction step.Neural markers MAP2 and TUBB3 reveals varied expressional changes, when VI protocol was favorable for MAP2 expression and IV protocol improved TUBB3 expression (Figure 6A).Differentiation was also assessed by examining gene expression of ion channels CACNA1D, KCNJ12, KCNJ2, and KCNH2 at late differentiation stage (Figure 6B).Comparing the gene expression between differentiation protocols with and without pretreatment step, the expression level of calcium ion channel CACNA1D was significantly upregulated when comparing protocol II and V, and protocol III and VI.Potassium ion channel KCNJ12 also showed great upregulation when preinduction stem was added to differentiation protocol, since expression level significantly increased comparing protocols I and IV, and protocols III and VI.Secretion of BDNF was analyzed at late differentiation stage (Figure 6C) and significant increase of BDNF can be observed with protocol I and IV when comparing to undifferentiated control.Neurogenic differentiation was confirmed by staining TUBB3, NCAM1, and Vimentin (Figure 7).Differentiated cells acquire more elongated morphology which is highlighted by reorganization of TUBB3, Vimentin and F-actin, and begin to form neurite growths with NCAM1 becoming more concentrated at the cell ends.Upregulation of neural marker genes and ion channel genes suggest the beneficial effect of adding small molecule treatment step to differentiation induction protocol. ",
"section_name": "Assessment of Neurogenic Differentiation",
"section_num": null
},
{
"section_content": "Ever since Takahashi and Yamanaka in 2006 discovered and developed iPSC technology (Takahashi and Yamanaka, 2006) alternative cell reprogramming approaches has been of great interest.Small molecules and their role in modifying cell fate is a rapidly developing field of study and they are an attractive alternative to viral and non-viral vectors for cellular reprogramming.One of the benefits of small molecules is their fast and mostly reversible effects that allows easy manipulation of cell treatment conditions.A vast assortment of different small molecules exists and their combination options are immeasurable. Small molecules have been widely used for differentiation induction of stem cells from various tissues of origin.Maioli et al. (2013) achieved cardiovascular phenotype of human AFSCs by using a mixture of hyaluronic, butyric, and retinoic acids.Another study used 5-azacytidine (AZA), RA, and dimethyl sulfoxide (DMSO) to induce cardiomyogenic differentiation of fetal liver-derived MSCs (Deng et al., 2016).Small molecules are also reported to facilitate transdifferentiation. TSA and AZA was used to induce hepatic differentiation with DMSO (Cipriano et al., 2017) or to enhance differentiation (Kim et al., 2016).Also, small molecules can be used to generate more specialized stem cells, such as MScs from iPSCs or ESCs.In the study by Chen et al. (2012) used TGF-β inhibitor SB431542 to initiate mesengenic differentiation and obtain MSCs.Results of previous studies reveal the broad potential of small molecules for applications in stem cell research. In this study, AFSCs were treaded with several selected small molecules and their combinations (Zhang et al., 2012;Qin et al., 2017;Yoshida and Yamanaka, 2017;De Angelis et al., 2018) and changes in cell and stem cell characteristics were observed.The tested combinations include HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaBut) that are shown to promote somatic cell reprogramming (Mali et al., 2010;Huang et al., 2011).It was proposed that HDAC inhibitors could replace CMYC and KLF4 factors during induction of pluripotency (Kretsovali et al., 2012).Vitamin C (vitC) was chosen based on evidence suggesting its role in DNA demethylation as a cofactor of Ten-Eleven Translocation (TET) enzymes (Esteban and Pei, 2012;Stadtfeld et al., 2012).By enhancing TET1 activity, vitC was found to indirectly promote reprogramming efficiency (Esteban et al., 2010;Blaschke et al., 2013).Retinoic acid (RA) signaling was linked to pluripotency reprogramming when enhancing effect of overexpression of RA receptor α (RARα) and γ (RARγ) was observed in iPSC derived using Yamanaka factors (Wang et al., 2011).And it has been demonstrated that the effects of RA are tightly related to used concentration.De Angelis et al. ( 2018) concluded that low concentrations of RA (0.5 µM) positively affected pluripotency state of hiPSCs while higher concentrations of RA (1.5 and 4.5 µM) promoted differentiation and downregulation of pluripotency markers OCT4, NANOG, and REX1.It was also demonstrated that a combination of retinoic acid and vitamin C act synergistically and boost cell reprogramming to pluripotency (Alexander et al., 2016). Our study was focused on short-term treatments with small molecules on AFSCs.When cells were treated with small molecules individually, stable or upregulated cell viability was observed.Many papers report tendencies that agree with our results with decitabine (Pang et al., 2019), TSA (Han et al., 2013), NaBut (Panta et al., 2019), RA (Pourjafar et al., 2017), vitC (Markmee et al., 2019).Decitabine is a wellknown hypomethylating agent which incorporates itself into host DNA, but is used as a drug to treat myelodisplastic syndrome and acute myeloid leukemia.And even though Öz et al. (2014) demonstrated that incorporation rates of decitabine at 100 nM are not genotoxic in myeloid leukemia cells, the effects of higher concentrations of decitabine and how its incorporation affect healthy cells are still unknown.Taking that into consideration, combinations with decitabine were excluded from further experiments. Certain gene expression and surface marker expression are an important characteristic of stem cells.In our experiments we obtained an interesting pattern of gene expression after treatment with small molecule combinations A (vitC, TSA, RA) and B (vitC, NaBut, RA).The obtained results show that combination C leads to more upregulated expression levels of OCT4, NOTCH1, SOX2, and NANOG after 24 h compared to 96 h treatments, while combination B promote higher upregulation after 96 h when compared to 24 h treatments.Increased gene expression of LIN28a and was observed with combination B and CMYC displayed slight upregulation with combination A after 96 h and combination B at both time points.Such differences in gene expression upregulation could be linked with different HDAC inhibitors present in used combinations of small molecules.Differential effects of TSA and NaBut were reported in breast cancer cells (Kalle and Wang, 2019), but more information on how these molecules influence pluripotency associated gene activation in lacking.At protein level investigated markers show different response to small molecule treatments.Oct4, Nanog, and c-Myc show minor changes in the expression levels with significant decrease of c-Myc with combination B comparing 96-24 h treatment and significant downregulation of Notch1 is induced by small molecule treatments. A set of surface markers related with multipotent mesenchymal stem cells were examined.The most profound effect was noted after 96 h incubation with B combination when significant decrease of CD positive cells were observed for such markers as CD44, CD73, CD105, CD117, CD146, SSEA4.Na et al. (2015) report that CD105 expression is regulated by Notch signaling pathway.They demonstrate that inhibition of Notch signaling leads to reduction of CD105 expression, in contrast, our results show NOTCH1 upregulation after 96 h with B combination, thus meaning that other mechanisms are in play of regulating the expression of this surface marker.One more factor that can influence expression of CD105 is the concentration of serum in culture media.A study by Mark et al. (2013) demonstrated that only 50% of bone marrow stem cells cultured under serum-free conditions were positive for CD105, when nearly 100% of cells cultured in media containing serum were positive for CD105. Cellular metabolism and metabolites are closely involved in regulating epigenetic state of the cell and epigenetics have a crucial role in regulating metabolic profile of the cell.Also, it has been established that during somatic cell reprogramming upregulation of genes associated with glycolytic metabolism occurs even before the expression of pluripotency genes (Folmes et al., 2011;Cao et al., 2015).In our experiment treatment with small molecule combinations result in more energetically active cells as evident by increased oxygen consumption (OCR) and extracellular acidification (ECAR) rates after 4 days.Also, both oxidative phosphorylation (NRF1, HIF1α, PPARGC1A) and glycolytic (ERRα, PKM, PDK1, LDHA) genes are upregulated after 96 h treatments.Small molecules are known to improve stem cell function by regulation of cellular metabolism (Son et al., 2018).And several studies suggest that the initiation phase of cell reprogramming could be characterized by a phenomenon called transient hyper-energetic metabolism, which is a hybrid of high OxPhos and high glycolysis.Cacchiarelli et al. (2015) report metabolism-related genes show peak levels of expression at an early stage of reprogramming.Also, Kida et al. (2015) have linked ERRα and ERRγ expression to iPSC generation.Their study identified transient upregulation of ERRα and ERRγ, which are typically expressed in oxidative tissues, in the early stages of reprogramming and showed that the transient OxPhos burst and increased glycolysis are essential for reprogramming (Kida et al., 2015).Additionally, we examined gene expression of NF-κB family, since it has been associated with cellular metabolism.It was shown that depletion of IKKa or RelB, which are important components of NF-κB signaling pathway, resulted in reduced mitochondrial content and function (Bakkar et al., 2012).Also, it was determined that p65 or RelA, another protein of NF-κB family, promotes the mitochondrial expression of cytochrome c oxidase 2 assembly factor and oxidative phosphorylation (Mauro et al., 2012).It was also proposed that glycolysis stimulates IKK/NF-κB activity, as revealed by reduced IKK activity in the presence of a glycolytic inhibitor and increased IKK activity after GLUT3 expression (Kawauchi et al., 2008).Increased levels of OCR and ECAR as well as upregulated expression of genes associated with cellular metabolism would suggest that small molecule treatments increase energetic needs of AFSCs, which could be similar to those linked to early reprogramming stages. Because of an increase in SOX2 and NOTCH1 expression, A combination was chosen for a preinduction step in neurogenic differentiation induction.SOX2 is a transcription factor known for its role in neuroectoderm development (Sarlak and Vincent, 2016) and NOTCH1 is a receptor and its signaling is crucial in neurogenesis (Lathia et al., 2008), thus the upregulation of these genes could potentially promote neurogenic differentiation of AFSCs.To test this theory, neurogenic differentiation was induced by using two commercially available supplements N2 and B27 and with the addition of RA (Janesick et al., 2015), without a preinduction step and with a 24 h preinduction, then expression of neurogenic differentiation associated genes was analyzed.In our experiment small molecule pretreatment improved the expression of astrocytic genes ALDH1L1 and GFAP, neural markers MAP2 benefited from preinduction step when induced with N2/B27 supplements and TUBB3 showed increased expression after small molecule treatment when induced using N2 supplement.Also, the preinduction step proved to benefit the expression levels of ion channel genes.Small molecule treatment boosted expression of calcium ion channel CACNA1D when differentiation was induced with B27 and N2/B27 supplements.While potassium ion channel gene KCNJ12 showed an increase in expression when N2 and N2/B27 supplements were used for neurogenic differentiation.Neurogenic differentiation was confirmed by significantly increased levels of secreted BDNF when using N2 supplement alone or with the preinduction step.Also, morphological changes were visualized by staining TUBB3, NCAM1, Vimentin and F-actin.Differentiated AFSCs become more elongated as evident by reorganization of structural cytoskeleton proteins TUBB3, Vimentin and F-actin, and possible neurites begin to form as NCAM1 is becoming more concentrated at the cell ends.Using small molecules to improve neurogenic differentiation by targeting signaling pathways (Song et al., 2018) or epigenetic regulation (Xu et al., 2019) has been reported, but evidence of similar effect when using the same small molecules as used in this study is scarce. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "To conclude, small molecules are an important tool in cell biology, cancer research, they are investigated as potential drugs for many disorders.Also, small molecules can be used to enhance cellular properties of stem cells.Our investigated small molecule combinations upregulated genes related to pluripotency, treatments lead to more energetically active cells, and pretreatment step deemed beneficial for neurogenic differentiation.A vast selection of small molecules exists and many different combinations could lead to different effects.Many studies are focused on establishing these effects and bringing small molecules closer to clinical use. ",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "We thank Natalija Krasovskaja (Vilnius University, Faculty of Medicine) for providing samples of human amniotic fluid and Veronika Dedonytė (Vilnius University, Life Sciences Center) for assistance in karyotype analysis.We acknowledge the COST Action CA17116 International Network for Translating Research on Perinatal Derivatives into Therapeutic Approaches (SPRINT), supported by COST (European Cooperation in Science and Technology) for the development of ideas and valuable discussions in scientific research in the field of perinatal derivatives. ",
"section_name": "ACKNOWLEDGMENTS",
"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 studies involving human participants were reviewed and approved by the Ethics Committee of Biomedical Research of Vilnius District.The patients/participants provided their written informed consent to participate in this study. AZ, VB, and RN: conception and design, collection of data, analysis, and interpretation, and writing original draft of manuscript.DŽ and IJ: data collection and interpretation. 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/fbioe.2021.623886/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": "The studies involving human participants were reviewed and approved by the Ethics Committee of Biomedical Research of Vilnius District.The patients/participants provided their written informed consent to participate in this study. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "AZ, VB, and RN: conception and design, collection of data, analysis, and interpretation, and writing original draft of manuscript.DŽ and IJ: data collection and interpretation. 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/fbioe.2021.623886/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:",
"section_num": null
}
] |
10.22625/2072-6732-2022-14-4-26-37
|
Immune system disturbances after a new coronavirus infection COVID-19
|
<jats:p>During the pandemic, a large number of works devoted to COVID infection have appeared, which have made it possible to understand the pathogenetic features of the disease and to accumulate significant clinical experience. However, the question remains about the degree of participation of humoral and cellular (primarily T-cell) immunity in the mechanisms of immune defense and resistance to COVID-19, the individual features of the immune response in different subjects. Post-COVID syndrome is currently a separate diagnosis included in the ICD-10 International Classification of Diseases, but the long-term effects of the SARS-CoV-2 on the immune system are not yet well established. At the same time, a long-term increased activity of the immune system can contribute to the development of autoimmune reactions. The review of the literature presents the results of studies, mainly devoted to immune system disorders after COVID infection. The changes in subpopulations of T-lymphocytes, B-lymphocytes, their functional properties, the complement system and other factors of humoral immunity, as well as the production of a number of cytokines are described. Data on immune disorders in post-COVID syndrome and during the convalescence period are presented in detail. Since COVID-19 is an infection that has a significant impact on the hematopoietic system and hemostasis, special attention is paid to the category of subjects with an increased risk of severe complications. Among the latter are elderly patients, persons suffering from diabetes mellitus, oncological and oncohematological patients, in particular, with hematopoietic and lymphoid tissue neoplasia, such as chronic lymphocytic leukemia, lymphoma, multiple myeloma. The review pays special attention to the peculiarities of the course of COVID-19 and the response of the immune system to vaccination in patients with oncohematological diseases. Deciphering the significance of individual links of cellular and humoral immunity in patients who have undergone COVID-19 is an important issue in creating effective vaccines and improving therapeutic methods.</jats:p>
|
[
{
"section_content": "Пандемия COVID-19, начавшись в 2019 г. и идя на спад в большинстве регионов, все же продолжается до настоящего времени и, по всей видимости, SARS-CoV-2 не исчезнет из человеческой популяции.За время пандемии появилось большое число работ, посвященных COVID-инфекции, позволивших достаточно глубоко понять патогенетические особенности течения заболевания, накопить значительный клинический опыт, но остаются и нерешенные вопросы, требующие дальнейшего изучения. Как и в случае других вирусных инфекций, иммунный контроль над SARS-CoV-2 достигается за счет согласованного взаимодействия гуморального и клеточного иммунитета [1][2][3].Однако при достаточно подробно изученном патогенезе заболевания остаётся открытым вопрос о степени участия гуморального и клеточного (прежде всего, Т-клеточного звена) иммунитета в механизмах иммунной защиты и невосприимчивости к инфицированию вирусом, особенностях иммунного ответа отдельных лиц. Известно также, что в течение 6 и более месяцев после перенесенного заболевания могут наблюдаться проявления постковидного синдрома при сохранении дисбаланса в иммунологических показателях [4][5][6].Постковидный синдром в настоящее время является самостоятельным диагнозом и включен в Международную классификацию болезней МКБ-10, а пациенты с постковидным синдромом нуждаются в его лечении.Если клинические постковидные проявления служат предметом активного изучения в последние годы, то отдаленные последствия воздействия вируса SARS-CoV-2 на иммунную систему еще недостаточно хорошо установлены.При этом длительно поддерживаемая повышенная активность иммунной системы может способствовать развитию аутоиммунных реакций и осложнений. В связи с вышесказанным актуальным является дальнейшее изучение особенностей состояния иммунной системы как в период заболевания, так и в постковидном и поствакцинальном периоде. ",
"section_name": "Введение",
"section_num": null
},
{
"section_content": "Коронавирусная инфекция COVID-19 является острым заболеванием, вызываемым РНКгеномным вирусом рода Betacoronavirus семейства Coronaviridae, который, попадая в организм через дыхательные пути, первоначально взаимодей-ствует с Толл-рецепторами (TLR) эпителиальных клеток бронхов, альвеол, кишечника, клеток сосудистого эндотелия с развитием эндотелиальной дисфункции, а также с рецепторами ангиотензинпревращающего фермента 2 (АСЕ2).Ключевым условием инфицирования SARS-CoV является связывание шиповидного белка вируса с рецепторами АСЕ2, имеющимися в большинстве тканей организма человека, включая мембраны пневмоцитов, энтероцитов, эндотелиальных клеток сосудов, гладкомышечных клеток в большинстве органов и даже в клетках коры и ствола головного мозга, гипоталамуса, следствием чего, вероятно, являются полиорганные поражения при инфекции COVID-19.Всё это приводит к разносторонним реакциям со стороны иммунной системы, при тяжелом течении -к образованию большого количества биологически активных веществ, так называемому «цитокиновому шторму», эндотелиальным васкулопатиям с микрокровоизлияниями и микротромбозами, дисрегуляцией свертывания крови, возникновению острого респираторного дистресс-синдрома.Поскольку развитие системного воспалительного ответа (цитокинового шторма, гипериммунных реакций) может вести к полиорганному поражению, существует мнение о том, что тяжесть течения COVID-19-инфекции обусловлена не столько самим вирусом, сколько гиперреактивностью иммунной системы [1,2,7]. Вопросу образования специфических антител к детерминантам SARS-CoV-2, отвечающих за противовирусный иммунитет, уделено достаточно большое внимание, и этот вопрос широко освещен в литературе.Однако в процессах противовирусной защиты важную роль играют не только антитела, но и факторы клеточного иммунитета.Для устранения начавшегося инфекционного процесса большое значение имеют Т-лимфоциты.Активированные Т-клетки-киллеры в ответ на SARS-CoV-2 обладают способностью предотвращать распространение вируса из верхних дыхательных путей.Следовательно, эффективность такого ответа будет определять тяжесть симптомов, вирусную нагрузку и скорость передачи инфекции.Предполагается, что развитие лейкопении и лимфопении является способом ускользания вируса от иммунного ответа хозяина [8,9].Показано, что инфекция COVID-19 приводит к истощению эффекторных Т-клеток, что отрицательно сказывается на их противовирусной защите от SARS-CoV-2, а функциональное истощение цитотоксических лимфоцитов (ЦТЛ) коррелирует с прогрессирова-нием заболевания [10,11].Эта утрата функции эффекторных Т-клеток происходит либо благодаря повышенной экспрессии ингибирующих рецепторов на поверхности Т-клеток из-за активности цитокинов, либо из-за снижения количества регуляторных Т-клеток.В то же время имеются данные о том, что истощение CD8+ Т-клеток в процессе развития инфекции SARS-CoV не влияло на клиренс или репликацию вируса, тогда как истощение CD4+ Т-клеток приводило к патологическим последствиям, представленным интерстициальным пневмонитом и задержкой клиренса инфекции из легких.Было обнаружено, что такое истощение CD4+ Т-клеток и задержка элиминации вируса из организма связаны со снижением выработки цитокинов, нейтрализующих антител и рекрутирования лимфоцитов в легочную ткань [12], что подчеркивает влияние COVID-19 на CD4+ Т-лимфоциты.Установлено, что общее количество NK и CD8+ Т-клеток у пациентов с инфекцией SARS-CoV-2 было заметно снижено.Функция NK и CD8+ Т-клеток была истощена при повышенной экспрессии CD94/NKG2A, ингибиторного рецептора, который распознает антигены класса HLA-E и экспрессируется на NK-клетках и субпопуляции Т-лимфоцитов.Важно отметить, что у выздоравливающих пациентов количество NK и CD8+ Т-клеток восстанавливалось на фоне сниженной экспрессии NKG2A.Эффективная терапия COVID-19 сопровождалась увеличением числа Т-клеток, ЦТЛ и NK-клеток [13]. Данными различных авторов было показано, что CD4+ Т-клетки формируют основную ось адаптивного иммунного ответа в отношении SARS-CoV-2 и способны дифференцироваться в несколько подтипов.Эти различные физиологические эффекторы позволяют CD4+ Т-клеткам выполнять различные роли в адаптивном иммунном ответе на SARS-CoV-2.О наличии классического противовирусного Th1-клеточного ответа при COVID-19 впервые сообщили еще в январе 2020 г., а в более поздних работах дополнительно описаны прямые противовирусные эффекты и профили экспрессии белков IFN-γ, ФНО и ИЛ-2 [14,15]. Вирус-специфические популяции Т-фолликулярных хелперных клеток (Tfh) участвуют в ряде функций, включая хелперную функцию в отношении В-клеток при инфекции SARS-CoV-2.Более высокие уровни циркулирующих клеток Tfh связаны с более легкой степенью тяжести заболевания SARS-CoV-2 и имеют решающее значение для стимуляции продукции В-клеток [15].Однако связь между тяжестью заболевания, количеством клеток Tfh и титрами нейтрализующих антител сложна и до конца не изучена Ключевыми эффекторами, продуцируемыми в ответ на ряд вирусных инфекций, являются CD8+ Т-клетки, а их повышенные уровни в периферической крови коррелируют со снижением тяжести заболевания.Роль данных клеток заключается в усилении цитотоксичности за счет экспрессии таких молекул, как IFN-γ, гранзим B, перфорин и CD107a.Подобно тому, как это показано для CD4+ T-клеток, было продемонстрировано существование SARS-CoV-2-специфических циркулирующих CD8+ эффекторных T-клеток, проявляющих иммунологическую память и перекрестную реактивность.В частности, описано наличие таких CD8+ Т-клеток уже через 1 день после появления симптомов, а также у 70% пациентов в периоде выздоровления [16]. У пациентов с COVID-19 по сравнению со здоровым контролем наблюдается иммунный ответ в виде выработки широкого спектра воспалительных цитокинов с участием Th1, Th2, Th17, а также противовоспалительных цитокинов.Цитокины, хемокины и факторы роста, отличающие заболевание средней тяжести от тяжелого или критического или коррелирующие с показателями тяжести заболевания легких, по крайней мере, по данным одного из исследований, включают IL1α, IL1-RA, MIP1α, MCSF, GCSF, HGF, IL-6, IL10, IL7 и IP10 [17].Уровень IP10 (CXCL10) также повышен при острых респираторных вирусных инфекциях, вызванных родственными коронавирусами SARS и MERS [18]. При тяжелой инфекции SARS-CoV необычная конверсия В-лимфоцитов в макрофагоподобные клетки приводит к неспособности гуморального и клеточного звена иммунной системы своевременно реагировать на нейтрализацию вирусной инфекции.В долгосрочной перспективе ответ В-клеток имеет решающее значение для развития надежной защиты хозяина от повторного заражения.Продолжительные исследования показали, что содержание В-клеток памяти, направленных против шиповидного белка SARS-CoV-2, увеличивается в интервале от 1 до 8 месяцев после инфицирования, и большинство из них представляют собой клетки, продуцирующие IgG, а меньшая часть -IgA [19]. Как и для большинства вирусов, ответ В-клеток на SARS-CoV-2 контролируется клетками Tfh и в итоге приводит к клональной пролиферации В-клеток с образованием субпопуляций плазмобластов для выработки специфических нейтрализующих антител.В-клеточный ответ на SARS-CoV-2 может происходить особенно быстро, во многих случаях без необходимости созревания аффинности, а в литературе имеются противоречивые сообщения относительно корреляции В-клеточного ответа с тяжестью заболевания [20]. Патогенные уровни иммунных комплексов (ИК) обычно наблюдаются при некоторых заболе-ваниях, таких как сывороточная болезнь или вирусные заболевания, при которых сообщалось об отложении ИК и чрезмерных воспалительных реакциях.Участие ИК в патогенезе тяжелых случаев инфекции SARS-CoV-2 подтверждается поздним развитием цитокинового шторма, особенно эндотелиита и диссеминированного микрососудистого тромбоза, поражающего многие органы, включая сердце, головной мозг и почки [21,22].Белки комплемента связываются с эритроцитами, которые затем переносят ИК к фагоцитам в печени и селезенке.Роль и механизмы активации путей комплемента и свертывания крови в условиях инфекции SARS-CoV-2 противоречивы, но несомненны [2,23].Кроме того, было обнаружено, что при активации зависимых от IL-6 воспалительных иммунных реакций система комплемента влияет на гуморальный иммунитет, восприимчивость к заболеванию и клинический исход, связанные с инфекцией SARS-CoV-2.Так, активация белка комплемента С3 происходит на ранних этапах и способствует протромботическим и провоспалительным состояниям, кульминацией которых является поражение органов-мишеней, наблюдаемое при тяжелом течении COVID-19.При этом высокие уровни маркеров воспаления коррелировали с патологически низкими уровнями компонентов комплемента С3 и С4.Сделан вывод, что эти низкие уровни являются результатом чрезмерной активации каскада комплемента с последующим развитием легочного поражения, что в итоге приводило к снижению уровня С3 [24]. Имеются сообщения о том, что инфекция SARS-CoV-2 вызывает типы иммунного ответа, которые связаны со значительным увеличением числа мегакариоцитов [25].Ряд цитокинов, которые активируются при инфекции SARS-CoV-2, также влияют на мегакариоцитопоэз.Одним из таких цитокинов является интерлейкин-6 (ИЛ-6), уровень которого повышен у пациентов с тяжелым течением инфекции COVID-19 [2,26].ИЛ-6, в свою очередь, повышает уровень тромбопоэтина (ТПО), основного фактора роста мегакариоцитов, ответственного за продукцию мегакариоцитов и тромбоцитов.При связывании со своим рецептором c-Mpl ТПО стимулирует продукцию клеток-предшественников мегакариоцитов в дополнение к ускорению их пролиферации.Ингибирование ТПО было связано с нейтрализацией тромбоцитоза и предотвращением тромбоэмболии, являющейся результатом повышения уровня ИЛ-6.Также было установлено, что у пациентов с положительным результатом теста на COVID-19 наблюдается активация IL-9 [27].Повышенные уровни этого цитокина также вызывают усиление передачи сигналов пути JAK-STAT, что приводит к более высоким концентрациям мегакариоци-тов [28].Цитокины, активируемые при инфекции SARS-CoV-2 и приводящие к увеличению содержания мегакариоцитов, включают, помимо ИЛ-6 и ИЛ-9, гранулоцитарно-макрофагальный колониестимулирующий фактор (GM-CSF), интерферонгамма (IFNγ) и IL-1b.Повышенные уровни ТПО и ИЛ-6 могут стимулировать передачу сигналов по пути JAK-STAT для увеличения выработки мегакариоцитов и тромбоцитов.С учетом повышенной стимуляции передачи сигналов JAK-STAT у пациентов с COVID-19, терапевтические стратегии для уменьшения клинических проявлений COVID-19 могут включать использование ингибиторов JAK [26]. ",
"section_name": "Характеристики изменений в иммунной системе при COVID-19",
"section_num": null
},
{
"section_content": "Одной из важнейших особенностей коронавирусной инфекции является полиорганность поражения, что во многом определяет долгосрочные последствия у значительной части пациентов с COVID-19 после выздоровления.Термин «Длительный COVID» (или «Long-COVID») начал широко использоваться с 2020 г. для описания различных симптомов, сохраняющихся либо проявляющихся через несколько недель или месяцев после заражения SARS-CoV-2, независимо от вирусного статуса.Позже появилось официальное название PASC (post-acute sequelae of COVID-19).Сегодня это уже самостоятельный диагноз, включенный в Международную классификацию болезней МКБ 10 с шифром U09.9, -состояние после COVID-19 (Post-COVID-19 condition).Частота постковидного синдрома точно неизвестна, но большинство исследователей сходятся во мнении, что у каждого пятого человека есть симптомы, которые сохраняются через 5 недель, а у каждого десятого есть симптомы в течение 12 недель или дольше после острой стадии COVID-19 [29].Данные наблюдений показали, что 20% людей, перенесших коронавирусную инфекцию, страдают от долгосрочных симптомов, длящихся до 12 недель и в 2,3% случаев дольше [30].Продолжаться такое состояние может 6, 12 месяцев, а у части наблюдаемых и больше года.Так, встречаются сведения, что более 70% людей, госпитализированных с коронавирусной болезнью, полностью не восстановились, и через 12 месяцев после выздоровления при этом отмечалась возможность длительного персистирования вируса в некоторых тканях [6,34]. Пациенты с постковидным синдромом нуждаются в соответствующем лечении и реабилитации после перенесенного COVID-19, и современная система здравоохранения во многих странах предус матривает создание так называемых постковидных центров -отделений по лечению постковидного синдрома с целью оказания специали-зированной помощи пациентам и сохранения качества жизни. Предметом дискуссии является вопрос, связано ли наличие симптомов с персистенцией вируса или же вирус запускает внутренние механизмы повреждения органов и тканей, тем более что у большей части пациентов с синдромом продолжающегося COVID-19 результаты ПЦРисследований отрицательны, что указывает на микробиологическое выздоровление.Вероятно, этиология Long-COVID носит многофакторный характер.Большую часть постковидных проявлений в настоящее время связывают не с активностью вируса, а с аутоиммунными реакциями в отношении центральной и автономной нервной системы, сердечно-сосудистой системы и т.д.[4].А длительное сохранение иммунного дисбаланса, по всей видимости, может служить предиктором остаточных поражений [5] Отдаленные последствия COVID- [32]. Было показано, что у больных, перенесших коронавирусную инфекцию легкой и тяжелой степени, отмечаются значительные изменения состава циркулирующих иммунных клеток, которые сохраняются длительное время (не менее 3-9 месяцев после острой фазы заболевания) и сходны с таковыми при аутоиммунных заболеваниях.Во-первых, это увеличение доли провоспалительных Т-хелперов 17 (Th17), циркулирующих в крови в течение длительного времени после заболевания, и снижение противовоспалительных Т-регуляторных клеток (Т reg ), сопровождающееся изменением баланса Th17/T reg [33], что характерно для широкого спектра аутоиммунных заболеваний [34].Во-вторых, наблюдался дисбаланс между субпопуляциями Т-фолликулярных хелперных (Tfh) клеток и регуляторных (Tfr) клеток [35], что также отмечается при аутоиммунных проявлениях, связанных с образованием аутоантител [36]. В-третьих, в периферической крови больных COVID-19 обнаружено повышенное количество короткоживущих высокодифференцированных CD8+ Т-лимфоцитов, сохраняющихся длительное время [5,33], что свидетельствует о продолжающихся процессах дифференцировки и циркуляции этих клеток с выходом из лимфоидной ткани.Эти клетки, по-видимому, больше не связаны с элиминацией SARS-CoV-2 и его антигенов, но могут быть еще одним признаком развития аутоиммунной реакции.Отмечено увеличение доли зрелых эффекторных клеток с фенотипом CD45RA+CCR7в периферической крови реконвалесцентов, а в случае CD8+ цитотоксических Т-клеток это также было связано с увеличением доли зрелых перфорин-и гранзим-экспрессирующих лимфоцитов, которые способны продуцировать TNFα и IFNγ [37].При этом наличие легочных осложнений было тесно связано с увеличением в циркуляции доли короткоживущих эффекторных CD27-CD62L-CD8+ Т-клеток, а также зрелых Т-клеток, способных продуцировать перфорин, гранзим В и IFNγ.Отмечено также, что через 1,5-2,5 месяца после острой фазы COVID-19 в периферической крови выздоровевших пациентов сохранялся высокий уровень гранзим+CD8+ Т-лимфоцитов [38]. Приводятся экспериментальные доказательства того, что у лиц с бессимптомным течением инфекции SARS-CoV-2 развивается вирус-специфический Т-клеточный ответ, который по силе неотличим от такового у пациентов с симптомами, но более полноценен функционально, характеризуясь усиленной секрецией Т-хелперами 1 типа (Th1) цитокинов IFNγ и IL-2, которая связана с пропорциональной и скоординированной продукцией провоспалительных (IL-6, TNF-α, IL-1β) и противовоспалительных (IL-10) цитокинов.Обсуждается значение этих результатов для патологии и разработки вакцин [39]. Результаты исследования ответа Т-клеток периферической крови COVID-19 реконвалесцентов в отдаленные сроки (20-26 недель) показали, что экспрессия маркера активации PD-1 на CD4+ T-клетках обследованной когорты сохранялась значительно сниженной по сравнению с таковой у здоровых доноров.Относительное содержание Tc2, Th2 и Th17 в когорте COVID-19 реконвалесцентов было сопоставимо с таковым у здоровых, но уровень Tc17 был выше.Также у COVID-19 реконвалесцентов было меньше Т-клеток, продуцирующих IFN-γ, но больше Т-клеток, секретирующих IL-2.В целом, Т-клеточный ответ в когорте COVID-19 реконвалесцентов в отдаленном периоде претерпел как фенотипические, так и функциональные изменения.Однако после клинического выздоровления специфические для SARS-CoV-2 Т-клеточные формы ответа могут сохраняться по крайней мере в течение 6 месяцев, что может быть полезным в борьбе с повторным заражением [40]. Специфичные для SARS-CoV-2 Т-клетки памяти, вероятно, могут быть критически важными для долгосрочной иммунной защиты от COVID-19.Авторы сопоставили функциональный и фенотипический ландшафт специфичных для SARS-CoV-2 Т-клеточных реакций у неинфицированных лиц, членов семьи заболевших и лиц с острой инфекцией или COVID-19 в стадии выздоровления.Специфичные для острой фазы SARS-CoV-2 Т-клетки обладали фенотипом высокоактивированных цитотоксических клеток, который коррелировал с различными клиническими маркерами тяжести заболевания, в то время как специфичные для фазы выздоровления от инфекции SARS-CoV-2 Т-клетки были полифункциональными и демонстрировали фенотип клеток памяти.Важно отметить, что специфичные к SARS-CoV-2 Т-клетки были обнаружены у серонегативных членов семьи заболевших и выздоравливающих лиц с бессимптомным и легким COVID-19 в анамнезе.Данные показывают, что SARS-CoV-2 вызывает широко направленные реакции Т-клеток памяти, что в дальнейшем может препятствовать возникновению повторных эпизодов инфекции COVID-19 [41]. ",
"section_name": "Период реконвалесценции и постковидный синдром",
"section_num": null
},
{
"section_content": "Антигенная мимикрия SARS-COV-2 с тканями человека привлекла внимание недавно.Было обнаружено, что несколько антигенов тканей хозяина проявляли сильную реактивность с антителами к SARS-CoV-2; это показало, что не только антигены легочной ткани, но и несколько других тканевых антигенов перекрестно реагировали с белками SARS-CoV-2 [42].В частности, взаимосвязь между шипом SARS-CoV-2, ядерными белками и аутоиммунными белками-мишенями высветила возможность аутоиммунной реакции против тканей человека, приводящей к обширному повреждению органов, тканей и клеток, наблюдаемому при тяжелых инфекциях SARS-COV-2.Чрезмерное реагирование потенциально может привести к аутоиммунной реакции против белков и тканей хозяина, таких как легочные сурфактантные белки, соединительная ткань, дыхательная, пищеварительная, сердечно-сосудистая и нервная системы [43].Стоит отметить, что недавнее исследование показало, что риск развития тяжелого COVID-19 среди пациентов с аутоиммунными заболеваниями не имеет различий по сравнению с таковым у лиц без аутоиммунной патологии [44].Это может быть связано с одновременным применением ими иммунодепрессантов, которые играют роль в модуляции цитокинового шторма в тяжелых случаях COVID-19. ",
"section_name": "Аутоиммунитет",
"section_num": null
},
{
"section_content": "Перенесенная коронавирусная инфекция, наряду с проблемами, связанными непосредственно с воздействием вируса SARS-CoV-2 и его последствиями, способствует выявлению скрытых, клинически неманифестных проблем в организме.Так как COVID-19 является инфекцией, оказывающей значительное влияние на кроветворную систему и гемостаз, то существуют категории лиц с повышенным риском развития тяжелых осложнений, в том числе пожилые больные, страдающие сахарным диабетом, онкологические и онкогематологические пациенты.Особенно это касается пациентов, имеющих опухолевые заболевания кроветворной и лимфоидной тканей, такие как хронический лимфолейкоз (ХЛЛ), лимфома, множественная миелома, острый лейкоз [45][46][47]. Существуют данные о том, что пожилой возраст является фактором повышенного риска смертности от COVID-19, а также инфекций, вызванных вирусами MERS-Cov и SARS, вирусом гриппа.Вероятной причиной этого могут быть некоторые возрастные хронические заболевания и/или более низкий уровень иммунитета.Кроме того, известно, что старение влияет на количественное содержание и функциональные особенности CD4 + T-клеток, CD8 + T-клеток, B-клеток [48].Возрастное снижение клонального разнообразия Т-клеток и В-клеток связано с нарушением ответа на вирусные инфекции, такие как грипп, а избыточная продукция цитокинов Тh2 может привести к формированию длительного провоспалительного иммунного ответа и, следовательно, возможно, способствовать ухудшению результатов лечения. Женщины с коронавирусом имеют более низкие показатели госпитализации и смертности, чем мужчины [49].Результаты метаанализа, выполненного H. Peckham et al. [50], также показали, что мужской пол, по-видимому, является фактором риска смертности от COVID-19.Ранее сообщалось о половых различиях как в адаптивной, так и врожденной иммунной системе, и они могут объ-яснить преимущество женщин при коронавирусной инфекции.В рамках адаптивной иммунной системы мужчины характеризуются меньшим количеством CD8 + T-клеток, CD4 + T-клеток и снижением продукции B-клеток по сравнению с женщинами [51].Более того, поскольку некоторые важные иммунорегуляторные гены расположены на Х-хромосоме, пациенты-женщины могут иметь преимущество из-за более высокой экспрессии TLR7. У части людей есть приобретенная невосприимчивость к SARS-CoV-2, вероятнее всего, обусловленная Т-клетками, которые ранее «активировались» другими коронавирусами и сохранили иммунологическую память.Так, в одном из исследований были обнаружены SARS-CoV-2-реактивные CD4+ Т-клетки у 40-60% людей, не имевших в анамнезе данных заболеваний или контактов с вирусоносителями, что свидетельствует о перекрестно-реактивном распознавании Т-клеток между циркулирующими коронавирусами «сезонных простудных заболеваний» и SARS-CoV [2,52].Поскольку Т-клетки являются инструментом «долговременной памяти» иммунной системы и могут оставаться в крови в течение длительного времени после заражения, то, возможно, этим объясняется устойчивость к вирусу COVID-19, поскольку в результате контакта с другими коронавирусами в прошлом иммунная система может сохранять способность распознавать вирусы с похожими поверхностными белками. Целый ряд факторов, ослабляющих иммунную защиту, влияет на развитие осложнений с фатальным исходом при инфицировании COVID-19 у онкологических больных.К таким факторам относятся особенности самих онкологических заболеваний и побочные эффекты проводимой терапии, включая развитие лейкопении и нейтропении, прямое иммуносупрессивное действие ряда применяемых средств терапии.Может играть роль неблагоприятный общесоматический статус у многих онкологических пациентов и сопутствующая патология, развитие мукозитов и нутритивная недостаточность [46,53]. Учитывая более пожилой возраст, сопутствующие заболевания и иммунную дисфункцию, пациенты с хроническим лимфолейкозом (ХЛЛ) могут подвергаться особенно высокому риску заражения и неблагоприятных исходов, связанных с COVID-19.Проведено международное исследование, включавшее пациентов с ХЛЛ, у которых был диагностирован симптоматический COVID-19, средний возраст на момент постановки диагноза COVID-19 составил 70,5 лет.В когортах пациентов, как не получающих терапию, так и на фоне лечения были одинаковые показатели госпитализации в отделения интенсивной терапии (35% против 36%), интубации (33% против 25%) и смертности (37% против 32%).Лечение ХЛЛ с помощью ингибиторов тирозинкиназы Брутона на фоне COVID-19 не влияло на выживаемость (летальность 34% против 35%), хотя большинство пациентов во время курса лечения COVID-19 получали лечение ингибиторами тирозинкиназы Брутона.Эти данные свидетельствуют о том, что категория пациентов с ХЛЛ, госпитализированных с COVID-19, независимо от фазы заболевания или статуса лечения, подвержена высокому риску смерти [53]. Затяжная инфекция COVID-19 является новой проблемой для пациентов с лимфомой или иммунодефицитом.Проведено изучение продолжительности пребывания в стационаре по поводу COVID-19 среди пациентов с лимфомой.В исследование включены 111 взрослых пациентов с лимфомой, поступивших для лечения COVID-19 в марте и апреле 2020 г.Медиана возраста составила 65 лет (диапазон от 19 до 92 лет), у 94 пациентов (85%) была В-клеточная неходжкинская лимфома.В течение 12 месяцев до госпитализации по поводу COVID-19 79 пациентов (71%) получали лечение, из них 63 (57%) -анти-CD20-терапию.При медиане наблюдения 191 день (3-260) 6-месячная общая выживаемость составила 69%.Авторами установлено, что возраст ≥ 70 лет, рецидивирующая/ рефрактерная лимфома и недавнее проведение анти-CD20-терапии являются факторами риска увеличения продолжительности госпитализации по поводу COVID-19 и смерти пациентов с лимфомой [47]. Проведенные исследования показали, что наибольшая степень риска осложнений COVID-19 характерна для больных, получающих противоопухолевую лекарственную или лучевую терапию в период инфицирования, а также при сочетании лейкопении с низким уровнем иммуноглобулинов.К группе повышенного риска относятся также пациенты, перенесшие трансплантацию гемопоэтических стволовых клеток менее 6 месяцев назад, лица, получающие иммуносупрессивную терапию длительное время, больные пожилого возраста, больные с рецидивом или прогрессией опухоли [46].Связанные с переносимой инфекцией вынужденные перерывы в противоопухолевой терапии, отказ от применения высокодозной химиотерапии, ограничения в использовании МКА и лучевой терапии дополнительно играют негативную роль в исходе сочетанной онкопатологии и COVID [45]. Инфекция COVID-19 приводит как к острой смертности, так и к длительной персистенции вируса и рецидивам заболевания у пациентов с гематологическими злокачественными новообразованиями, но факторы, способствующие персистированию инфекции в этой популяции, неизвестны. В публикации 2022 г. C. Lee et al. [54] [55]. Также было проведено исследование 551 пациента с гематологическими злокачественными новообразованиями: лейкемией, лимфомой и множественной миеломой с определением титров анти-SARS-CoV-2 IgG антител и их нейтрализующей активности через 1 и 3 месяца после первоначальной вакцинации.По сравнению с группой контроля (лиц без злокачественных новообразований системы крови) у пациентов с гематологическими злокачественными новообразованиями титры антител через 1 и 3 месяца были снижены.Кроме того, у пациентов с гематологическими злокачественными новообразованиями была заметно снижена вирус-нейтрализующая способность антител -26,3% через 1 месяц и 43,6% через 3 месяца, несмотря на положительную сероконверсию, частота которой составляла 51,5% и 68,9% соответственно.Лица контрольной группы имели 93,2% и 100% нейтрализующую способность через 1 и 3 месяца соответственно.Пациенты с лейкемией, лимфомой и множественной миеломой в процессе наблюдения имели одинаково сниженный ответ на вакцинацию.Лечение ингибиторами тирозинкиназы Брутона, венетоклаксом, ингибиторами фосфоинозитид-3-киназы, анти-CD19/CD20 терапией и ингибиторами CD38/антигена созревания В-клеток существенно замедляло ответ на вакцину, тогда как однокомпонентные иммуномодулирующие агенты не давали такого эффекта [56]. Результаты нескольких исследований показали, что пациенты с гематологическими злокачественными новообразованиями, особенно лимфоидной природы, имеют более низкий ответ антител на вакцины против COVID-19, чем население в целом, и могут оставаться незащищенными от инфекции SARS-CoV-2 [57].Cамый плохой ответ отмечается у пациентов с ХЛЛ.В исследовании, включавшем 67 пациентов с гематологическими злокачественными новообразованиями, только у 23% пациентов с ХЛЛ выработались антитела к SARS-CoV-2 после полной вакцинации по сравнению с 61,1% пациентов с другими гематологическими злокачественными новообразованиями, хотя 69,2% пациентов с ХЛЛ не получали активной противоопухолевой терапии.Терапия ибрутинибом и антителами к CD20, по-видимому, также сильно влияет на выработку антител в результате вакцинации [58].Сходные результаты получены и в другом исследовании, показавшем, что почти у всех обследованных пациентов с лимфомой, получавших препараты моноклональных антител против CD20 отдельно или в сочетании с химиотерапией, не наблюдалось серопозитивного ответа после вакцинации против COVID-19.Количество CD19+ клеток у преобладающего большинства пациентов с лимфомой было низким.В течение периода наблюдения в 3 месяца только у одного из этих пациентов развилась легкая форма инфекции COVID-19.Результаты убедительно свидетельствуют о том, что у большинства пациентов с низким количеством лимфоцитов CD19+, вызванным лечением моноклональными антителами против CD20, не достигается серопозитивный ответ после вакцинации против COVID-19 [59]. В ходе проспективного пролонгированного мультиомного исследования, в котором приняли участие 398 взрослых, было проведено сравнение особенностей выработки антител и специфического Т-клеточного ответа на 35-й и 120-й день после вакцинации против SARS-CoV-2 у пациентов с гематологическими злокачественными новообразованиями и контрольной когорты (лиц без гематологических злокачественных новообразований).Хотя процент сероконверсии на 35-й день в контрольной группе (98%) был выше по сравнению с пациентами с миелоидными (82%) и лимфоидными (48%) новообразованиями, на 120-й день сущес твенное увеличение сероконверсии наблюдалось в когорте пациентов как с миелоидными (97%), так и с лимфоидными (66%) новообразованиями.Примечательно, что специфические к белку «шипа» CD4+ и CD8+-клетки и когорте в лимфоидными (71%/31%) новообразованиями и контрольной группе (74%/42%) были сопоставимы.Таким образом, предоставлены убедительные доказательства индуцированной вакциной иммуногенности у большинства пациентов с гематологическими злокачественными новообразованиями, а кинетика сероконверсии и клеточные ответы имеют решающее значение для определения того, у каких пациентов с гематологическими злокачественными новообразованиями выработается иммунитет [60]. В связи с этим пациентам с онкологическими заболеваниями особенно важно соблюдать все меры по профилактике COVID-19 и проводить вакцинацию при первой же возможности.При этом следует учесть, что для многих пациентов с опухолями, преимущественно лимфоидной природы (особенно при моноклональных гаммапатиях), выработка антител нарушена, и это предрасполагает как к более тяжелому течению, так и к более частым случаям реинфекции [55]. ",
"section_name": "COVID-19 у отдельных категорий больных и онкогематологических пациентов",
"section_num": null
},
{
"section_content": "Несмотря на значительное число работ, посвященных данной проблеме, иммунный ответ при COVID-19 еще не полностью изучен, а окончательные данные о постинфекционном иммунитете отсутствуют.Дальнейшие исследования и точные научные данные будут иметь важное значение для более глубокого понимания патологических процессов при инфицировании вирусом SARS-CoV-2, для планирования и практики общественного здравоохранения, создания новых подходов к формированию восстановительного лечения пациентов, перенесших COVID-19. Дальнейшие исследования актуальны также для понимания механизмов иммунной защиты, разработки программ иммунореабилитации при постковидном синдроме, создания новых подходов к формированию восстановительного лечения пациентов, перенесших COVID-19.Расшифровка значимости отдельных звеньев клеточного и гуморального иммунитета является важным вопросом при создании эффективных вакцин и методов терапии. ",
"section_name": "Заключение",
"section_num": null
}
] |
[] |
10.3389/fgene.2023.1148126
|
Development and validation of an immune infiltration/tumor proliferation-related Notch3 nomogram for predicting survival in patients with primary glioblastoma
|
<jats:p><jats:bold>Background:</jats:bold> Notch receptors (Notch 1/2/3/4), the critical effectors of the Notch pathway, participate in the tumorigenesis and progression of many malignancies. However, the clinical roles of Notch receptors in primary glioblastoma (GBM) have not been fully elucidated.</jats:p><jats:p><jats:bold>Methods:</jats:bold> The genetic alteration-related prognostic values of Notch receptors were determined in the GBM dataset from The Cancer Genome Atlas (TCGA). Two GBM datasets from TCGA and Chinese Glioma Genome Atlas (CGGA) were used to explore the differential expression between Notch receptors and IDH mutation status, and GBM subtypes. The biological functions of Notch Receptors were explored by Gene Ontology and KEGG analysis. The expression and prognostic significance of Notch receptors were determined in the TCGA and CGGA datasets and further validated in a clinical GBM cohort by immunostaining. A Notch3-based nomogram/predictive risk model was constructed in the TCGA dataset and validated in the CGGA dataset. The model performance was evaluated by receiver operating curves, calibration curves, and decision curve analyses. The Notch3-related phenotypes were analyzed via CancerSEA and TIMER. The proliferative role of Notch3 in GBM was validated in U251/U87 glioma cells by Western blot and immunostaining.</jats:p><jats:p><jats:bold>Results:</jats:bold> Notch receptors with genetic alterations were associated with poor survival of GBM patients. Notch receptors were all upregulated in GBM of TCGA and CGGA databases and closely related to the regulation of transcription, protein-lysine N-methyltransferase activity, lysine N-methyltransferase activity, and focal adhesion. Notch receptors were associated with Classical, Mesenchymal, and Proneural subtypes. Notch1 and Notch3 were closely correlated with IDH mutation status and G-CIMP subtype. Notch receptors displayed the differential expression at the protein level and Notch3 showed a prognostic significance in a clinical GBM cohort. Notch3 presented an independent prognostic role for primary GBM (IDH1 mutant/wildtype). A Notch3-based predictive risk model presented favorable accuracy, reliability, and net benefits for predicting the survival of GBM patients (IDH1 mutant/wildtype and IDH1 wildtype). Notch3 was closely related to immune infiltration (macrophages, CD4<jats:sup>+</jats:sup> T cells, and dendritic cells) and tumor proliferation.</jats:p><jats:p><jats:bold>Conclusion:</jats:bold> Notch3-based nomogram served as a practical tool for anticipating the survival of GBM patients, which was related to immune-cell infiltration and tumor proliferation.</jats:p>
|
[
{
"section_content": "Results: Notch receptors with genetic alterations were associated with poor survival of GBM patients.Notch receptors were all upregulated in GBM of TCGA and CGGA databases and closely related to the regulation of transcription, protein-lysine N-methyltransferase activity, lysine N-methyltransferase activity, and focal adhesion.Notch receptors were associated with Classical, Mesenchymal, and Proneural subtypes.Notch1 and Notch3 were closely correlated with IDH mutation status and G-CIMP subtype.Notch receptors displayed the differential expression at the protein level and Notch3 showed a prognostic significance in a clinical GBM cohort.Notch3 presented an independent prognostic role for primary GBM (IDH1 mutant/wildtype).A Notch3-based predictive risk model presented Glioma is the most common of all primary central nervous system tumors.It was graded from Ⅰ to Ⅳ depending on the tumor's malignant status (Louis et al., 2021a).Glioblastoma (GBM) is the major subtype of grade Ⅳ glioma and one of the deadliest cancers, with only a 3% 5-year survival rate (Ostrom et al., 2013).Surgical resection, radiotherapy, and chemotherapy are the main methods for glioblastoma therapy.Despite advances in treatment, disease recurrence usually occurs within 6 months, and patients generally die of disease progression in a little over a year (Stupp et al., 2005).Therefore, it is urgent to explore effective tools to predict the survival of patients with GBM.Even though some prognostic biomarkers have been widely validated for predicting GBM survival, these indicators cannot fully elucidate the individual variants and benefit clinical practice well. Notch signaling is highly conserved in humans and is involved in regulating a variety of cellular processes throughout life, including cell proliferation, stem cell maintenance, cell fate decisions, and differentiation (Aster et al., 2017a).It has been identified that there are four Notch receptors (Notch receptors, Notch1-4) and five types of Notch binding ligands (Delta-like-1, -3, and -4 (DLL-1, 3, 4) and Jagged-1, and -2 (JAG-1, -2) in mammals.After Notch receptors bind to their ligands, Notch intracellular domain (NICD) is transported to the nucleus and regulates the expression of downstream genes, which contributes to the determination of cell fate (Zhou et al., 2022).The dysregulation of Notch signaling is confirmed in multiple cancers and is closely related to cancer progression (Aster et al., 2017b).Particularly in glioma, the Notch pathway participates in tumor development and growth, as well as cancer invasion and recurrence.This is due to the roles of aberrant activation of Notch components in GBM, such as overexpression of DLL-4 and JAG-1 were detected in GBM endothelial cells and promoted the maintenance and differentiation of glioma stem cells via activating the downstream Hes1 in tumor cells (Zheng et al., 2021).Furthermore, high expression of DLL-4 has been shown to be correlated with glioma angiogenesis (Nandhu et al., 2014), and Notch activation via gp130/STAT3 signaling confers resistance to chemoradiotherapy (Koerdel et al., 2021).Although the Notch pathway has been confirmed to play a critical role in glioma progression in vitro and in vivo, the values and application of Notch receptors in clinical practice for primary GBM have not yet been completely elucidated. In this study, we comprehensively explored the expression pattern and biological functions of each Notch receptor in GBM at gene and mRNA levels using TCGA and CGGA databases and validated their expression at protein level in clinical GBM samples.Moreover, the relationships between the Notch receptors and GBM subtypes were further explored to test their application for clinical classification.We also performed survival analysis to screen out the independent clinicopathologic factors with Kaplan-Meier and Cox regression analyses.A novel nomogram and prognostic risk model were constructed and validated in GBM cohorts (IDH1 mutant/wild type and IDH1 wild type) from TCGA and CGGA databases.Finally, we examined the role of Notch3 expression in tumor immune infiltration and tumor cell proliferation. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "2.1 Ethics statement and GBM samples 70 primary GBM samples with complete clinical follow-up data were collected from the department of neurosurgery of the First Affiliated Hospital of Soochow University and Sanbo Brain Hospital of Capital Medical University from 2013 to 2015 (Table S1).All the patients undergo standard therapy treatment of maximum surgical resection combined with radio/chemotherapy.Informed consent was obtained from the GBM patients for the use of brain tissue and for access to medical records for research purposes.The brain tumors were confirmed by neurosurgeons, radiologists, and pathologists based on a physical examination, neuroimaging, and histological examination.The method was in accordance with the World Medical Association's Declaration of Helsinki.The study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.The experimental protocols were also supported by the Ethics Committee of the First Affiliated Hospital of Soochow University. ",
"section_name": "Materials and methods",
"section_num": "2"
},
{
"section_content": "WHO IV primary GBM samples (IDH1-wild type and IDH1 mutant) from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases were used in this study.The mRNA sequencing data of Notch receptors and clinical patients' information were downloaded and tidied from GlioVis (http://gliovis.bioinfo.cnio.es/)(Bowman et al., 2017) and the official CGGA website (www.cgga.org.cn)(Zhao et al., 2021).Then we explored the prognostic and diagnostic values of Notch receptors in GBM, including differential expression analysis, survival analysis, and correlation analysis between Notch receptors and GBM subtypes.The mRNA expression z-scores relative to diploid samples were set at 1.5.The data was analyzed by SPSS software and the online bioinformatic analysis tools (https://www.xiantao.love/)by R (V3.6.3). ",
"section_name": "TCGA and CGGA GBM databases",
"section_num": "2.2"
},
{
"section_content": "cBioPortal (http://www.cbioportal.org/)was widely used for genetic alteration analysis based on the TCGA database (Gao et al., 2013).We included 592 GBM samples (Pan Cancers Atlas, 378 cases with complete mutation and CNA data) to analyze the different genetic alterations type of Notch receptors in GBM.Moreover, we also explored the relationship between the genetic alteration of Notch receptors and the prognosis of GBM patients with Kaplan-Meier survival analysis. ",
"section_name": "Genetic alteration analysis of notch receptors",
"section_num": "2.3"
},
{
"section_content": "We screened out the top 50 genes that were most similar to each Notch receptor in GBM from GEPIA (http://gepia2.cancer-pku.cn/)(Tang et al., 2017) (Supplementary File S1).The protein-protein interaction network was performed using the STRING database (https://string-db.org)(Szklarczyk et al., 2019) (Supplementary Figure S1).We performed and visualized the analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) by Cytoscape software. ",
"section_name": "Biological function analysis of notch receptors",
"section_num": "2.4"
},
{
"section_content": "Paraffin-embedded tissue sections were dewaxed (xylene, graded ethanol), peroxidase activity quenched (0.3% hydrogen peroxide), and antigen-retrieved; The U87 and U251 glioma cells were fixed in 4% paraformaldehyde.Then they were further blocked with 5% goat serum incubated with primary antibodies (Notch1:100, Abcam, ab52301; Notch2: 1:50, Santa Cruz Biotechnology, sc-518169; Notch3 1:50, Santa Cruz Biotechnology, sc-518169; Notch4 1: 50 dilutions, Santa Cruz Biotechnology, sc-32613, Ki67, abcam15580) at 4 °C overnight.Subsequently, specific secondary antibodies were used to incubate the sections or glioma cells for 1 hour at 37 °C.Then the sections were immersed in ABC peroxidase with diaminobenzidine (Beyotime, China) and counterstained with Mayer hematoxylin for 2 min (Beyotime, China).The U87 and U251 glioma cells were stained and covered with a DAPI solution.A microscope (Nikon, Tokyo, Japan) was used to photograph the staining signals.ImageJ Pro (Media Cybernetics, Rockville, Maryland, United States) was used by two observers (blinded to the experimental groupings) for statistical analysis. ",
"section_name": "Immunostaining",
"section_num": "2.5"
},
{
"section_content": "All GBM tissue sections were evaluated by two independent observers.The semi-quantitative evaluation for Notch receptors expression was referred to in the previous studies (Dell 'Albani et al., 2014).The percentage of positive cells in the five random areas was assessed and scored under a 400x light microscope.The frequency of Notch1-4 staining was evaluated on a scale of 0-4 (0: <5%, 1: 5%-25%, 2: 25%-50%, 3 50%-75%, 4:>75%).The expression of Notch receptors scored≤1 was the low expression, while Notch receptors scored >1 was the high expression.All areas of the specimen were examined.The score presented the predominant cell staining intensity in each case. ",
"section_name": "Evaluation of immunostaining",
"section_num": "2.6"
},
{
"section_content": "Human glioma cell lines (U87MG, U251) were purchased from the American Type Culture Collection (Virginia, USA).The U87 and U251 glioma cell lines were incubated in 10% FBS DMEM mediums.A lentiviral packaging kit was purchased from GeneChem (Shanghai, China) to generate stable Notch3knockdown (Notch3 shRNA) glioma cell lines.According to the manufacturer's protocol, before we began the knockdown, the U251 and U87 were thawed and incubated at a concentration of 5*10 5 per well (six-well plate) overnight.The next day we changed the medium and added lentiviral vectors that encoded Notch3 small hairpin RNA (shNotch3) and nontargeting shRNA (shNT) to the medium for a 3-day infection.Finally, the U251 and U87 cell lines were harvested for protein extraction.Western blot was used to detect the knockdown efficiency of Notch3.Cell proliferation was determined by Ki-67staining.The primers for shNotch3 and shNT are provided in Supplementary Materials. ",
"section_name": "Cell culture with Notch3 knockdown",
"section_num": "2.7"
},
{
"section_content": "By Kaplan-Meier method with a log-rank test, we analyzed the relationship between Notch receptors and overall survival (OS) of GBM patients (IDH1-wild type and IDH1 mutant) in TCGA, CGGA, and our clinical GBM cohort.Univariate and Multivariate Cox analysis was applied to analyze the influence of Notch3 expression on GBM prognosis along with other clinicopathologic factors (Age, Gender, IDH mutation status, chemotherapy status, radiotherapy status).The Median of the Notch3 expression level was used as the cut-off value.In all tests, p < 0.05 was defined as statistically significant.The TCGA GBM database was set as a training cohort (IDH1-wild type and IDH1 mutant, n = 247, Supplementary Table S2), and the CGGA GBM database was used for an external validation cohort (IDH1wild type and IDH1 mutant, n = 190, Supplementary Table S3).Acquired from multivariate Cox analysis, we used the independent prognostic factors to construct a nomogram and predictive risk score model to assess the GBM prognosis for 1, 2, and 3 years, respectively.The following equation was utilized to compute the predictive risk score: Y = 0.488*Notch3+0.472*age-0.998*idh-0.810*chemo-2.237*radio.The TCGA GBM database (IDH1-wild type, n = 229) and the CGGA GBM (IDH1-wild type, n = 159) were used to test the performance of the predictive risk score in IDH1wild type GBM.To determine the accuracy, reliability, and clinical benefits of the predictive risk model, time-dependent receiver operating curves (ROC), calibration curves, and decision curve analysis were utilized, respectively.All the data were analyzed and visualized using the online bioinformatic analysis tools (https://www.xiantao.love/)by R (V3.6.3). ",
"section_name": "Survival analysis, model construction and evaluation",
"section_num": "2.8"
},
{
"section_content": "GBM single-cell sequencing data (GSE57872) were used to explore the correlation between Notch3 expression and the functional state of glioma cells.All the data were analyzed and visualized on Cancer SEA (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp)(Yuan et al., 2019).The workflow has been summarized in Supplementary Figure S2. ",
"section_name": "Single-cell analysis and functional state analysis",
"section_num": "2.9"
},
{
"section_content": "The data were statistically analyzed and visualized using GraphPad Prism 8 (GraphPad, San Diego, CA, United States).Data from two groups were analyzed by Student's unpaired twosided t-test.Other statistical comparisons between more than two groups were performed using one-way ANOVA and post hoc least significant difference tests for multiple comparisons.R (V3.6.3) and SPSS software (version 24.0, IBM, SPSS, Chicago, IL) was used to perform Kaplan-Meier survival analyses with log-rank tests.Cox proportional hazards regression was used for the multivariate analysis.Receiver operating curves (ROC) analysis was performed using probabilities to calculate the area under the curve (AUC).p < 0.05 was considered statistically significant for all statistical analyses. ",
"section_name": "Statistical methods",
"section_num": "2.10"
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3"
},
{
"section_content": "To determine the genetic alteration of Notch receptors in GBM, we included 592 GBM samples with 378 mutation and CNA data in TCGA (Pan Cancers Atlas).The genetic alteration rates of Notch receptors were analyzed via cBioPortal.A total alteration rate (25/378,6.6%) of Notch receptors was detected in the GBM patients, and the alteration rate of Notch receptors ranged from 1.9% to 2.9%. As is shown in Figure 1A,7 (1.9%) GBM patients presented Notch1 genetic alterations, including missense mutation and amplification.Notch2 showed the highest rate of genetic alteration, and 11 (2.9%)GBM cases indicated missense mutation, splice mutation, truncating mutation, and amplification.Notch3 displayed the same types of genetic alterations as Notch1.There are 7 (1.9%)GBM samples that displayed missense mutation and amplification in Notch3.Only 7 GBM samples (1.9%) presented missense mutation in Notch4.Additionally, the mutation frequencies in different sites of notch receptors were close to 1% and most types of gene mutations in Notch receptors (like missense mutation) were sparsely distributed from 0 to 2000 sites of the amino acid (Figure 1B). To further explore the clinical value of genetic alteration of each Notch receptor in GBM, we performed the corresponding survival analysis.We found that GBM patients with the genetic alteration of Notch1 and Notch4 have a shorter median overall survival and median disease-specific survival than the unaltered group, especially Notch3 presented the shortest median overall survival and median disease-specific survival (Figure 1C), suggesting an essential role of Notch receptors in the GBM progression. ",
"section_name": "The alteration of notch receptors in GBM at the genetic level",
"section_num": "3.1"
},
{
"section_content": "To further examine the expression variation of Notch receptors at the mRNA level, we downloaded the mRNA-seq sequencing data from TCGA and CGGA databases.The differential expression analysis was performed to evaluate the expression patterns of Notch receptors in GBM.As is shown in Figure 2, All Notch receptors (including Notch1, Notch2, Notch3, and Notch4) presented significantly higher expression at mRNA level in GBM tissues than in normal tissues both in TCGA and CGGA databases (Figures 2A,B), suggesting that all Notch receptors might have oncogenic roles in the tumorigenesis and progression of GBM. To further explore the oncogenic roles of Notch receptors in GBM, we screened out the top 50 × 4 genes significantly associated with Notch receptors in GBM from TCGA data (Supplementary Material).The protein-protein interaction network showed that nucleic acid binding genes, including U2AF2, HNRNPM, and SETD1A, and transcription factor binding genes, such as CREBBP and SIN3A, were most related to Notch receptors (Supplementary Figure S1).We also performed GO and KEGG analysis to explore the biological functions of the top 50 genes by Cytoscape software.The results of biological process (BP) and cellular component (CC) indicated that Notch receptors were related to the positive regulation of transcription of the Notch receptor target and exerted an effect on the transcription regulator complex, as indicated by the red box (Figure 2C).Moreover, the molecular function (MF) analysis demonstrated that the function of Notch receptors was associated with proteinlysine N-methyltransferase activity and lysine N-methyltransferase activity.Notably, Notch receptors related genes were enriched in focal adhesion of tumor cells except for the classical Notch signaling pathway (Figure 2D).These findings suggested that Notch receptors were related to transcription regulation of the Notch target gene during the malignant GBM progression and expansion. ",
"section_name": "The expression patterns and biological roles of notch receptors in GBM at the mRNA level",
"section_num": "3.2"
},
{
"section_content": "GBM was divided into three subtypes, including Proneural, Classical, and Mesenchymal types, based on the pathological features, which indicated different survival of GBM patients (Wang et al., 2017).Thus, we tested whether Notch receptors were associated with different GBM subtypes in the TCGA database.As shown in Figure 3A, Notch1 in Classical and Proneural subtypes of GBM showed significantly higher expression than that in the Mesenchymal subtype, while Notch2 and Notch3 displayed high expression in Classical and Mesenchymal subtypes of GBM and low expression in Proneural subtype.Notch4 in GBM showed a notable differential expression between Classical and, Proneural subtypes. Additionally, CpG Island Methylator Phenotype (G-CIMP) is another type of GBM classification.Patients with G-CIMP tumors are usually younger at the time of diagnosis and experience significantly improved outcomes (Noushmehr et al., 2010).Thus, we examine the possible relationship between Notch receptor expression and G-CIMP types in GBM.As is indicated in Figure 3B, Notch1 and Notch3 showed increased expression in non-G-CIMP subtypes of GBM, suggesting that Notch1 and Notch3 served as important markers for GBM G-CIMP classification. Isocitrate dehydrogenase (IDH) mutation, a significant event in GBM progression, has been widely used as a diagnostic and prognostic marker for GBM patients (Yan et al., 2009).GBM patients with IDH mutation possess a relatively favorable clinical outcome.Next, we explored the possible relationship between Notch receptors and IDH mutation status in GBM from CGGA and TCGA databases.The TCGA result indicated a higher expression of Notch1 in IDH1-mutant GBM as compared to the IDH1 wild-type GBM.On the contrary, Notch3 and Notch4 showed a higher expression in IDH1 wild-type samples as compared to IDH1-mutant samples (Figure 3C).Interestingly, the Notch receptors analysis from CGGA databases presented similar results to the TCGA database for Notch1 and Notch3 (Figure 3D).Together, these data indicated that Notch1 and Notch3 were correlated with IDH mutation status, suggesting they might act as promising markers for predicting GBM survival and GBM IDH classification. ",
"section_name": "The relation between notch receptors and IDH mutational status and GBM subtypes",
"section_num": "3.3"
},
{
"section_content": "To determine the prognostic values of Notch receptors in GBM, we conducted a comprehensive Notch receptors survival analysis in GBM patients with clinical follow-up data in TCGA and CGGA databases.We found that GBM patients with high expression of Notch3 displayed a shorter survival time, while the expression of Notch1, Notch2, and Notch4 failed to show a prognostic significance in GBM (Figures 4A,B). To verify the expression and clinical prognostic values of Notch receptors in GBM at the protein level, we collected 70 cases of GBM samples with complete clinical follow-up data from our medical center.Immunohistochemical staining was used to detect the Notch receptors' expression, and the semi-quantitative assessment method was used to evaluate the levels of Notch receptors' expression.As indicated in Figures 4C-F, positive staining of Notch receptors was mainly displayed in the cytomembrane of tumor cells (red arrowhead) and minorly presented in the nucleus of tumor cells (blue arrowhead) in GBM samples.The mildly positive staining of Notch receptors (Notch2/4) was also observed in the cytomembrane of endothelial cells (green arrowhead).70 cases were divided into the high expression of Notch receptors and low expression of Notch receptors based on the Notch receptors' semi-quantitative score.The result indicated that 14% (10/70) of GBM cases presented a high expression of Notch1.Furthermore, over half of all cases displayed a high expression of Notch2 (80%, 56/70), Notch3 (51.4%, 36/70), and Notch4 (88.5%, 62/70).These data indicated that a large number of GBM samples presented a high expression of Notch receptors at the protein level, which was parallel to their expression at mRNA levels, suggesting a critical role of Notch receptors expression in the GBM initiation and progression. Similar to the mRNA expression-based survival analysis, we also performed a Kaplan-Meier analysis based on the protein expression levels of Notch receptors in GBM tissue.The results demonstrated that GBM patients with high expression of Notch3 at protein levels presented with a poor prognosis, while the expression of Notch1, Notch2, and Notch4 showed no relation to GBM patient survival, which was consistent with the results in TCGA and CGGA at mRNA levels (Figures 4C-F).Together, these data demonstrate that Notch3 may present a prognostic implication for GBM patients. ",
"section_name": "The expression patterns and prognostic values of notch receptors expression in GBM",
"section_num": "3.4"
},
{
"section_content": "To further confirm the independent prognostic role of Notch3 in primary GBM, we included age, gender, IDH mutation status, GBM subtypes, chemotherapy status, radiotherapy status, and Notch3 expression to perform Cox survival regression analysis in the TCGA database.The univariate analysis results indicated that age, IDH mutation, Notch3 expression, GBM subtypes, chemotherapy status, and radiotherapy status were significant for GBM patients' survival (Figure 5A).These variables were further included to perform the multivariate analysis.The results indicated that Notch3 showed an independent prognostic value for GBM patients.GBM patients with high Notch3 expression demonstrated a higher risk for poor survival than that with low Notch3 expression (HR = 1.63, p < 0.028, Figure 5B). Except for the Notch3 expression, the age, IDH mutation status, chemotherapy status, and radiotherapy status also presented independent prognostic values for GBM patients' survival.Thus, we further developed a practical nomogram constructed by these independent clinicopathologic variables to predict 1-, 2-, and 3-year survival probability for the GBM patients (Figure 5C).The point scale of this nomogram was used to assign points to each variable based on the results of multivariate Cox regression.With the adjusted range from 1 to 100, total scores were calculated by adding up the points of each variable.By delineating a direct line down from the total score line to the outcome line, the survival probabilities of each GBM patient at 1-, 2-, and 3-year were defined. To further explore the prognostic values of Notch3 and other independent clinicopathologic factors for GBM patients' survival, we selected these variables to construct a prognostic risk score model (PRSM) based on the analysis of multivariate Cox regression (forward likelihood ratio).The TCGA data were used as the training cohort, and the risk score of each GBM patient was calculated by combining the coefficient weighting score of each variable.We divided the TCGA GBM population into high-and low-risk score groups (Figure 5D).Kaplan-Meier survival analysis demonstrates that the GBM patients with high-risk scores presented short survival (HR = 2.31, p < 0.001) (Figure 5E). To verify the prognostic performance of the Notch3-based risk score model in the different populations, we included the CGGA as the external validation cohort and used the same formula to calculate the risk score for each CGGA patient (Figure 5F).Kaplan-Meier survival analysis presented a similar result as those observed for TCGA GBM patients (Figure 5G), suggesting that the Notch3-based risk score model may function as a valuable tool for predicting the GBM patients' survival. ",
"section_name": "Construction of a Notch3-based nomogram and risk score model to predict the GBM patients' survival",
"section_num": "3.5"
},
{
"section_content": "To further assess the predictive accuracy of the Notch3-based prognostic risk score model (PRSM) for primary GBM patients' survival, we calculated the area under the curve (AUC) using analysis of the time-dependent receiver operator characteristic curve (t-ROC).As shown in Figure 6 A, the AUC values of PRSM in the training TCGA cohort and validating CGGA cohort both exceeded 0.6, indicating that the Notch3-based prognostic risk score model (PRSM) was moderately accurate. Moreover, to assess the authenticity of the Notch3-based prognostic risk score model (PRSM), the calibration curve analysis was performed and demonstrated that survival probability at 1-,2-, and 3-year presented an optimal consistency between the prediction and observation both in the training and external validation cohorts (Figure 6B).Additionally, we also evaluated the clinical benefits of PRSM using the decision curve analysis (DCA).The results demonstrated that Notch3-based PRSM could achieve more net benefits compared with other prognostic variables for almost all threshold probabilities in both the training and validation cohorts (Figure 6C), suggesting that Notch3-based PRSM showed a better-predictive performance for primary GBM patient's survival. According to the 2021 WHO classification, only GBM with IDH1 wild type (WT) was defined as GBM (Louis et al., 2021b).To assess the performance of our Notch3-based PRSM in the IDH1-WT GBM, we excluded the previous GBM with IDH1 mutant from the GBM datasets in TCGA and CGGA and assessed the IDH1-WT GBM using Notch3-based PRSM.As is indicated by Kaplan-Meier survival analysis, the IDH1-WT GBM patients with high-risk scores presented shorter survival than that with high-risk scores (Figure 6D).Moreover, the analyses of t-ROC, calibration curve, and DCA indicated a good performance of Notch3-based PRSM in predicting IDH1-WT GBM patients' survival (Figures 6E-G).These data suggested that our Notch3-based PRSM presented a stable value in predicting the GBM patients' survival. ",
"section_name": "Evaluation of Notch3-based risk score model in primary GBM and primary IDH1wild type (WT) GBM",
"section_num": "3.6"
},
{
"section_content": "As immune-cell therapy has a positive effect on GBM clinical outcomes (Sokratous et al., 2017), we aimed to explore the correlation between Notch3 and various immune cells.Based on the TIMER, we found that arm-level gain alteration of Notch3 was significantly associated with immunological of macrophages, CD4 + T cells, and Dendritic cells (Figure 7A); while on the mRNA levels, Notch3 expression is negatively related to B cells and positively correlated to the CD4 + T cells and dendritic cells (Figure 7B), which implies that Notch3 present a promising potential in assessing the efficacy of GBM immunotherapy. ",
"section_name": "Notch3-related immune infiltrates",
"section_num": "3.7"
},
{
"section_content": "To explore the effect of Notch3 expression on the functional state of GBM, we next performed the single-cell analysis in the primary GBM dataset (GSE57872) and used CancerSEA (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp) to analyze and visualize the results.The distribution and range of Notch3 expression are shown in Figures 8A,B.We explored the possible relationship between notch3 expression and GBM phenotypes.We found that Notch3 expression is positively related to tumor proliferation (Figure 8C).To validate this relationship, we knocked down the Notch3 expression in GBM cell lines (U87MG and U251) to detect the alteration of cell proliferation by Ki-67 staining.The results demonstrated that Notch3 knockdown significantly reduced the propagation of glioma cells (Figures 8D-F), suggesting that Notch3mediated GBM progression is closely related to tumor cell proliferation. ",
"section_name": "Single-cell analysis and validation of the correlation between Notch3 and tumor proliferation",
"section_num": "3.8"
},
{
"section_content": "Notch receptors, the critical components of Notch signaling pathways, have been confirmed to participate in tumor initiation, progression, and recurrence (Aster et al., 2017b).However, the clinical values and application of Notch receptors for primary GBM have not yet been completely elucidated.In this study, we comprehensively analyzed the changes in mRNA expression and structural variation of Notch receptors using TCGA and CGGA databases and further confirmed the protein expression and clinical value in our clinical GBM database.We found that the Notch receptor with genetic alteration in GBM was correlated with poor prognosis.All the Notch receptors presented high expression at the mRNA level in GBM tissue.We pinpointed that high expression of Notch3 showed an independent prognostic value in GBM.Based on the Cox regression results, we constructed and validated a novel Notch3-based nomogram for predicting the survival of patients with primary GBM.Finally, we The expression of Notch receptors and their clinical roles in GBM remains controversial among various reports.Previous studies have shown that Notch1 displayed a high expression in GBM (El Hindy et al., 2013) and was correlated with the patient's poor prognosis (Li et al., 2011;Hai et al., 2018).This is related to the promoting effects of Notch1 in the phenotype of glioma stem cells (Wang et al., 2019;Yi et al., 2019).Furthermore, high expression of Notch1 has been observed in Classical and Proneural Subtypes of GBM (Hai et al., 2018), which was in line with our results, suggesting a critical role of Notch1 in these two GBM subtypes.Whereas in our study, Notch1 both presented a high expression in TCGA and CGGA, but it showed no relation to GBM patient survival, which may be ascribed to the different number of GBM samples in the study. Notch2 has been confirmed to play an oncogenic role in many malignancies (Xiu and Liu, 2019).However, the exact role of Notch2 in GBM has not been reported.In particular, Notch2 was reported to display a weak expression at the protein level in 11 cases of GBM samples (Dell'Albani et al., 2014), which is different from our results that over half of GBM samples (80%, 56/70) presented with a high expression of Notch2.Moreover, the high expression of Notch2 in our study was closely related to the Classical subtype of GBM and showed no association with IDH1 mutation status.These data were partly consistent with previous results that Notch signaling is highly expressed in the Classical subtype (Verhaak et al., 2010) Additionally, our results further demonstrated that the expression of Notch2 at mRNA or protein levels showed no relationship with patients' prognosis, suggesting that Notch2 may play a minor role in the GBM initiation and progression. Notch 3 was also reported to be activated in glioma and played a significant role in glioma cell proliferation, cell migration, invasion, and apoptosis based on the in vitro experiment (Alqudah et al., 2013).As GBM is the highest grade and most malignant tumor in glioma, our results were consistent with the partial phenotype of previous results.Based on the GBM single-cell analysis, we confirmed that notch3 expression was positively correlated with tumor proliferation in primary GBM samples and in vitro experiments.Furthermore, Notch3 expression was closely associated with poor-prognosis-related GBM subtypes, including IDH1 wild-type, Classical and Mesenchymal subtypes, suggesting that Notch3 may be a promising marker for GBM prognosis. Moreover, we performed the Kaplan-Meier survival analysis of Notch3 expression within TCGA and CGGA databases and validated its expression and prognostic value at protein levels in our GBM cohort.The results demonstrated that GBM patients with high expression of Notch3 indicated a shorter survival period, which was similar to the previous reports (Shen et al., 2015) that Notch3 gene polymorphism is associated with the prognosis of gliomas.Additionally, we also confirmed the independent prognostic roles of Notch3 in two primary GBM cohorts with univariate and multivariate Cox regression analysis, suggesting that notch3 may serve as a useful biomarker for primary GBM prognosis. To further explore the clinical value and application of notch3 in primary GBM, we constructed a novel nomogram using Notch3 expression and other independent clinicopathological factors based on the Cox regression analysis, which could serve as a practical tool for predicting the survival of GBM patients with considering individual variation.Furthermore, based on these independent prognostic factors, we developed and validated a prognostic risk model for GBM patients that contributes to assessing their prognosis.Time-dependent ROC curves, calibration curves, and decision curve analysis all presented that our prognostic risk models presented good accuracy and reliability, which might provide clinical benefit in assessing survival in GBM patients.Despite our prognostic risk model being established in the previous GBM cohorts (IDH1 mutant and IDH1-WT), it showed a high performance in predicting the prognosis of GBM patients with IDH-WT that was redefined as GBM in the 2021 WHO classification of CNS tumors (Louis et al., 2021b). Notch4 signaling not just affects tumor cell biological behaviors but also is responsible for tumor angiogenesis (Xiu et al., 2021).Interestingly, our data demonstrated a similar result that Notch4 showed positive staining both in tumor cells and endothelial cells, suggesting Notch4 may play dual roles respectively in tumor cells and endothelial cells.Despite that Notch 4 presented a differential expression in GBM at mRNA and protein levels.Its expression showed no clinical significance in predicting patients' survival. Notwithstanding the practicability of our findings, some limitations in our study need to be addressed.First, despite strict inclusive and exclusive criteria, selection and recall bias are unavoidable because of the retrospective design.Second, although we have included two of the large population of GBM cohorts, the prognostic model could be improved in a larger dataset to ensure its robustness in the future.Third, due to the limitation of molecular pathological diagnosis in our GBM sample and differences in clinicopathological information, some essential molecular features like IDH1 mutation status were not available in 70 clinical samples.Fourth, as we confirmed the relation between notch3 expression and tumor cell proliferation, the potential mechanisms need to be explored in the future.Anyway, this study provided a practical nomogram and prognostic risk model for assessing the survival of patients with GBM based on multi-omics and multi-database analysis. ",
"section_name": "Discussion",
"section_num": "4"
}
] |
[
{
"section_content": "The authors thank Prof Lin Zhi-xiong (Department of Neurosurgery, Sanbo Brain Hospital of Capital Medical University, Beijing, China) for his kind support in GBM samples and clinical information.The authors also thank the Helixlife Corporation for helping to visualize the data with their online bioinformatic analysis tools by R (V3.6.3). ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by the Chinese Government Scholarship from the Chinese scholarship council (CSC NO.202206920038). ",
"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 human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.The patients/participants provided their written informed consent to participate in this study. ZW performed the study concept and design; Z-QZ and G-GZ performed most of the experiments; Z-QZ and G-GZ provided acquisition, analysis, and interpretation of data and statistical analysis; G-QY, J-HH, Q-QN, and M-CZ provided technical and material support.Z-QZ and G-GZ conducted the writing, review, and revision of the paper; ZW supervised the project.All authors read and approved the final paper.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. 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/fgene.2023.1148126/full#supplementary-material ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.The patients/participants provided their written informed consent to participate in this study. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "ZW performed the study concept and design; Z-QZ and G-GZ performed most of the experiments; Z-QZ and G-GZ provided acquisition, analysis, and interpretation of data and statistical analysis; G-QY, J-HH, Q-QN, and M-CZ provided technical and material support.Z-QZ and G-GZ conducted the writing, review, and revision of the paper; ZW supervised the project.All authors read and approved the final paper.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. ",
"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/fgene.2023.1148126/full#supplementary-material ",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.1158/1078-0432.22841375.v1
|
Table S1 from Novel BCL-2 Inhibitor Lisaftoclax in Relapsed or Refractory Chronic Lymphocytic Leukemia and Other Hematologic Malignancies: First-in-Human Open-Label Trial
|
<jats:p><p>Representativeness of study participants.</p></jats:p>
|
[
{
"section_content": "Patients had disease that relapsed or was refractory/intolerant to prior standard therapy, and most patients with CLL/SLL had intermediate, high, or very high-risk disease. Patients with hairy cell leukemia (HCL) and Castleman disease-which are rare lymphoproliferative diseases-were also enrolled, and the study drug showed benefit. Overall The patients in this study were heavily pretreated, and most had high-risk disease. ",
"section_name": "Other considerations",
"section_num": null
}
] |
[] |
10.21203/rs.3.rs-1014087/v1
|
Genetic Alterations of Esophageal Squamous Cell Carcinoma in Korean Patients
|
<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background</jats:bold>:<jats:bold> </jats:bold>Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers in Korea, although its incidence is lower in Korea than in China and Japan. There are no data on genetic alterations associated with ESCC in Korea. Our study is the first report of the mutational landscape of ESCC in a Korean cohort.<jats:bold>Methods</jats:bold>: We performed whole exome sequencing analysis of 43 ESCC tumor samples and germline DNA from normal esophageal mucosa or whole blood. Using electronic medical records, the patients’ clinical characteristics were reviewed carefully.<jats:bold>Results</jats:bold>: We found a mutually exclusive mutation pattern in <jats:italic>NFE2L2</jats:italic>–<jats:italic>ZNF750</jats:italic> gene pairs. Non-silent variants of <jats:italic>KMT2D</jats:italic> (<jats:italic>MLL2</jats:italic>) were associated with a poor prognosis. An analysis to identify potentially druggable genes revealed <jats:italic>NOTCH1</jats:italic> as a potential therapeutic target. We observed a high frequency of C:G → T:A transitions regardless of smoking history. In our cohort, deconstruction of the mutation signature revealed enrichment of COSMIC single base substitution (SBS) 13, SBS 39, SBS 2, SBS 40, and SBS 1.<jats:bold>Conclusion</jats:bold>: We did not find a Korean-specific signature of genetic alterations, but our data suggest that <jats:italic>KMT2D</jats:italic> is a molecular prognostic marker. Further studies need to examine the role of <jats:italic>KMT2D </jats:italic>in ESCC prognosis due to our small sample size.</jats:p>
|
[
{
"section_content": "Worldwide, esophageal cancer causes 544,076 deaths annually and is the sixth leading cause of cancer death. 1 East Asia has the highest regional incidence rates for both men and women, in part because of the large burden in China.In 2017, there were 232,255 new cases of cancer in Korea, of which 2483 (1.1% of all cancers) were esophageal cancer. 2The crude incidence rate of esophageal cancer was 4.8 cases per 100,000 population in 2017.The age-standardized incidence rate decreased from 4.0 per 100,000 population in 1999 to 2.6 in 2017, with annual percent changes of -2.8% in men and -1.3% in women. 2 The incidence of esophageal cancer in Korea is relatively low compared with that in Japan and China, but the reasons for this are not clear. 3In 2013, 90.2% of esophageal carcinoma cases was squamous cell carcinoma (ESCC) histologically, followed by adenocarcinoma (3%). 4The proportions of localized and regional cancer tended to increase compared with that of distant cancer, and the 5-year relative survival rate of esophageal cancer improved from 14.0% in 1993-1995 to 38.0% in 2013-2017. 2][7][8][9][10] For instance, a study of ESCC in a Chinese ESCC cohort of 139 reported known mutated genes, such as TP53, PIK3CA, and NOTCH1, and previously uncharacterized mutated genes including FAT1, FAT2, ZNF750, and KMT2D. 7Song et al. reported mutual exclusivity between mutations in NOTCH1 and PIK3CA, and that patients with NOTCH1 mutations had shorter survival times than patients without mutations. 10In a whole exome sequencing (WES) analysis of 144 Japanese patients with ESCC, Sawada et al. identi ed mutations in EP300 and TET2 that correlated with a shorter survival time.8 In a WES analysis of 28 Indians with ESCC, Kiran et al. reported higher frequencies of C:G → A:T transversion and mutation signature 4 compared to smokers and non-users of tobacco. 5However, the genomic alterations associated with ESCC in Korea are not well-characterized.In this study, we used WES to characterize the mutational landscape of ESCC in Korea. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Tumor and paired normal DNA from 43 ESCC patients was subjected to WES.The mean read depth of the target regions was 134.4⊆, and 99.0% of the target bases were covered by >10 independent reads after alignment.This identi ed 6,557 variants in 4,752 genes: 2,090 silent variants (synonymous and deep intronic variants) and 4,467 non-silent variants (Fig. 1).The non-silent variants include 3807 missense, 288 nonsense, 222 frameshift, 84 splicing, 51 in-frame, eight nonstop, and seven start codon variants.We found a median of 74 non-silent variants (range 18-307) per sample with a median tumor mutation burden of 2.47 (range 0.60-10.23)non-silent variants/MB. ",
"section_name": "Results of whole exome sequencing analysis",
"section_num": null
},
{
"section_content": "Our ESCC cohort included the recurrently mutated genes TP53 (non-silent variants, 88% of samples), TTN (37%), NFE2L2 (30%), ZNF750 (23%), NOTCH1 (21%), FSIP2 (19%), KMT2D (16%), and LRP1B (16%) (Fig. 1, Supplementary Fig. S1).The top ve affected oncogenic signaling pathways were TP53 (88% of samples), NOTCH (58%), RTK-RAS (53%), Hippo (44%), and NRF2 (37%) (Supplementary Fig. S2).We identi ed NFE2L2 as a cancer driver gene with a single cluster, and all seven mutations were in that cluster based on the OncodriveCLUST algorithm run in maftools (p <0.05). 13We performed Pfam annotation using maftools, and the top ve mutated protein domains were COG5048, 7tm_1, FN3, Cadherin_repeat, and P53 (Supplementary Table S1).We observed a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs in ESCC (Fig. 2).In our cohort, NFE2L2 was mutated in 30% (13/43) of the samples and ZNF750 in 23% (10/43) (Supplementary Fig. S1B,C).(19/43), con rmed pathologically.All patients were followed until either death or the last follow-up date. ",
"section_name": "Somatic mutation spectrum of esophageal squamous cell carcinoma",
"section_num": null
},
{
"section_content": "During the follow-up period, 7 (16.3%)patients died and 13 (30.2%)suffered recurrence (Table 1). In the clinical enrichment analysis, there were no genes signi cantly associated with sex, a history of alcohol or tobacco consumption, the grade of tumor differentiation, the grade of tumor invasion, the presence of nodal metastasis, lymphovascular invasion, or perineural invasion. In the overall survival analysis of the top 30 genes, non-silent variants of KMT2D (MLL2) were associated with a poor prognosis (hazard ratio 6.18, p <0.05) (Fig. 3A).KMT2D was mutated in 16.3% (7/43) of the samples in our cohort (Supplementary Fig. S1D).The mortality rates in the mutated and non-mutated KMT2D groups were 42.9% and 11.1%, respectively (p = 0.072).In the disease-free survival analysis of the top 30 genes, non-silent variants of KMT2D (MLL2) were associated with poor disease-free survival (hazard ratio 2.07) (Fig. 3B), but this was not signi cant (p = 0.191). In the disease-free survival analysis of the top 30 genes, non-silent variants of LRP1B were associated with a greater likelihood of recurrence (hazard ratio 2.6), without statistical signi cance (p = 0.083) (Supplementary Fig. S3).The recurrence rates in the mutated and non-mutated LRP1B groups were 71.4% and 22.2%, respectively (p <0.05).LRP1B was mutated in 16.3% (7/43) of the samples in our cohort, and all non-silent variants of LRP1B were missense variants (Supplementary Fig. S1E). ",
"section_name": "Clinical and prognostic relevance of each mutation in esophageal squamous cell carcinoma",
"section_num": null
},
{
"section_content": "To identify potential druggable targets, we screened genes bearing non-silent variants detected in at least 20% of the samples against FDA-approved antineoplastic drugs in DGIdb. 14This revealed NOTCH1 as a potential therapeutic target.In our cohort, NOTCH1 was mutated in 21% (9/43) of the samples (Supplementary Fig. S1F). ",
"section_name": "Identi cation of potentially druggable genes",
"section_num": null
},
{
"section_content": "In this study, seven samples were from non-smokers.The median mutation burden was 2.47 and 2.43 non-silent variants/MB in smokers and non-smokers, respectively.We observed a high frequency of C:G → T:A transitions regardless of smoking history.Further, deconstruction of the mutation signature of our cohort revealed the enrichment of COSMIC single base substitution (SBS) 13, SBS 39, SBS 2, SBS 40, and SBS 1 (Fig. 4).We did not observe distinct signatures associated with ESCC in patients with a history of smoking or alcohol consumption. ",
"section_name": "Mutation signatures associated with smoking history",
"section_num": null
},
{
"section_content": "This study investigated the mutational landscape of ESCC in a Korean cohort.We identi ed several known and novel variants in ESCC.7][8][9]15 We identi ed several frequently mutated genes, such as TP53, TTN, NFE2L2, ZNF750, NOTCH1, FSIP2, and KMT2D, which have been reported in ESCC.Mutated genes commonly found in over 10% of the ESCC samples in previous reports and this study are described in Supplementary Table S2.Except for TTN, NFE2L2, and FSIP2, most of these genes had nonsense, frameshift, or splicing variants, suggesting their tumor suppressor roles (Fig. 1). The NRF2 pathway consisting of NFE2L2, KEAP1, and CUL3 was genetically deregulated in 37.2% of our cohort (Supplementary Fig. S1).NFE2L2 encodes a transcription factor that induces the production of a cytoprotective enzyme in response to oxidative stress, whereas the KEAP1/CUL-dependent proteasomal mechanism degrades the NFE2L2 protein under non-stressed circumstances. 16All NFE2L2 variants in our cohort involved two hot spots within the DLG and ETGE motifs that bind to KEAP1, as observed in a Japanese cohort. 8These mutations are thought to contribute to tumor development by stabilizing the NFE2L2 protein. 17There were NFE2L2 mutations in 9.6-16.7% of the cases in previous reports. 6,8,18ecently, in a Chinese cohort of 508 patients, NFE2L2 mutations were reported to be signi cantly associated with a worse prognosis of ESCC. 19But there was no signi cant association between the prognosis of ESCC and NFE2L2 mutations based on our data. ZNF750, an epidermal differentiation regulator, is thought to be an ESCC tumor suppressor gene, 7,8,15 as supported by our data, which showed that most ZNF750 variants were null variants (Fig. 1).ZNF750 is mutated in 3.9-16.7% of ESCC cases. 6,18,20In Japanese ESCC datasets, ZNF750 variants were positively associated with the APOBEC signature, 8 but not in our study (p = 0.069). We observed a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs in ESCC, although this mutual exclusivity has not been implicated in any type of cancer. 21Mutual exclusivity has been widely observed in cancer genomes and there are two major hypotheses associated with mutually exclusive mutated genes in cancers: the functional redundancy in downstream pathways and synthetic lethality hypotheses. 22Because these two genes do not share the same pathway, the mutually exclusive mutation in NFE2L2-ZNF750 gene pairs supports the synthetic lethality hypothesis. NOTCH1 is frequently disrupted by loss-of-function mutations, implying that the loss of NOTCH pathway activity is critical for the growth of tumor cells with squamous differentiation characteristics. 23NOTCH1 is mutated in about 16% of cases. 6,8,18There are several reports that NOTCH1 is associated with a poor prognosis, and its mutations are mutually exclusive with PIK3CA mutations. 5,10,24However, there was no signi cant association between the prognosis of ESCC and NOTCH1 mutations based on our data.Nevertheless, the analysis to identify potentially druggable genes revealed NOTCH1 as a potential therapeutic target. KMT2D is tumor suppressor gene that encodes histone methyltransferase and promotes the transcriptional activation of target genes by modifying histone H3 lysine 4 trimethylation (H3K4me3).LRP1B is putative tumor suppressor gene that encodes low-density lipoprotein receptor-related protein 1b 27 .LRP1B is frequently deleted in various tumors and was deleted in 20.8% of a Japanese cohort 8 and mutated in 25% of an Indian cohort. 5A study of LRP1B gene expression in ESCC showed that 42.9% of primary esophageal cancer cases have homozygous LRP1B deletions, and LRP1B mRNA expression was frequently silenced in cell lines without homozygous deletions (37.8%). 28Bisul te-PCR analysis and sequencing showed that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a LRP1B CpG island.Recently, Brown et al. reported better immune checkpoint inhibitor treatment responses in a group with likely pathogenic/pathogenic variants in LRP1B genes compared with a group carrying variant of unknown signi cance, indicating that mutations in the LRP1B gene have signi cant implications for the prognosis and treatment of multiple cancer types. 27All LRP1B variants in our study were missense variants.Further studies of their expression and methylation are needed to clarify the roles of these LRP1B missense mutations. Kiran et al. showed that tobacco chewers had a higher frequency of mutation signature 4 than did smokers and non-users of tobacco in an Indian ESCC cohort. 5There were no tobacco chewers in our study and no distinct signatures associated with ESCC in patients with a smoking history. Our study is the rst report of the mutational landscape of ESCC in a Korean cohort.It provides insight into molecular alterations in ESCC in Koreans and reveals potential candidates for therapeutic targeting. Our data suggest that KMT2D is a molecular prognostic marker.Further studies need to examine the role of KMT2D in ESCC prognosis due to the small size of our cohort.Additionally, copy number and expression studies of ESCC in Koreans are needed, as none were conducted here. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "ESCC is one of the deadliest cancers in Korea, although its incidence in Korea is lower than in China and Japan.There are no data on genomic alterations associated with ESCC in Korea.Our study is the rst report of the mutational landscape of ESCC in a Korean cohort.We found a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs.Non-silent variants of KMT2D (MLL2) were associated with a poor prognosis.An analysis to identify potentially druggable genes revealed NOTCH1 as a potential therapeutic target.We observed a high frequency of C:G → T:A transitions regardless of smoking history. Further, deconstruction of the mutation signature of our cohort revealed the enrichment of COSMIC SBS 13, SBS 39, SBS 2, SBS 40, and SBS 1.In summary, this study characterized the genomic alterations of ESCC with highly mutational heterogeneity, which provides the understanding of the molecular pathophysiology of ESCC, and it can be useful for nding potential therapeutic target in the aspect of precision medicine. ",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials And Methods",
"section_num": null
},
{
"section_content": "We performed WES analysis of 43 ESCC tumor samples and germline DNA from normal esophageal mucosal tissue or whole blood obtained during surgical resection.The patients' clinical characteristics were reviewed using electronic medical records (Table 1).The study protocol was approved by the Institutional Review Board of Yonsei University College of Medicine (approval no.IRB 4-2018-1210).All subjects gave informed consent when they were enrolled and all methods were performed in accordance with the relevant guidelines and regulations of Institutional Review Board of Yonsei University College of Medicine. ",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "A WES DNA library was constructed using an Agilent SureSelect V6-Post kit (Agilent Technologies, Santa Clara, CA) with genomic DNA extracted from fresh-frozen tissue using the Qiagen DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany).Genomic DNA was sheared into 150-200-bp fragments using an ultrasonic sonicator.These sheared fragments were end-repaired, phosphorylated, adenylated at the 3' ends, and ligated using paired-end adaptors.The hybridization reaction was done at 65℃ for 16 hours using a DNA library with added biotinylated RNA library bait (Agilent Technologies).Cluster ampli cation was performed according to the manufacturer's protocol (Illumina, San Diego, CA).Paired-end sequencing was performed on an Illumina NovaSeq 6000 system (Illumina) by Macrogen Inc. (Seoul, South Korea). Raw reads acquired in FASTQ format were trimmed using the Trimmomatic tool in paired-end mode to increase the mapping accuracy and speci city. 11These were mapped against the reference genome hg19 (GRCh37) using the Burrows-Wheeler aligner with default parameters.Binary alignment map (bam) les were further processed using GATK (Genome Analysis Toolkit, Broad Institute, Cambridge, MA), which included the removal of duplicates using MarkDuplicates in Picard, IndelRealigner, and BaseRecalibrator.High-con dence somatic single-nucleotide variants (SNVs) were ltered using HaplotypeCaller, Mutect2, VarScan, and Pindel and annotated using AnnoVar and VEP.The variant call format (vcf) les were converted into mutation annotation format (maf) les using vcf2maf tools [Cyriac Kandoth (2020); mskcc/vcf2maf: vcf2maf v1.6.19,doi:10.5281/zenodo.593251]. Oncoplot, lollipop plots of somatic SNVs, the identities of genes with mutually exclusive variants and cancer driver genes, and actionable therapeutic target and mutational signatures were generated using the R package maftools. 12To predict clinically actionable therapeutic targets, the Drug-Gene Interaction Database (DGIdb) was accessed, and the \"Druggable Genome\" category was used to identify potentially druggable targets in ESCC. 5 ",
"section_name": "Whole exome sequencing analysis",
"section_num": null
},
{
"section_content": "The overall and disease-free survival distributions were described with Kaplan-Meier curves, and statistical signi cance was calculated using the log-rank test in STATA 16.Overall survival was evaluated from the time of diagnosis to death or the last follow-up.Censored cases were de ned as patients who lost contact during follow-up and who were still alive at the end of the study.Disease-free survival was evaluated from the time of diagnosis to death, the recurrence of ESCC, or the last follow-up.Censored cases were de ned as patients who were still alive and had no recurrent tumors at the end of the study. Clinical enrichment analysis was conducted using the R package maftools, with which pairwise and group-wise Fisher's exact tests can be performed to nd differentially enriched genes for clinical features such as sex and determine whether metastasis has occurred, the patient survives, or the cancer has recurred. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Oncoplots ",
"section_name": "Declarations Figures",
"section_num": null
}
] |
[
{
"section_content": "This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1I1A1A01055513). ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The data that support the ndings of study are available from the corresponding author upon reasonable request. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "S.P curated, interpreted the data, and wrote the paper.S.Y.P and S.T.L conceptualized this research, review, editing this paper, and supervised entire project.S.Y.P acquired the funding.All authors reviewed the results and approved the nal version of the manuscript. The authors declare no competing interests. This is a list of supplementary les associated with this preprint.Click to download.Supplementalinfromation20211013.docx ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "S.P curated, interpreted the data, and wrote the paper.S.Y.P and S.T.L conceptualized this research, review, editing this paper, and supervised entire project.S.Y.P acquired the funding.All authors reviewed the results and approved the nal version of the manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "This is a list of supplementary les associated with this preprint.Click to download.Supplementalinfromation20211013.docx ",
"section_name": "Supplementary Files",
"section_num": null
}
] |
10.1038/nature14508
|
HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease
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Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.
|
[
{
"section_content": "The protein coding sequence of (mouse) codon optimised Sf3b1 Supplementary Data 1│Coding and amino acid sequence of (mouse) codon optimised Sf3b1. ",
"section_name": "",
"section_num": ""
}
] |
[] |
10.3390/ijms23031094
|
Detecting Bacterial–Human Lateral Gene Transfer in Chronic Lymphocytic Leukemia
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<jats:p>Chronic lymphocytic leukemia (CLL) is a very common and mostly incurable B-cell malignancy. Recent studies revealed high interpatient mutational heterogeneity and worsened therapy response and survival of patients with complex genomic aberrations. In line with this, a better understanding of the underlying mechanisms of specific genetic aberrations would reveal new prognostic factors and possible therapeutic targets. It is known that chromosomal rearrangements including DNA insertions often play a role during carcinogenesis. Recently it was reported that bacteria (microbiome)–human lateral gene transfer occurs in somatic cells and is enriched in cancer samples. To further investigate this mechanism in CLL, we analyzed paired-end RNA sequencing data of 45 CLL patients and 9 healthy donors, in which we particularly searched for bacterial DNA integrations into the human somatic genome. Applying the Burrows–Wheeler aligner (BWA) first on a human genome and then on bacterial genome references, we differentiated between sequencing reads mapping to the human genome, to the microbiome or to bacterial integrations into the human genome. Our results indicate that CLL samples featured bacterial DNA integrations more frequently (approx. two-fold) compared to normal samples, which corroborates the latest findings in other cancer entities. Moreover, we determined common integration sites and recurrent integrated bacterial transcripts. Finally, we investigated the contribution of bacterial integrations to oncogenesis and disease progression.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a very common B-cell malignancy that mostly affects elderly people [1].CLL patients show a vastly heterogeneous mutational profile, comprising a high number of passenger and driver mutations, which forward clonal evolution and the course of disease [2,3].Several somatic mutations and chromosomal aberrations were identified as prognostic factors, and are now being used in clinics for therapy choice.For example, IGHV mutated patients have a better prognosis and respond better to fludarabine-cyclophosphamide-rituximab (FCR) chemoimmunotherapy, which leads to prolonged progression-free survival [4,5].Moreover, mutations in TP53 and ATM genes or complex chromosomal alterations such as del17q, del11q and tri12 correspond with worsened prognosis and survival [2,3].Moreover, a complex karyotype (≥3 distinct chromosomal abnormalities) leads to impaired therapy response [6].Alongside deletions, duplications and translocations, integrations of foreign DNA represent another type of genomic aberration that can influence carcinogenesis.Various transposon types are well described and are known to contribute to oncogenesis once they become mobile.In the context of leukemia, Alu and SINE-VNTR-Alu (SVA) retrotransposon insertions 2 of 10 play a major role [7].Furthermore, integrations of mitochondrial DNA into the nuclear genome represent a recurrent genetic alteration in cancer cells [8].The causative effects of some viral integrations on cancer development have been known for two decades, and investigated intensively since.The most prominent examples are human papillomavirus (HPV) integrations in cervical malignancies [9] and hepatitis B virus (HBV) integrations in hepatocellular carcinoma [10].Often these viral integrations occur in oncogenes or tumor-suppressor genes, resulting in increased cell proliferation and downregulation of apoptotic pathways [11].A recent survey assesses that over two million cancer cases per year are infection-associated (data for 2018) [12].While most of the described diseases are caused by viruses including HPV and HBV, over a third of reported cases are attributed to infections with Helicobacter pylori.However, the known role of bacteria during tumorigenesis was restricted to their inflammatory properties and to the production of DNA-damaging agents [13,14].A paper published by Riley et al. revealed that somatic bacterial integrations into the human genome are detectable in several cancer types, suggesting bacteria-to-human lateral gene transfer (LGT) by pathologic or commensal bacteria to be a novel mechanism possibly contributing to cancerogenesis [15].These data prompted us to investigate bacteria-to-human LGT in a cohort of clinically well-characterized CLL patients and to examine whether the magnitude of LGT associates with prognostically relevant parameters or treatment outcome. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "Based on a recent study that described bacterial-human lateral gene transfer in several cancer entities [15], we customized the analysis workflow to examine the bacterial integration rate in CLL patients.The paired-end RNA sequencing data from 45 CLL samples and 27 healthy B-cell samples (naïve, non-class-switched and class-switched memory B-cell subsets from 9 healthy donors) were processed.First, the RNA sequencing reads were aligned to the human genome using the BWA method (Figure 1). Int. J. Mol.Sci.2022, 23, x FOR PEER REVIEW 2 In the context of leukemia, Alu and SINE-VNTR-Alu (SVA) retrotransposon inserti play a major role [7].Furthermore, integrations of mitochondrial DNA into the nuc genome represent a recurrent genetic alteration in cancer cells [8].The causative effect some viral integrations on cancer development have been known for two decades, investigated intensively since.The most prominent examples are human papillomav (HPV) integrations in cervical malignancies [9] and hepatitis B virus (HBV) integrati in hepatocellular carcinoma [10].Often these viral integrations occur in oncogenes or mor-suppressor genes, resulting in increased cell proliferation and downregulation apoptotic pathways [11].A recent survey assesses that over two million cancer cases year are infection-associated (data for 2018) [12].While most of the described diseases caused by viruses including HPV and HBV, over a third of reported cases are attribu to infections with Helicobacter pylori.However, the known role of bacteria during tum igenesis was restricted to their inflammatory properties and to the production of DN damaging agents [13,14].A paper published by Riley et al. revealed that somatic bacte integrations into the human genome are detectable in several cancer types, sugges bacteria-to-human lateral gene transfer (LGT) by pathologic or commensal bacteria to a novel mechanism possibly contributing to cancerogenesis [15].These data prompted to investigate bacteria-to-human LGT in a cohort of clinically well-characterized CLL tients and to examine whether the magnitude of LGT associates with prognostically r vant parameters or treatment outcome. ",
"section_name": "Detecting Bacterial Integrations into the Human Genome in CLL Patients",
"section_num": "2.1."
},
{
"section_content": "Based on a recent study that described bacterial-human lateral gene transfer in eral cancer entities [15], we customized the analysis workflow to examine the bacte integration rate in CLL patients.The paired-end RNA sequencing data from 45 CLL s ples and 27 healthy B-cell samples (naïve, non-class-switched and class-switched mem B-cell subsets from 9 healthy donors) were processed.First, the RNA sequencing re were aligned to the human genome using the BWA method (Figure 1).RNAseq data were aligned to the human genome (hg19) and subsequently to bacterial genome references via BWA.The reads supporting LGT were defined as reads featuring only one mate mapping exclusively to human genome and only one mate mapping exclusively to bacterial genome. An output for each read pair indicated if both mates, only the first, or only the second mate mapped to the human genome.For further analysis, the read pairs mapping with only one mate to the human genome were selected and aligned to bacterial genome references.The reads which mapped with one mate to human and with another mate to bacterial reference were defined as reads potentially supporting bacterial-human lateral gene transfer.In the next filtering step, we excluded all reads with <90% alignment coverage and low-complexity reads containing long poly-A/-T/-G/-C sequences.Furthermore, we excluded duplicated junctions, which were defined by the genomic position of the integration and the genomic position on respective bacterial reference of the integrated fragment, to rule out possible technical artefacts. ",
"section_name": "Detecting Bacterial Integrations into the Human Genome in CLL Patients",
"section_num": "2.1."
},
{
"section_content": "In total, we analyzed 2,320,158,744 read pairs originating from CLL samples and 1,051,860,898 from healthy B-cell samples.Although we could detect at least three LGT events per healthy sample, each CLL sample featured 23 or more unique LGT events.Most of the CLL patients had >50 reads supporting LGT (Figure 2A, Supporting Figure S1, Supplementary Table S1).Furthermore, the number of reads supporting LGT was normalized to the total read number.This revealed that CLL patients had 2.2-times higher frequency of bacterial integrations into the human genome compared to healthy donors (Figure 2B; p-value = 0.0011).Notably, all three outliers in the healthy cohort with elevated numbers of LGT events originated from class-switched B cells.To corroborate these results, we accessed and analyzed additional RNAseq data from in-vitro-EBV-transformed B cells (n = 15), naïve B cells (n = 10) as well as memory B cells (n = 3).Thereby, we could confirm low-level LGT in naïve and EBV-transformed B cells and a high LGT level in memory B cells (Supporting Figure S2, Supplementary Table S2). An output for each read pair indicated if both mates, only the first, or only the second mate mapped to the human genome.For further analysis, the read pairs mapping with only one mate to the human genome were selected and aligned to bacterial genome references.The reads which mapped with one mate to human and with another mate to bacterial reference were defined as reads potentially supporting bacterial-human lateral gene transfer.In the next filtering step, we excluded all reads with <90% alignment coverage and low-complexity reads containing long poly-A/-T/-G/-C sequences.Furthermore, we excluded duplicated junctions, which were defined by the genomic position of the integration and the genomic position on respective bacterial reference of the integrated fragment, to rule out possible technical artefacts. ",
"section_name": "Reads Supporting Bacterial-Human LGT were Enriched in CLL versus Non-Leukemic B Cells",
"section_num": "2.2."
},
{
"section_content": "In total, we analyzed 2,320,158,744 read pairs originating from CLL samples and 1,051,860,898 from healthy B-cell samples.Although we could detect at least three LGT events per healthy sample, each CLL sample featured 23 or more unique LGT events.Most of the CLL patients had >50 reads supporting LGT (Figure 2A, Supporting Figure S1, Supplementary Table S1).Furthermore, the number of reads supporting LGT was normalized to the total read number.This revealed that CLL patients had 2.2-times higher frequency of bacterial integrations into the human genome compared to healthy donors (Figure 2B; p-value = 0.0011).Notably, all three outliers in the healthy cohort with elevated numbers of LGT events originated from class-switched B cells.To corroborate these results, we accessed and analyzed additional RNAseq data from in-vitro-EBV-transformed B cells (n = 15), naïve B cells (n = 10) as well as memory B cells (n = 3).Thereby, we could confirm lowlevel LGT in naïve and EBV-transformed B cells and a high LGT level in memory B cells (Supporting Figure S2, Supplementary Table S2). ",
"section_name": "Reads Supporting Bacterial-Human LGT were Enriched in CLL versus Non-Leukemic B Cells",
"section_num": "2.2."
},
{
"section_content": "According to gene coding structure annotation, the majority of bacterial integration events (36%) occurred in intronic regions, 22% in exonic regions and 17% in UTR3 (Figure 3A).While we detected bacterial integrations throughout the human genome, several genes were identified as hot spots for LGT, which recurrently appeared in a number of patients.Figure 3B summarizes the top 30 genes with a bacterial integration in both CLL and healthy cohorts.Interestingly, three of the four top genes (SNORD141A/B, MIR4507 and MALAT1) are non-coding RNA transcripts, which are reported to be disease associated, among others in lung adenocarcinoma [16] and B-cell malignancies [17].Notably, LGT in CD74 (here, the top sixth gene) was also described for stomach adenocarcinomas [15].CD74, or MHC class II-associated invariant chain, plays an important role in antigen presentation.It also modulates several survival pathways, including the NF-κB pathway, which are associated with oncogenesis [18,19].Intriguingly, the top 10 genes featuring LGT in our analysis also comprised HLA-B and HLA-C genes, which encode two of three main types of class I major histocompatibility complex (MHCI), whereas the HLA-A gene is known to be affected by retrotransposons, particularly by SVA integration, in the leukemic context [7]. The graph shows the percentage of samples featuring indicated number of reads supporting LGT.(B) The figure displays a comparison between CLL samples from CLL patients and B-cell samples from healthy (H) donors, whereas percentage of reads supporting bacterial-human lateral gene transfer is shown.Median is indicated.Significance was calculated using unpaired two-tailed t-test.Below, key information is summarized for both groups. ",
"section_name": "Bacterial Integrations Occurred Recurrently in Several Genes",
"section_num": "2.3."
},
{
"section_content": "According to gene coding structure annotation, the majority of bacterial integration events (36%) occurred in intronic regions, 22% in exonic regions and 17% in UTR3′ (Figure 3A).While we detected bacterial integrations throughout the human genome, several genes were identified as hot spots for LGT, which recurrently appeared in a number of patients.Figure 3B summarizes the top 30 genes with a bacterial integration in both CLL and healthy cohorts.Interestingly, three of the four top genes (SNORD141A/B, MIR4507 and MALAT1) are non-coding RNA transcripts, which are reported to be disease associated, among others in lung adenocarcinoma [16] and B-cell malignancies [17].Notably, LGT in CD74 (here, the top sixth gene) was also described for stomach adenocarcinomas [15].CD74, or MHC class II-associated invariant chain, plays an important role in antigen presentation.It also modulates several survival pathways, including the NF-κB pathway, which are associated with oncogenesis [18,19].Intriguingly, the top 10 genes featuring LGT in our analysis also comprised HLA-B and HLA-C genes, which encode two of three main types of class I major histocompatibility complex (MHCI), whereas the HLA-A gene is known to be affected by retrotransposons, particularly by SVA integration, in the leukemic context [7]. ",
"section_name": "Bacterial Integrations Occurred Recurrently in Several Genes",
"section_num": "2.3."
},
{
"section_content": "Throughout the data from the CLL cohort, we identified 216 bacteria genera integrated into the human genome at distinct locations (Supporting Figure S3A, Supplementary Table S1), compared to 113 genera for healthy controls (Supporting Figure S3B, Supplementary Table S1).However, there was a large variance in the abundancies of different genera (Figure 4A).Whereas Pseudomonas sp., the second most common genus detected, was present in almost all analyzed samples with mostly comparable counts, Mesorhizobium of 10 sp., the most common genus detected, was highly abundant in one group of patients (22 patients) and absent in the others.Our analysis showed no specific integration sites preferably used by specific bacteria.To visualize the insertion map, we generated circos plots, which exemplarily show the integrations of three bacteria genera, Pseudomonas sp., Mesorhizobium sp. and Acinetobacter sp., which are distributed across the whole human genome (Figure 4B).Interestingly, Pseudomonas was reported to be the most prevalent integrated taxonomic unit in stomach adenocarcinoma [15].Infection with Pseudomonas aeruginosa was further reported to stimulate gastric cancer development in animal experiments [20].Moreover, integrations from the genus Acinetobacter, which had the ninth highest abundance in our cohort, were determined as the most frequent in acute myeloid leukemia by Riley et al. [15].Until now, there was no particular evidence of DNA integrations from the genus Mesorhizobium into the human genome or of its role during carcinogenesis.Nevertheless, it was described that Mesorhizobium sp. was increased in the microbiome of patients suffering from benign biliary pathology, although it was barely present in a healthy human bile duct microbiome [21]. Furthermore, the multidimensional scaling analysis (MDS) of genus-specific bacterial integration counts divided CLL patients into two groups (group 1, n = 21 and group 2, n = 22, Figure 4C).This classification nicely correlated with a general frequency of LGT, with group 2 showing 2.8 times more LGT events than group 1 (Figure 4C, Supporting Figure S4). ",
"section_name": "Some Bacteria Genera Appear to Integrate into Human Genome with Higher Likelihood",
"section_num": "2.4."
},
{
"section_content": "Finally, we examined if patient classification based on LGT events corresponded to clinically relevant parameters.We compared progression-free survival (PFS) and overall survival (OS) time upon treatment between LGT group 1 and group 2 patients (Figure 5A) and between patients grouped according to the number of LGT events (cutoffs were calculated by Cox regression analysis; for PFS: LGTlow < 5.82947 × 10 -5 % LGT events, n = 5; LGThigh > 5.82947 × 10 -5 % LGT events, n = 38; for OS: LGTlow < 0.000309331% LGT events, n = 36; LGThigh > 0.000309031% LGT events, n = 7; Figure 5B).We did not observe any significant differences in survival between the respective groups, although there was a trend towards prolonged PFS in group 2 patients (Figure 5A).Associating LGT events with prognostically relevant parameters such as IGVH mutation status, RAI staging or common cytogenetic aberrations determined by FISH also did not reveal any significant correlations (Figure 5C-I, Supporting Figure S5). ",
"section_name": "Correlation of LGT Events with Clinically Relevant Parameters",
"section_num": "2.5."
},
{
"section_content": "Lateral (or horizontal) gene transfer describes DNA integrations from one organism into the genome of another. LGT is considered an evolutionary mechanism, and is especially important for bacteria to acquire new properties not only from other related bacteria, but also from eukaryotes, including humans [22,23].Lateral gene transfer in the opposite direction as an evolutionarily relevant event is evident as well, considering that many genes within the human genome seem to originate from bacterial genomes [24,25].On the other hand, bacterial integrations into somatic genomes may, on an individual level, lead to gene recoding or to the overexpression of oncogenes, thus altering cellular homeostasis and causing disease development.Accordingly, a study by Riley et al. confirmed the presence of LGT events in various cancers [15].In our study, we for the first time analyzed the load and nature of bacterial integrations in chronic lymphocytic leukemia patients using sequencing data and stratified patients according to LGT events and clinical parameters.Although the cohort of 45 patients was rather small, the patients were clinically well characterized and allowed a thorough correlation of LGT grouping with prognostic and predictive factors.Intriguingly, although the overall frequency of LGT events was fairly low, we detected a more than two times higher integration rate in CLL samples compared to healthy controls.Of note, a high integration rate was also detected in classswitched memory B cells, showing that LGT may increase with the lifespan of a cell and with high activity of the DNA repair machinery required for immunoglobulin class-switch recombination.In this regard, CLL patients often feature a high number and wide range of different mutations, among others, complex chromosomal aberrations [3], possibly resulting from an intrinsically lower ability to precisely repair DNA damage [26].Likely, the higher propensity to acquire DNA aberrations may also contribute to the integration of foreign DNA into the somatic genome of tumor cells.While in some other cancer types (e.g., in cancers arising in the gastrointestinal tract) the interaction with the microbiome is certainly higher than in CLL, the contact of malignant B cells with bacterial DNA could occur in the blood due to the presence of minute amounts of cell-free DNA originating from current or past infections [27], or from the microbiome [28].In addition, bacterial DNA could be transported to lymphoid tissue-whereupon it is available for CLL cells-during constant processing and presentation of microbiome-derived antigens by innate immune cells [29].Although our study did not reveal any robust association of LGT events with clinically relevant prognosticators such as IGVH mutation status, chromosomal aberrations or RAI staging, we noticed a trend towards differential PFS upon treatment in patients grouped according to LGT events.The fact that LGT was also detected in healthy cells could point to LGT as an inevitable by-product of DNA repair rather than a strong cancer-driving process.However, although it is conceivable that in most cases LGT has no effect on cell homeostasis, it may contribute to the clonal evolution of cancer cells in individual cases. In summary, we were able to show that bacteria-to-human LGT frequently occurs in CLL.However, as LGT likely has only a subtle impact on disease pathogenesis, its contribution to disease development, progression and treatment outcome remains to be analyzed in the longitudinal tracking of large patient cohorts. ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "The LGT detection method was based on a workflow published by Riley et al. [15].Paired-end RNA sequencing data from 45 untreated CLL patients sampled prior treatment with lenalidomide in combination with fludarabine and rituximab (AGMT-REVLIRIT trial, ClinicalTrials.govIdentifier: NCT00738829 and NCT01703364) [30] and 9 healthy B-cell donors (each in triplicates, 27 samples; downloaded from the European Genome-phenome Archive: EGAS00001000374) [31] were aligned to the human genome (bwa mem hg19; bwa v0.7.12-r1039).Reads that mapped with only one mate to hg19 were extracted (SAMtools view -h -f 4 -F 8 -F 2048, SAMtools view -h -f 8 -F 4 -F 2048; SAMtools merge; SAMtools v1.10), reformatted to FASTQ (Picard SamToFastq; Picard v2.21.9) and realigned to bacterial genome references (RefSeq, NCBI) using the same parameters.The reads supporting LGT were defined as reads featuring only one mate mapping exclusively to the human genome and only one mate mapping exclusively to the bacterial genome.Thus, the integration junction sequence lies in-between the two mates (within the unsequenced insert) of the same RNA fragment.The post-alignment filtering and analysis were performed on a Linux Redhat system using custom Bash and R (v4.0.4) scripting.All reads with <90% alignment coverage and low-complexity reads with long poly-A/-T/-G/-C sequences were excluded.Additionally, the integration sites were intersected with RepeatMasker file, available online: https://genome.ucsc.edu/cgi-bin/hgTables(accessed 15 December 2021) using Bedtools v2.26.0 in order to identify integration sites in low-complexity regions.Moreover, duplicated integrations (integrations of the same bacterial transcript region at the same genomic position) were excluded from further analysis.For gene and gene structure ",
"section_name": "Materials and Methods",
"section_num": "4."
}
] |
[
{
"section_content": "Acknowledgments: Figure 1 was created with BioRender.comall authors accept it: Open Access Funding by the Austrian Science Fund (FWF). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Funding: This work was supported by the Austrian Science Fund (FWF) FWF P32762-B (N.Zaborsky), the SCRI-LIMCR, the City of Salzburg, the Province of Salzburg, WISS 2025 (Cancer Cluster Salzburg, CCSII-IOS to R. Greil). Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Funding: This work was supported by the Austrian Science Fund (FWF) FWF P32762-B (N.Zaborsky), the SCRI-LIMCR, the City of Salzburg, the Province of Salzburg, WISS 2025 (Cancer Cluster Salzburg, CCSII-IOS to R. Greil). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Data Availability Statement: Customized bioinformatics scripts are available upon request. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1038/s41408-023-00784-z
|
Risk of second primary malignancies in patients with chronic lymphocytic leukemia: a population-based study in the Netherlands, 1989-2019
|
<jats:title>Abstract</jats:title><jats:p>The longevity of patients with chronic lymphocytic leukemia (CLL) has improved progressively over the past decades, making it essential to understand long-term health outcomes, such as second primary malignancies (SPMs). Therefore, this nationwide, population-based study assessed the risk of SPM development in CLL patients diagnosed during 1989-2019 in the Netherlands compared to the expected number of malignancies in an age-, sex-, and period-matched group from the general Dutch population. In 24,815 CLL patients followed for 162,698.49 person-years, 4369 SPMs were diagnosed with a standardized incidence ratio (SIR) of 1.63 (95% confidence interval [CI] 1.59–1.68). This elevated risk was observed for solid (SIR, 1.67; 95% CI, 1.65–1.75) and hematological SPMs (SIR 1.42; 95% CI, 1.24–1.62). The highest risk for SPMs was noted beyond five years post-diagnosis (SIR, 1.70; 95% CI, 1.62–1.77), for male individuals (SIR, 1.70; 95% CI, 1.64–1.77), and patients aged 18–69 years (SIR, 1.92; 95% CI, 1.79–2.05). The risk of SPMs was higher in CLL patients who received anti-neoplastic therapy (SIR, 2.12; 95% CI, 1.96–2.28), as compared with those who did not (SIR, 1.58; 95% CI, 1.53–1.63). Routine surveillance activities and tailored interventions to counteract the increased morbidity and excess mortality associated with SPMs are essential for improving long-term outcomes in CLL patients.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most frequently diagnosed leukemia among adults in the Western world, with an age-standardized incidence rate ranging from 3.8 to 5.0 per 100,000 person-years as of the 2000s [1][2][3][4][5].The clinical behavior of CLL is heterogeneous, ranging from an indolent disease with a tendency to remain stable for many years without therapy to a more aggressive illness that rapidly relapses after initial treatment. The past decades have witnessed significant progress in managing patients with CLL.More specifically, the most notable therapeutic breakthrough was the introduction of chemoimmunotherapy, which translated into improved outcomes for CLL patients at the population level [1,[6][7][8].More recently, novel agents such as ibrutinib and venetoclax entered the therapeutic realm of CLL.Due to the availability of more efficacious therapies in the upfront and relapsed setting, the longevity of CLL patients improved progressively over time [1,8].Nevertheless, excess mortality among long-term CLL survivors persists and remains a threat in modern times [8,9]. As the population of long-term CLL survivors is rapidly expanding, it is essential to understand long-term health outcomes.The development of second primary malignancies (SPMs) -i.e., cancers diagnosed after CLL-may contribute to morbidity and offset the improved longevity of CLL patients.Therefore, awareness of the nature and magnitude of SPMs in CLL is essential for health-related planning and surveillance activities [10][11][12][13][14].The relative scintilla of population-based studies in CLL has shown an increased risk of SPM development compared to the general population [15][16][17][18].However, most of these studies have not investigated SPM development with long-term follow-up since the widespread use of chemoimmunotherapy in the 2010s and the most recent availability of novel targeted approaches.Also, most of these studies have included comparatively small cohorts with a short follow-up time.Therefore, to complement and extend the currently sparse literature on SPM development in CLL, this nationwide, population-based study aimed to assess temporal trends in SPM development -compared with an age-, sex-and period-matched group of the general populationin various subgroups of CLL patients in the Netherlands during a 30year period that takes into account the treatment advances of CLL during that period. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Established in 1989, the Netherlands Cancer Registry (NCR), which is maintained and hosted by the Netherlands Comprehensive Cancer Organisation (IKNL), has an overall coverage of at least 95% of all newly diagnosed malignancies in the Netherlands [19].The NCR relies on comprehensive case notification via the Nationwide Network and Registry of Histopathology and Cytopathology and the National Registry of Hospital Discharges (i.e., inpatient and outpatient discharges).Basic information on dates of birth and diagnosis, sex, primary therapy, and disease stage, topography, and morphology of all newly diagnosed malignancies are routinely ascertained in the NCR by trained registrars of IKNL through retrospective review of medical records.Topography and morphology are coded as per the International Classification of Diseases for Oncology (ICD-O) [20].Information on vital status (i.e., alive, dead, or emigration) is obtained through an annual linkage with the Nationwide Population Registries Network that holds this information for all residents in the Netherlands. ",
"section_name": "PATIENTS AND METHODS The Netherlands Cancer Registry",
"section_num": null
},
{
"section_content": "All patients diagnosed with CLL between January 1, 1989, and December 31, 2019, were selected from the NCR using the ICD-O morphology code 9823.Patients diagnosed at autopsy (n = 71) were excluded from the analysis.Through cross-linkage with the NCR, SPMs diagnosed between 1989 and 2019 were identified.The ICD-O morphology and topography codes used to categorize specific SPM groupings are depicted in Supplemental Table 1 [20].Basal cell carcinomas of the skin were excluded from the analysis because these malignancies were not standardly ascertained throughout the study period.Also, diffuse large B-cell lymphomas (DLBCL) and Hodgkin lymphomas were excluded since these lymphomas might have been misclassified as SPMs when they may be transformations of CLL (i.e., Richter's syndrome).Finally, synchronous SPMs diagnosed within six months after CLL diagnosis were excluded to minimize surveillance bias since these SPMs might be incidental findings rather than true SPMs.Patients with multiple and metachronous SPMs were counted only once in the analysis for all sites and all solid and hematological SPMs combined.However, these subsequent cancers contributed to the cancer site-specific analysis regardless of whether it was preceded by a malignancy from another site [21]. According to the Central Committee on Research involving Human Subjects (CCMO), this type of observational, non-interventional study does not require approval from an ethics committee in the Netherlands.The Privacy Review Board of the NCR approved the use of anonymous data for this study. ",
"section_name": "Study population",
"section_num": null
},
{
"section_content": "The NCR generally ascertains information on primary therapy initiated within one-year postdiagnosis.For the overall analysis (i.e., 1989-2019), primary therapy was grouped into (i) no anti-neoplastic therapy, including a watch-and-wait approach, and (ii) anti-neoplastic therapy.The latter group was subdivided into chemotherapy alone and chemoimmunotherapy.Of note, the NCR ascertains the use of rituximab as of January 1, 2007.However, the use of rituximab before 2007 in the frontline management of CLL is presumed to be neglectable, as rituximab was initially introduced around 2010 for previously untreated CLL patients in combination with fludarabine and cyclophosphamide [22]. As of January 1, 2014, information on the exact therapeutic regimens was registered in the NCR.These regimens were categorized as fludarabine, cyclophosphamide and rituximab (FCR), bendamustine and rituximab (BR), rituximab, cyclophosphamide, vincristine and prednisone (R-CVP), rituximab or obinutuzumab with chlorambucil (R-or O-Clb), chlorambucil monotherapy, ibrutinib, venetoclax, and other less frequently applied modalities.Of note, ibrutinib was reimbursed in the Netherlands as of late 2014 and venetoclax as of 2017 for previously untreated CLL patients harboring TP53 aberrations [23][24][25][26]. ",
"section_name": "Primary therapy",
"section_num": null
},
{
"section_content": "Person-years at risk were calculated from the date of CLL diagnosis until SPM diagnosis, death, or end of follow-up (December 31, 2019), whichever occurred first.The risk time ended at the diagnosis of the first SPM of interest in the case of multiple SPMs within one patient.Standardized incidence ratios (SIRs) were computed as the ratio of observed SPMs to expected SPMs from the general population.The expected number of malignancies was based on age-, sex-, calendar, and site-specific cancer-incidence rates in the Dutch population, which were multiplied by the corresponding person-years at risk.The absolute excess risk (AER) represents the additional incidence of SPMs measured beyond the background incidence of SPMs found in the Dutch general population.The AER was calculated as the expected number of SPMs subtracted by the observed number of SPMs, divided by the person-years at risk and multiplied by 10,000, resulting in an AER per 10,000 person-years [27,28].Poisson distribution for the number of observed SPMs was assumed to calculate the 95% confidence intervals (CIs) for the SIR and AER.Unless otherwise stated, the SIRs and AERs were presented overall and according to age category (18-69 and ≥70 years), sex, the latency period for SPM development defined as the years from CLL diagnosis until SPM development (0.5-5 and ≥5 years), calendar period (1989-1995, 1996-2002, 2003-2009, and 2010-2019) and the receipt of antineoplastic therapy (no versus yes).The calendar periods were used as a proxy for the evolution of therapeutic modalities over time.The criteria of non-overlapping CIs were used to show statistically significant differences between subgroups [27]. The cumulative incidence of SPMs was evaluated, with death treated as a competing risk.The expected cumulative incidence in the general population was derived from the expected cancer incidence rates and expected overall mortality rates in the Dutch general population.The cumulative incidence was estimated for all sites, all solid and hematological SPMs, and individual SPM subtypes (Supplemental Table 1). A multivariable analysis was performed using the Fine and Grey method to analyze the effect of age (18-59, 60-69, 70-79, and ≥80 years), sex, calendar period (1989-1995, 1996-2002, 2003-2009, and 2010-2019), and receipt of anti-neoplastic therapy on the cumulative incidence of SPMs [29]. All statistical analyses were performed with STATA Statistical Software version 17.0 (StataCorp, College Station, TX) and SAS version 9.4 (SAS Institute Inc, Cary, North Carolina, USA). ",
"section_name": "Statistical analyses",
"section_num": null
},
{
"section_content": "Our analytic cohort included 24,815 CLL patients (61% males; median age 69 years; interquartile age range [IQR], 61-67 years) diagnosed in the Netherlands between 1989 and 2019.The baseline and primary treatment characteristics are presented in Table 1 according to the calendar period of diagnosis.Overall, the median follow-up period was 6.2 years (IQR, 3.2-10.6years), with 28% of the patients being followed for at least ten years.This overall follow-up period resulted in a total follow-up of 162,698.49person-years. In the overall series, most patients did not receive antineoplastic therapy, including a watch-and-wait approach, within one year post-diagnosis (84%; Table 1).The use of anti-neoplastic therapy decreased with each successive calendar period, following a broader institution of a watch-and-wait approach.The gradual increase in the age-standardized incidence rate between 1989-1995 (3.23 per 100,000 person-years) to 2003-2009 (4.54 per 100,000 person-years) was followed by a stabilization during 2010-2019 (4.53 per 100,000 person-years).This finding may suggest that the initial increase might be attributed to higher detection of early-stage CLL and consequently might explain the higher proportion of watch-and-wait approaches over time (Table 1).Of the patients receiving anti-neoplastic therapy as of 2007, 595 (37%) patients received chemotherapy and 893 (55%) chemoimmunotherapy.For patients diagnosed as of 2014 and receiving anti-neoplastic treatment, the majority of the patients received FCR (27%) followed by R-or O-Clb (26%), chlorambucil monotherapy (10%), R-CVP (9%), BR (9%), other, less frequently applied treatments (9%), venetoclax-based (5%), and ibrutinibbased treatment (4%). ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "During the follow-up period, 4,700 SPMs were diagnosed in 4,369 CLL patients.SPMs were diagnosed after a median follow-up period of 4.2 years post-CLL diagnosis (IQR, 1.6-7.9years) and at a median age of 74 years (IQR, 68-82 years).The cumulative incidence of SPM development was 29.45% (95% CI, 28.66%-30.26%),50.84% (95% CI, 49.20%-52.52%)and 69.18% (95% CI, 63.50%-74.71%) at 10, 20, and 30 years respectively (Table 1).The cumulative incidence of all SPM subtypes is depicted in Supplemental Fig. 1. Overall, the risk of developing an SPM was significantly higher among CLL patients compared to the general population (SIR, 1.63; 95% CI, 1.59-1.68),resulting in 125.06 excess malignancies per 10,000 person-years (Table 2).This increased risk of SPM development was observed for both solid (SIR, 1.67; 95% CI, 1.65-1.75)and hematological SPMs (SIR, 1.42; 95% CI, 1.24-1.62;Table 2).The risk was more than two-fold increased among CLL patients compared to the general population for squamous cell carcinomas of the skin (SIR, 4.82; 95% CI, 4.57-5.07),acute myeloid leukemia (AML; SIR, 2.75; 95% CI, 2.08-3.58),melanomas of the skin (SIR, 2.74; 95% CI, 2.43-3.08),soft-tissue sarcomas (SIR, 2.39; 95% CI, 1.70-3.27),and thyroid cancers (SIR, 2.12; 95% CI, 1.26-3.35;Table 2).Although relative risks were doubled, the absolute risk for AML, soft-tissue sarcomas and thyroid cancers remained comparatively low-reflected by a 30-year cumulative incidence of 1.90%, 1.12%, and 0.17%, respectively-owing to the low background risk for these malignancies in the general population (Table 2 and Supplemental Fig. 1). Squamous cell carcinomas of the skin contributed most to the overall excess risk (AER, 80.31/10,000 person-years), representing 64% of the total excess risk, followed by melanomas of the skin (9%; AER, 11.72/10,000 person-years), lung and bronchus cancer (8%; AER, 9.98/10,000 person-years), colon and rectum cancer (5%; AER, 6.13/10,000 person-years), and kidney cancer (3%; AER, 3.36/ 10,000 person-years; Table 2). ",
"section_name": "Risk of second primary malignancies as compared to the general population",
"section_num": null
},
{
"section_content": "The SIRs for any SPM were statistically higher for males (SIR, 1.70; 95% CI, 1.64-1.77)than for females (SIR, 1.55; 95% CI, 1.46-1.63).Also, the AER was nearly 2-fold higher for males than females (155.20 versus 85.10 per 10,000 person-years).The spectrum of SPMs for male and female CLL patients is depicted in Fig. 1.Generally, the spectrum of SPMs was comparable across the sexes except for the risk of soft-tissue sarcomas (SIR, 2.74; 95% CI, 1.73-3.75)and non-Hodgkin lymphomas excluding DLBCLs (SIR, 1.47; 95% CI, 1.01-1.93),which was only heightened in males.Although the SIRs for squamous cell carcinoma of the skin were increased for both sexes, the SIRs for males (SIR, 5.22; 95% CI, 4.91-5.54)were significantly higher than for females (SIR, 3.97; 95% CI, 3.57-4.36). ",
"section_name": "Relative and absolute excess risk according to sex",
"section_num": null
},
{
"section_content": "Overall, the SIRs were the highest for patients aged 18-59 years (SIR, 1.92; 95% CI, 1.79-2.06)compared with those aged 60-69 years (SIR, 1.60; 95% CI, 1.52-1.68),70-79 years (SIR, 1.57; 95% CI, 1.49-1.65)and ≥80 years (SIR, 1.57; 95% CI, 1.44-1.71;Supplemental Fig. 2A).However, the absolute risk increased with advancing age, ultimately reaching 155.24 excess malignancies per 10,000 person-years in patients aged ≥80 years (Supplemental Fig. 2B).This inverse correlation is probably due to a lower background risk for developing SPMs in younger individuals. For the site-specific analysis, the SIRs and AERs were reported for patients aged 18-69 years and ≥70 years (Fig. 2).The risk of colon and rectum carcinomas (SIR, 1.30; 95% CI, 1.01-1.58)and urinary bladder and renal pelvis carcinomas (SIR, 1.76; 95% CI, ).This elevation was noticeable from 2003 onwards and was attributed to a significantly higher risk for AML and myelodysplastic syndromes (MDS) in the latter period.Also, the risk of thyroid cancer was significantly higher in the most recent calendar period only.Conversely, the risk of lung and bronchus cancer, kidney cancer, soft-tissue sarcomas, and cancers with an unknown origin was higher among CLL patients in the earlier studied periods and lost significance in the latter calendar period.Of note, the risk of squamous cell carcinomas of the skin and melanomas of the skin remained significantly elevated throughout the entire study period. Trends of second primary malignancies according to the latency period Overall, the SIRs for any SPMs remained significantly higher throughout different latency periods among CLL patients than in the general population, even after 21-30 years post-diagnosis (SIR, 1.86; 95% CI, 1.47-2.34;Supplemental Fig. 3A).Also, the SIRs remained comparatively stable throughout different latency periods.However, the absolute risk was significantly lower for patients with a latency time of 0.5-5 years (AER, 109.00;95% CI, 101.77-116.90)compared to the overall excess risk (AER, 125.06; 95% CI, 119.25-131.23).The absolute risk increased steadily over the latency time, reaching 212.04 excess malignancies per 10,000 person-years after more than 20 years of follow-up (Supplemental Fig. 3B). For the site-specific analysis, the SIRs and AERs were reported for a latency period of 0.5-5 years and ≥5 years (Fig. 3).The spectrum of SPM subtypes varied across the latency periods.More specifically, the risk of colon and rectum cancers (SIR, 1.31; 95% CI, 1.15-1.46),urinary bladder and renal pelvis cancers (SIR, 1.31; 95% CI, 1.03-1.60),and thyroid cancers (SIR, 3.08; 95% CI, 1.27-4.90)was significantly higher among patients with CLL that developed an SPM within 0.5-5 years, as compared with the general population.On the other hand, the risk of developing a soft-tissue sarcoma (SIR, 2.78; 95% CI, 1.58-3.99)was only elevated after a latency time of ≥5 years. ",
"section_name": "Relative and absolute excess risk according to age",
"section_num": null
},
{
"section_content": "The SIRs and AERs were significantly higher in the patients who received anti-neoplastic therapy within one year post-diagnosis (SIR, 2.12; 95% CI, 1.96-2.28)as compared to those who did not (SIR, 1.57; 95% CI, 1.52-1.62),attributing to 204.45 and 113.36 excess cases per 10,000 person-years, respectively (Fig. 4).The risk of developing any hematological cancer (SIR, 3.11; 95% CI, 2.34-4.03),AML (SIR, 7.04; 95% CI, 3.45-10.62),MDS (SIR, 3.75; 95% CI, 1.20-6.30),and non-Hodgkin lymphomas (SIR, 2.90; 95% CI, 1.32-4.48)was significantly higher among patients who received anti-neoplastic therapy as compared to those who did not.Also, the risk of squamous cell carcinomas of the skin was significantly higher and more than 2-fold higher among CLL patients receiving treatment (SIR, 9.38; 95% CI, 8.27-10.49)as compared with those that did not (SIR, 4.32; 95% CI, 4.08-4.57).Of note, the risk of colon and rectum carcinomas was only elevated in untreated patients (SIR, 1.21; 95% CI, 1.10-1.32). The SIRs for patients treated with chemotherapy (SIR, 2.16; 95% CI, 1.98-2.35)and chemoimmunotherapy (SIR, 2.09; 95% CI, 1.72-2.52)were comparable.As for patients diagnosed with CLL between 2014 and 2019, the spectrum of SPM development per treatment category is listed in Supplemental Table S3.Of note, these data do not show the SIRs and AERs due to the comparatively low number of SPMs. Multivariable effects on second primary malignancy development within the cohort Next, we fitted a multivariable competing risk model for evaluating the effect of baseline patient characteristics and primary therapy on the risk of developing an SPM within our cohort (Table 3).The sHR of developing an SPM was higher in males (sHR, 1.45; 95% CI, 1.37-1.54)and in patients who received anti-neoplastic therapy within one-year postdiagnosis (sHR, 1.19; 95% CI, 1.09-1.27).The sHR of developing an SPM was higher in individuals aged 60-69 (sHR, 1.39; 95% CI, 1.29-1.50)and 70-79 years (sHR, 1.40; 95% CI, 1.29-1.51)as compared with those aged 18-59 years.Conversely, elderly patients ≥80 years had a Fig. 1 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to sex.Statistically significant standardized incidence ratios are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude.Abbreviations: AER, absolute excess risk; CI, confidence interval; and SIR, standardized incidence ratio. ",
"section_name": "Effect of primary therapy on standardized incidence ratios",
"section_num": null
},
{
"section_content": "In this large, nationwide, population-based study with long-term follow-up, we observed that CLL patients have a 63% higher risk of developing any SPM than an age-, sex, and calendar periodmatched group from the general Dutch population.This risk for developing solid and hematological SPMs was 67% and 42% higher, respectively.This finding aligns with previously reported estimates from an Australian, Danish and U.S. study [15,16,18].The spectrum of SPMs was also broadly comparable and mainly consisted of squamous cell carcinomas of the skin, melanomas of the skin, lung and bronchus cancer, colon and rectum cancer, softtissue sarcomas, AML, and thyroid cancer [14,15,18,30].The study from the U.S., which was based on data from the Surveillance, Epidemiology, and End Results (SEER) Program, reported the highest risk for Kaposi sarcomas with a SIR of 3.82 (95% CI, 2.19-6.21).Since the number of patients diagnosed with Kaposi sarcoma within our cohort was less than 10, we did not incorporate it in our analysis.Differences in HIV incidence might cause a lower incidence of Kaposi sarcoma in the Netherlands than in the U.S. Indeed, the incidence of HIV in 2020 was 9.2 and 2.3 per 100,000 person-years in the U.S. and the Netherlands, respectively [31,32].Also, as shown in the U.S. study, we could not objectify the decreased risk of hepatobiliary, breast, and uterine cancers [15].Since there is currently no clear pathophysiological explanation for these associations, this warrants further validation in forthcoming studies. The SEER-based analysis reported the highest incidence of SPMs to be diagnosed between 2 and 6 months after the CLL diagnosis; thereafter, the incidence of SPMs was lower and remained comparatively stable [15].Notably, we observed that the magnitude of the SIRs remained stable across the different latency periods.However, a significantly lower AER for SPMs was observed within 0.5-5 years after the CLL diagnosis.This observation is probably related to excluding synchronous malignancies diagnosed within six months after the CLL diagnosis to minimize surveillance bias due to heightened medical care.Also, this finding highlights that a longer follow-up time is needed to capture the effect of impaired immune surveillance, environmental exposures, and chemotherapeutics on SPM development [33]. In line with previous studies, we observed that the SIRs were the highest in younger individuals while the AER increased with advancing age, the latter being attributed to a greater background incidence of SPMs in the general population.On the other hand, we observed an increased cumulative incidence of SPMs in CLL patients aged 60-79 years.This finding suggests that the SPM risk progressively increases with age, which might be explained by the improved longevity and the accumulation of risk factors [15,34,35]. The SIRs and AERs were higher in male CLL patients than in female CLL patients.The greater SPM risk in males is attributed to a higher risk of squamous cell carcinomas of the skin, which might be explained by a higher likelihood of males working outdoors with concomitant higher UV exposure [36,37]. We noted an increasing cumulative incidence of SPMs in the most recent calendar periods, driven mainly by a higher number of hematological cancers rather than an increase in solid cancers, Fig. 2 Risk of second primary malignancies among patients with chronic lymphocytic leukemia to age.Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude.AER absolute excess risk, CI confidence interval, SIR standardized incidence ratio. which actually remained relatively stable over time.Among the hematological the risk of AML and MDS increased as of the 2000s, likely due to a higher application of fludarabine-based therapies from that time onwards.Indeed, this trend was previously described in the SEER-based analysis and in a singlecenter study in which patients were uniformly treated with FCR [12,15].In addition, the German CLL study group (GCLLSG) registry study demonstrated a higher-than-expected incidence of hematological SPMs (SIR, 3.64; 95% CI, 1.66-6.90) in treated versus untreated CLL patients within prospective studies as compared with the German general population [38].An excess of skin malignancies, including squamous cell carcinomas and melanomas, seems to characterize CLL patients, and the burden is known to increase with therapy [39][40][41].Lastly, apart from surveillance bias, it is proposed that CLL-related therapy and the immune dysfunctional nature of CLL might enhance the effect of common carcinogens, such as UV exposure and smoking, in increasing the probability of skin and respiratory cancers [14,33]. The advent of targeted treatment approaches has transformed CLL management, which, in turn, has improved patient survival.As for the latter, long-term health risks, including SPMs, are becoming increasingly important because the improved longevity of CLL patients may be offset by these unwanted risks [42].In patients treated with Bruton's tyrosine kinase inhibitors, the risk and the spectrum of SPMs were similar to that reported following chemotherapy or chemoimmunotherapy [11,43].In our cohort we could not calculate the SIRs for patients receiving novel approaches due to the detection of only two SPMs and a short follow-up time in the last calendar period (2014-2019) in which these agents have become available in the Netherlands [44].When using the calendar period as a proxy for the evolution of treatment over time, the risk and the spectrum of SPMs were comparable for the 2003-2009 and 2010-2019 periods, suggesting that both the introduction of chemoimmunotherapy and, in part, targeted therapies did not dramatically alter the SPM landscape [45].Therefore, future research is warranted to assess whether the broader application of targeted therapies might alter the SPM spectrum of solid and hematological cancers in patients with CLL.Also, future research with more detailed patient-level data on CLL-specific characteristics, the entire treatment landscape, and non-treatment-related exposures that may increase the risk of cancer development (e.g., tobacco use and UV exposure) should adopt multi-state modeling to explore sequences of these exposures on SPM development [46]. The strength of our study is the use of population-based data from a comprehensive, long-running, and well-established cancer registry, enabling us to accurately quantify the risk of developing an SPM over a 30-year period post-diagnosis.Limitations concern the lack of information on the exposure to well-known carcinogens and the exact therapeutic regimens during the overall course of the disease. Collectively, identifying and managing SPMs is an essential part of the longevity of patients with cancer, especially in diseases with therapeutic advances that contribute to a noticeable improvement in survival, such as in CLL.Our findings can be used in shared decision-making about appropriate surveillance activities and interventions to counteract the increased morbidity and excess mortality associated with SPMs.The current study serves as a benchmark to assess how the spectrum of SPMs may alter with a broacher application of targeted therapies. Fig. 3 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to the latency time.Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude.AER absolute excess risk per 10,000 person-years, CI confidence interval, SIR standardized incidence ratio. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "The data that support the findings of this study are via The Netherlands Comprehensive Cancer Organisation.These data are not publicly available, and restrictions apply to the availability of the data used for the current study.However, these data are available upon reasonable request and with permission of The Netherlands Comprehensive Cancer Organisation. Fig. 4 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to the receipt of antineoplastic therapy.Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude.AER absolute excess risk, CI confidence interval, SIR standardized incidence ratio. ",
"section_name": "DATA AVAILABILITY",
"section_num": null
}
] |
[
{
"section_content": "The authors would like to thank the registration clerks of the Netherlands Cancer Registry (NCR) for their dedicated data collection.The nationwide population-based NCR is maintained and hosted by the Netherlands Comprehensive Cancer Organisation (IKNL). ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "A.G.D. and Lvd.S. designed the study; Lvd.S. and M.A.W.D. analyzed the data; O.V. was responsible for the data collection; Lvd.S. wrote the manuscript with contributions from all authors, who also interpreted the data, and read, commented, and approved the final version of the manuscript. The authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s41408-023-00784-z. Correspondence and requests for should addressed to Lina van der Straten. 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": "A.G.D. and Lvd.S. designed the study; Lvd.S. and M.A.W.D. analyzed the data; O.V. was responsible for the data collection; Lvd.S. wrote the manuscript with contributions from all authors, who also interpreted the data, and read, commented, and approved the final version of the manuscript. ",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-023-00784-z. Correspondence and requests for should addressed to Lina van der Straten. 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
}
] |
10.1007/s11605-011-1718-0
|
“Surgical” Abdomen in a Patient with Chronic Lymphocytic Leukemia: A Case of Acquired Angioedema
|
Acquired angioedema (AAE), an acquired deficiency of C1esterase inhibitor, is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen. This disorder is strongly associated with chronic lymphocytic leukemia (CLL) and other indolent lymphoplasmacytic disorders.We describe a patient with known CLL who developed incapacitating, recurrent severe abdominal pains, culminating in partial bowel resection. Signs, symptoms, laboratory and pathologic findings demonstrated AAE.Wider appreciation of the possibility of AAE, particularly in patients with lymphoproliferative disorders, could lead to preventive therapy and spare unnecessary surgery. This is more important now that more effective medical therapies are available.
|
[
{
"section_content": "Acquired angioedema (AAE) is due to acquired deficiency of C1 inhibitor (C1Inh), resulting in excessive complement and bradykinin activities.Blood vessel permeability is increased; thus, angioedema occurs.Just as with hereditary angioedema (hereditary C1Inh deficiency; HAE), common clinical manifestations are skin swelling, laryngeal edema, and/or abdominal pain. 1,2AE often occurs in the context of lymphoplasmacytic disorders, such as monoclonal gammopathy of unknown significance (MGUS), non-Hodgkin's lymphoma, or chronic lymphocytic leukemia (CLL). 1,3Among 32 patients with AAE, Castelli found that 13 (40%) had MGUS and 9 (28%) had lymphoproliferative disease. 3erefore, all cases of AAE should be evaluated for the possibility of underlying lymphoplasmacytic disorder.Conversely, when patients with known lymphoproliferative disease manifest compatible symptoms, AAE should be expeditiously considered.This is important because AAE can be effectively treated medically, but delayed diagnosis can lead to unnecessary diagnostic procedures, therapeutic interventions, or life-threatening complications, well-illustrated by our case. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "A 78-year-old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy.One year earlier, she had been diagnosed with Rai Stage I CLL which had been observed without treatment.Two months earlier, she presented with severe abdominal pain, nausea, and vomiting.Over the next 8 weeks, she had six emergency room and/or hospital admissions for identical symptoms.The episodes left the patient weak and incapacitated.Pains would begin at rest in the lower abdomen, spread to the upper abdomen, described as \"gas-like\", non-radiating, constant.There was no association with meals, exertion, or bowel movements.Subsequent vomiting was nonbloody, nonbilious, and did not relieve the pain.Episodes resolved spontaneously within 2-3 days with intravenous hydration and pain control.Extensive evaluation included colonoscopy, endoscopic retrograde cholangiopancreatography, magnetic resonance angiography, and abdominal aortography.Benign colon polyps and small gallstones were removed, mesenteric stenoses ruled out, yet pains recurred unabated.She had lost 12 lbs.There was no dysphagia, change in bowel habits, GI bleeding, fever, or sweats.Medications included baby aspirin, clopidogrel, benazepril, hydrochlorothiazide, and glipizide. Physical examination on transfer revealed blood pressure 130/88 mmHg, pulse 88 beats per minute, respiratory rate 20 cycles per minute, pulse oximetry of 95% on ambient air, and temperature 97.0°F.The patient was in acute distress due to abdominal pain.Left cervical and axillary lymph nodes were enlarged to 1.5 cm in diameter.Cardiopulmonary examination was unremarkable.Abdomen was slightly distended, diffusely exquisitely tender with guarding and rebound.Bowel sounds were hypoactive.Rectal examination showed guaiac-negative brown stool. Hemoglobin was 18.1 g/dL, hematocrit 55%, platelets 146×10 9 /L, and leukocytes elevated to 34,500 cells/μL with 47% neutrophils and 48% lymphocytes.Chemistries and liver function tests were normal.Abdominal X-ray showed no free air, nor air-fluid levels.CT scan of abdomen and pelvis with IV contrast showed multiple abnormal loops of small bowel with contrast-enhanced bowel wall edema (Fig. 1a). At laparotomy, massively swollen small bowel was encountered and resected.Pathologic examination revealed massive submucosal edema (Fig. 1b).There was no leukemic infiltration visible. A hematology consultant, called postoperatively, suspected AAE.C4 was 3 mg/dL (normal 17-46), C3 66 mg/dL (85-200), and C1Inh activity reportedly 83% (68-200%).Serum protein electrophoresis revealed two faint bands immunofixing as monoclonal IgM kappa and IgG kappa.Chlorambucil was started for CLL and danazol to raise C1Inh.Lymphocytosis and lymphadenopathy improved and C1Inh activity increased to 110%.Over 3 years of follow-up, abdominal symptoms never recurred. ",
"section_name": "Case Presentation",
"section_num": null
},
{
"section_content": "Approximately 145 cases have been reported of AAE, 3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from AAE with associated CLL.Table 1 shows characteristics of patients with CLL and AAE we have seen (some briefly mentioned in a prior report). 4This is our only case to undergo surgery, allowing unique and dramatic demonstration of massive bowel edema visible radiographically, on surgical inspection and on histopathology.An initial C1Inh activity was reported low normal, but there can be no doubt about the diagnosis based on radiologic/surgical/histologic findings, further laboratory results, and the clinical course.Characteristic are very low C4 level and low C3; diagnostic recommendations currently add C1q and anti-C1Inh antibody levels.Autoantibody is demonstrable in up to 70% with AAE. 1 Our patient had monoclonal gammopathy, which frequently corresponds to the C1Inh autoantibody.Patients with the hereditary form (HAE) Angioedema should be borne in mind among \"medical\" illnesses that can mimic acute surgical abdomen, along with such other disorders as porphyria, Familial Mediterranean fever and sickle cell disease (Fig. 2).One may need to discern whether a true surgical emergency might supervene even when one of these disorders is present.Our patient had typical symptoms of acute bowel edema, including diffuse abdominal pain, occasionally rebound tenderness and vomiting, with spontaneous resolution within 1-5 days. 1,2Some patients with AAE have cutaneous or upper respiratory edema in addition, or instead of, bowel symptoms.Because of cardiovascular comorbidities, there was a high suspicion for ischemic bowel in our patient, but radiography and endoscopy did not support this.In the differential diagnosis, angiotensin-converting enzyme inhibitors rarely precipitate angioedema, but our patient had taken benazepril many years and continued it without incident after AAE therapy. In 2009, the US FDA approved the C1Inh concentrate Berinert P® and the kallikrein inhibitor ecallantide (Kalbitor®) for treatment of acute attacks, and the C1Inh concentrate Cinryze® for prophylaxis in severely affected patients. 5resh frozen plasma can be given when these are unavailable.6][7][8][9] Doses used in the clinical HAE trials may need to be higher for AAE because of increased enzyme clearance.Ecallantide is subcutaneous, facilitating patient self-administration out of hospital.A bradykinin B2 receptor antagonist icatibant (Firazyr®) is under investigation.For long-term control, the underlying cause should be addressed, CLL in our case.Commonly used for prophylaxis are anti-fibrinolytic drugs or the attenuated androgen danazol, which increases C1Inh synthesis at low cost. 10 ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Recurrent episodes of nausea, vomiting, abdominal pain or diarrhea May have rebound tenderness on exam Spontaneous recovery in 1-5 days CT scan with PO/IV contrast: may reveal bowel wall edema-(\"donut sign\") May have concurrent or history of cutaneous or upper respiratory tract edema ",
"section_name": "Symptoms & Signs",
"section_num": null
},
{
"section_content": "",
"section_name": "Especially consider Acquired AE when",
"section_num": null
},
{
"section_content": "",
"section_name": "Prophylaxis",
"section_num": null
},
{
"section_content": "C1Inh concentrate (Cinryze ® ) Androgen (eg.danazol) Anti-fibrinolytic drugs (eg.tranexamic acid) Fig. 2 Algorithm for diagnosis and treatment for suspected AAE In conclusion, earlier suspicion for AAE in our known CLL patient could have spared her the morbidities of recurrent abdominal pains, hospitalizations, morbid interventions, and bowel resection.Wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. ",
"section_name": "Acute attack",
"section_num": null
}
] |
[] |
10.3324/haematol.2014.122069
|
Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide-B
|
RNA splicing plays a fundamental role in human biology. Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematologic malignancies. We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from patients with chronic lymphocytic leukemia and leukemia-lymphoma cell lines. We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention - spliceosome modulation. This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes. The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment. In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model. Overall, these findings give evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide an additional rationale for the development of spliceosome modulators for cancer therapy.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. 1Despite improvements in the survival of patients who are treated with chemoimmunotherapy, 2 there is still no cure for this disease, except allogeneic bone marrow transplantation.High-risk patients, such as those with deletions in chromosome 17 -Del(17p) or TP53 mutations, generally fail to respond to chemotherapy and have a very poor prognosis. 3As such, there is a need for development of therapeutic agents that target novel pathways in CLL. 4 Splicing, the removal of introns and joining of exons from nascent pre-mRNA, has gained attention as a target for cancer therapy given the distinct splicing patterns identified both in tumor cells and metastatic tumor populations. 5,6Recently, a series of studies identified heterozygous missense mutations in U2AF1 and splicing factor 3B subunit 1 (SF3B1) genes associated with myelodysplastic syndromes, and have shown that SF3B1 is frequently mutated in myelodysplastic syndromes, 7,8 and CLL. 9,10This, combined with the identification of small molecules that target the spliceosome, motivated us to explore the application of these agents to CLL. Identified in 1994, 11 FD-895 was the first member of a large family of polyketides isolated from related strains of Streptomyces platensis, which includes pladienolide-B (PLAD-B) and pladienolide-D (PLAD-D) (Online Supplementary Figure S1). 12PLAD-B targets the splicing factor subunit SF3b and this interaction is postulated to be responsible for its mechanism of antitumor activity. 13Pladienolide-resistant clones from WiDr and DLD1 colorectal-cancer cell lines shared an identi-cal mutation at Arg 1074 (R1074H) in the SF3B1 gene suggesting that this mutation is critical for its anti-cancer activity via spliceosome modulation. 14,15dditional work has also identified other small molecules with splicing modulator activity including spliceostatin A, herboxidiene, isoginkgetin, and E7107, a compound that has been tested in phase I clinical studies in which it has shown clinical activity, albeit with unexpected visual toxicities. 16dditional data will be required to define the role of this and other related compounds as potential anti-cancer agents. Here, we present studies using FD-895 and PLAD-B on primary leukemia cells derived from CLL patients and leukemia and lymphoma cell lines.We anticipate that these studies will provide the foundation for future development of pharmacologically-optimized spliceosome modulators. 17 ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "For in vivo study, PLAD-B was purchased from Santa Cruz Biotechnology (catalog # sc-391691).For in vitro studies, FD-895 was prepared by total synthesis in our laboratories, 17 and PLAD-B was obtained as a gift from Merlion Pharmaceuticals.Fludarabine (F-ara-A) (catalog # F9813) and Bendamustine (catalog # B5437) were obtained from Sigma-Aldrich. Peripheral blood mononuclear cells from CLL patients were obtained from the CLL Research Consortium tissue bank.After the diagnosis of CLL had been confirmed, 18 patients provided written informed consent to collection of blood samples in a protocol approved by the Institutional Review Board of the University of California, San Diego (USCD) in accordance with the Declaration of Correspondence: [email protected] or [email protected] splicing plays a fundamental role in human biology.Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematologic malignancies.We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from patients with chronic lymphocytic leukemia and leukemia-lymphoma cell lines.We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention -spliceosome modulation.This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes.The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment.In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model.Overall, these findings give evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide an additional rationale for the development of spliceosome modulators for cancer therapy. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Analysis of RNA-seq data from CLL cells and NBC showed differences in splicing efficiency between the two.In order to assess splicing efficiency quantitatively, the intron retention (IR) ratios were calculated for each gene by comparing the RNA-seq read density in exons, measured in fragments per kilobase per million fragments mapped, relative to the RNA-seq density of introns (Online Supplementary Table S2).Comparison of IR ratios showed that several genes have widespread changes in splicing efficiency, resulting in a basal increase of intron/exon RNA ratios in untreated CLL cells compared to NBC (1.4-fold average increase, Figure 1A and Online Supplementary Figure S2A,S2B).An average 71% of sequenced genes showed increased IR in CLL cells compared to NBC (Online Supplementary Table S3).Moreover, after incubation of CLL cells with FD-895 or PLAD-B there was an additional increase in IR ratios (2.2-fold average increase, Figure 1B, Online Supplementary Figures S2C, S2D, and3A), but this was not observed after F-ara-A treatment (Figure 1C, 1D).We observed that treatment with FD-895 or PLAD-B induced IR more significantly in genes with the lowest basal IR suggesting a de novo spliceosome modulation induced by these compounds rather than exacerbation of an existing abnormal splicing process (Online Supplementary Figure S4).The DNAJB1 gene encodes a chaperone which is a member of the DnaJ or Hsp40 (heat shock protein 40 kD) family of proteins.DNAJB1 was one the genes that showed an increased IR ratio after treatment with FD-895 or PLAD-B (7-fold increase, Figure 1A-C) and because of that, this gene was selected for the validation studies described below. We performed an unsupervised cluster analysis of 3,500 highly expressed genes using IR ratios of CLL cell and NBC samples treated with FD-895, PLAD-B, or F-ara-A or untreated controls.We observed that IR ratios from untreated samples clustered together with F-ara-A samples, indicating minimal IR changes induced by F-ara-A regardless of the cell type (CLL or NBC).Distinctively, FD-895 and PLAD-B treated samples showed higher IR ratios compared to untreated controls or F-ara-A-treated samples (Figure 1D, Online Supplementary Figure S5).Additionally, we analyzed gene expression profiles of the same samples and we found that unlike IR ratios, gene expression profiles were segregated by cell type forming NBC and CLL clusters.Untreated cells and F-ara-A-treated samples clustered together while samples treated with FD-895 or PLAD-B showed similar gene expression profiles (Figure 1E).We selected the top 50 genes that were affected by IR induced by FD-895 and found that the majority of those belong to pathways representing RNA splicing and gene regulation, signal transduction, endoplasmic reticulum stress and apoptosis (representative genes from those pathways include DNAJA1, DNAJB1, ABT1, NFKB1, GPX1, SLC2A3, HERPUD1, RBM4-RBM14, RBM8A, and WTAP) (Figure 1F, Online Supplementary Figure S5).There was a similar pathway distribution when the gene sampling was expanded to the top 900 targets affected by IR (Online Supplementary Figure S3B). Using gene ontology (GO) functional enrichment analysis we found that incubation with FD-895 and PLAD-B induced IR mainly in pathways that are critical for RNA editing/processing, cell survival and cell cycle regulation (Figure 1G, Online Supplementary Figure S3B).Splicing abnormalities in these pathways were present as a baseline finding in untreated CLL cells compared with NBC, although with higher P values (Online Supplementary Figure S3C).We also analyzed the GO enrichment of differential gene expression induced by these compounds and found different pathways that were enhanced, including immune regulation and cell survival, while gene expression of the RNA processing and editing pathway was significantly down-regulated (Figure 1G). ",
"section_name": "FD-895 and PLAD-B induce intron retentionspliceosome modulation",
"section_num": null
},
{
"section_content": "Because IR is a process considered to be a surrogate marker of spliceosome modulation, 20 and because the RNA-seq data suggested a process of IR, we decided to perform a series of validation studies in cells treated with FD-895 and PLAD-B under different conditions.For these experiments, we selected DNAJB1, a gene that has been used in previous studies, 9 and one of the highest ranked genes showing an IR pattern (Figure 1B, 1C, and1F). CLL samples were incubated with a concentration gradient of FD-895, PLAD-B, bendamustine, or F-ara-A for 4 h (Figure 2A).After treatment, the levels of spliced and unspliced gene expression were evaluated by reverse transcriptase PCR and real-time quantitative PCR.While CLL cells treated with nanomolar concentrations of FD-895 or PLAD-B showed IR, this was not observed in samples treated with bendamustine or F-ara-A, even at supra-physiological concentrations (Figure 2A).FD-895 or PLAD-B did not induce IR in the housekeeping gene GAPDH (Figure 2A and Online Supplementary Figure S6A).IR was observed in cells treated with FD-895 or PLAD-B for all three genes examined, including: DNAJB1, RIOK3 (Figure 2B, Online Supplementary Figure S6B), and BRD2 (data not shown).The process of IR was time-dependent and occurred within 15 min of treatment (Figure 2B). We quantified IR using real-time quantitative PCR and observed a time-dependent increase of unspliced DNAJB1 in samples treated with FD-895 or PLAD-B, but not with Fara-A (Figure 2C).In addition, we found that FD-895 and PLAD-B induced ~4 fold increases in IR for both DNAJB1 and RIOK3 in CLL cells when compared to NBC (P<0.0001, Figure 2D,2E). ",
"section_name": "Validation of FD-895 and PLAD-B induced intron retention/spliceosome modulation in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "The RNA-seq data showed IR in the MCL-1 gene after Targeting the spliceosome in leukemia and lymphoma haematologica | 2015; 100 (7) 947 S6C).We, therefore, investigated whether or not spliceosome modulation/IR by FD-895 and PLAD-B was associated with regulation of alternative splicing using MCL-1 and BCL-X, two genes involved in apoptosis, which are known to depend on alternative splicing for their functions, pro-apoptotic (short isoform) and anti-apoptotic (long isoform). 21We observed that treatment of CLL cells with FD-895 or PLAD-B induced expression of the short/pro-apoptotic alternatively spliced isoforms for both MCL-1 (Figure 3A) and BCL-X (Figure 3B).This process was time-dependent, occurring within 30 min of treatment.In contrast, there was no evidence of alternative splicing in NBC (Figure 3A, 3B).In addition, we did not observe IR or alternative splicing in GAPDH (Figure 3C and Online Supplementary Figure S6A). Using gel electrophoresis and densitometric analysis we calculated the ratio of small to large (S/L) isoforms for MCL-1 and BCL-X, which is used as a marker for apoptosis. 21We found a higher S/L ratio (P<0.0001) for MCL-1 (Figure 3D) and BCL-X (Figure 3E) in CLL cells treated with FD-895 or PLAD-B, but there were no changes in NBC.S/L ratios remained unchanged in F-ara-A-treated cells. ",
"section_name": "The activity of FD-895 and PLAD-B in chronic lymphocytic leukemia cells is associated with regulation of alternative splicing",
"section_num": null
},
{
"section_content": "We cultured CLL cells in 100 nM FD-895 or 100 nM PLAD-B, and examined apoptosis over time.The minimum time for the irreversible development of apoptosis was determined by exposing the CLL cells to FD-895 or PLAD-B over increasing incubation times up to 48 h, followed by washing twice with media, and continuing to culture for a total of 48 h.We found that the effect of both FD-895 and PLAD-B is irreversible and at least 2 h of incubation was required to induce apoptosis (Figure 4A). ",
"section_name": "FD-895 and PLAD-B induce early irreversible commitment to apoptosis in chronic lymphocytic leukemia cells within 2 hours of treatment",
"section_num": null
},
{
"section_content": "FD-895 and PLAD-B induced apoptosis in CLL cells at nanomolar concentrations with an IC 50 in the range of 5.1-138.7 nM (Figure 4B,4C).In contrast, NBC and normal T cells were resistant to the activity of both compounds, and the IC 50 value was not achieved even when concentrations >1 μM were used (Figure 4D). In order to assess the activity of FD-895 and PLAD-B under stringent conditions that resemble the anti-apoptotic tumor microenvironment, 16 we incubated CLL cells with increasing concentrations of FD-895, PLAD-B (0.01 -1 μM) and F-ara-A (1-10 μM) alone or with stroma-NK-tert cells.Stroma-NKtert cells increased the viability of CLL cells and inhibited the activity of F-ara-A by more than 50%.However, FD-895 and PLAD-B were able to overcome the anti-apoptotic effect of the stroma cell support without causing direct cytopathic effects on stroma-NK-tert cells (Figure 4E).S1).The untreated controls (CLL cells or NBC) for DNAJB1 and RIOK3 were set to a value of 1. GAPDH was used as a control for normalization. ",
"section_name": "Determination of FD-895 and PLAD-B IC 50 values and effect of co-culture conditions that resemble the tumor microenvironment",
"section_num": null
},
{
"section_content": "We evaluated the anti-leukemia activity of FD-895 or PLAD-B in cells derived from CLL patients with Del(17p) and/or inactivating mutations in TP53.The samples were cultured alone or with stroma-NK-tert cells and harvested after treatment, at 48 h, and apoptosis was measured by flow cytometry.Cells derived from CLL patients with Del(17p) and/or TP53 mutations [% Del(17p) by fluorescence in situ hybridization, 66-99.5%]displayed resistance to F-ara-A, with IC 50 values >10 μM (Figure 4C), while wild-type CLL samples underwent apoptosis when treated with F-ara-A at IC 50 values of ~1 μM (Figure 4B).In contrast to the differential sensitivity to Fara-A based on TP53 status, we observed that CLL samples underwent apoptosis with FD-895 or PLAD-B at IC 50 values of 10-50 nM, regardless of the presence of Del(17p) or TP53 mutations (Figures 4B,4C, Figure 5).Moreover, B and T lymphocytes from healthy volunteers were resistant to the pro-apoptotic activity of FD-895 or PLAD-B, and showed significantly lower levels of apoptosis after 48 h in culture compared with CLL cells (Figure 4D, Figure 5). ",
"section_name": "FD-895 and PLAD-B selectively induce apoptosis in chronic lymphocytic leukemia cells but not in normal lymphocytes in a TP53-independent manner",
"section_num": null
},
{
"section_content": "",
"section_name": "Evaluation of the activity of FD-895 and PLAD-B across different human leukemia and lymphoma cell lines",
"section_num": null
},
{
"section_content": "We also tested whether treatment with FD-895 or PLAD-B could induce apoptosis in cells from CLL patients with wild-type or mutant SF3B1 (Online Supplementary Table S4).We found that treatment with 100 nM FD-895 (Figure 5A) or 100 nM PLAD-B (Figure 5B) induced cell death in CLL cells regardless of their SF3B1 mutational status with levels of apoptosis that were significantly higher compared to those of normal lymphocytes (Figure 5). ",
"section_name": "FD-895 and PLAD-B induce apoptosis in chronic lymphocytic leukemia cells independently of TP53 and SF3B1 mutational status",
"section_num": null
},
{
"section_content": "We performed a colorimetric-proteolytic assay to determine if FD-895 or PLAD-B induced apoptosis through a caspase-dependent mechanism.Treatment of CLL cells with FD-895 or PLAD-B induced activation of caspases 3, 6, 8, and 9 (Figure 6A).There was no caspase activation in NBC treated with either FD-895 or PLAD-B (Figure 6A).gesting that caspase activation is necessary for FD-895and PLAD-B-induced apoptosis (Figure 6B). ",
"section_name": "FD-895 and PLAD-B induce apoptosis via a caspase-dependent pathway",
"section_num": null
},
{
"section_content": "We analyzed the levels of PARP and Mcl-1 by western blot analysis in CLL cells after treatment with 100 nM FD-895, 100 nM PLAD-B or 10 μM F-ara-A.CLL cells were treated for 6 h and 24 h for Mcl-1 and PARP, respectively.We observed increased levels of cleaved PARP (89 kDa) in CLL cells treated with FD-895, PLAD-B or the control Fara-A.However, as observed in the Mcl-1 alternative splicing experiments, only samples treated with FD-895 or PLAD-B showed down-regulation of the anti-apoptotic isoform of Mcl-1 (48 kDa) (Figure 6C). ",
"section_name": "Apoptosis induced by FD-895 or PLAD-B in chronic lymphocytic leukemia is associated with regulation of PARP and Mcl-1",
"section_num": null
},
{
"section_content": "FD-895 and PLAD-B both induce apoptosis and IR at nanomolar concentrations in a wide range of cell lines including A20 murine lymphoma (Figure 6D).To assess whether the in vitro activity of these compounds correlates with antitumor activity in vivo, we treated BALB/c mice bearing subcutaneous A20 lymphoma tumors with intraperiotoneal injections of PLAD-B for 5 consecutive days.We observed tumor regression and improved survival (P<0.001) in the group of mice treated with PLAD-B.There was a dose-dependent effect that favored higher doses of this compound (3 mg/Kg/day versus 10 mg/Kg/day).In contrast, mice injected with vehicle or dexamethasone control continued to show tumor progression (Figure 6E).After 35 days of follow up, none of the vehicle-or dexamethasone control-treated mice had survived while 33% (low dose) and 83% (high dose) of PLAD-B-treated mice were still alive (Figure 6F).None of the mice treated with PLAD-B showed evidence of toxicity, behavioral changes, diarrhea, rough coat, withdrawal, weight loss or gross visual impairment. 22 ",
"section_name": "Induction of intron retention and apoptosis correlates with tumor regression in vivo",
"section_num": null
},
{
"section_content": "Gene expression consists of several steps, including transcription, pre-mRNA processing (capping, splicing, and poly-adenylation), mRNA surveillance, and mRNA export.These steps are extensively coupled to form 'gene expression factories', 23 whose modulation, if selective, offers new avenues into cancer therapy.Among these events, the regulation of mRNA maturation by the spliceosome has gained significant recognition as a potential therapeutic target due to the recent discovery of muta- ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "M.K. Kashyap et al.",
"section_num": null
},
{
"section_content": "tions in genes associated with the spliceosome.These include mutations in U2AF35, ZRSR2, SRSF2 and SF3B1, which have been reported in solid tumors as well as hematologic malignancies including CLL and myelodysplastic syndromes. 9,10,24n CLL, SF3B1 mutations are found in ~10-15% of cases and constitute an independent prognostic factor associated with rapid progression and short survival with a frequency that increases after exposure to chemotherapy, suggesting clonal selection of SF3B1 mutant cells after treatment. 9,10,25SF3B1 is upregulated in CLL cells compared with NBC, possibly due to epigenetic regulation through hypomethylation. 26In addition, in silico data strongly suggest that hot spot mutations of SF3B1 in CLL are in fact oncogenic gain-of-function mutations conferring SF3B1 attributes of a proto-oncogene. 27It is, therefore, plausible that the expression of mutated SF3B1 could result in \"hyper-activation\" of the spliceosome system leading to the generation of oncogenic/leukemogenic alternatively spliced mRNA isoforms that participate in cell survival, proliferation and possibly chemoresistance.Although the causative link between SF3B1 mutations and CLL pathogenesis remains unclear, recent findings suggest that SF3B1 mutations might be linked not only to deregulation of the spliceosome but also to genomic instability and epigenetic alterations. 28n this project, we studied FD-895 and PLAD-B, two polyketides, whose biological activity has been associated with the ability to modulate splicing by targeting SF3b. 15e evaluated the effects of these two compounds in primary leukemia cells from CLL patients as well as normal lymphocytes and leukemia-lymphoma cells lines. RNA-seq transcriptome analysis allowed us to analyze the effect of FD-895 and PLAD-B from a global perspective.We used fragments per kilobase per million fragments mapped intron/exon ratios as a surrogate marker of IR/spliceosome modulation. 13,29By calculating IR ratios, we can estimate the relative efficiency of splicing for each gene and indirectly assess the functional status of the spliceosome system. Our initial experiments showed an increased pattern of IR in untreated CLL cells compared with NBC.This was somewhat unexpected, although amplified IR has been recently reported in solid tumors including lung and breast cancers. 30,31To our knowledge, this is the first time that a basal increase in IR has been reported in CLL or hematologic malignancies.This suggests that baseline abnormalities that involve the RNA processing/spliceosome system could be implicated in CLL leukemogenesis. We observed that FD-895 and PLAD-B very rapidly induced a generalized process of IR in which the majority of genes (>82%) showed intronic sequences after incubation with this agent but no IR in a housekeeping gene (GAPDH) or intronless genes.The majority of genes (25%) with the highest levels of IR belonged to the gene regulation/RNA splicing pathway.This strongly suggests that the IR effect mediated by FD-895 and PLAD-B involves a broad process that is not stochastic in nature and that certain pathways, mainly RNA processing/editing, are highly sensitive to the effect of these compounds and may provide an explanation for their mechanism of action.In addition, genes with a high basal IR ratio showed the lowest rates of IR increase after treatment.Contrariwise, genes that initially had low levels of IR were the ones that showed the highest post-treatment increase in IR (Online Supplementary Figure S4).Overall, this suggests that the activity of the compounds used in our experiments induced a de novo pattern of spliceosome inhibition more than exacerbation of an existing abnormal splicing process. Our data show that structurally similar macrolides (PLAD-B and FD-895) have common potent pro-apoptotic Targeting the spliceosome in leukemia and lymphoma haematologica | 2015; 100 (7) 951 FD-895 and PLAD-B induced early and irreversible commitment to apoptosis within 2 h of treatment (Figure 4A).These nanomolar concentrations compare favorably with pharmacokinetic data of E7107, a compound used in clinical trials. 16In addition, these polyketides not only produced effects in vitro but also demonstrated encouraging clinical activity in vivo using the A20 lymphoma mouse model.Overall, this suggests the potential applicability of these compounds in cancer therapy.We found a striking difference between the activity of FD-895 and PLAD-B compared with chemotherapy agents such as bendamustine and F-ara-A.Even though CLL cells ultimately die after incubation with these latter, we never observed evidence of IR induced by these agents.In contrast, a hallmark of the apoptosis induced by FD-895 and PLAD-B was early induction of IR.IR after incubation with FD-895 and PLAD-B was not only very rapid, occurring within minutes of incubation, but also took place at nanomolar concentrations and was at least 50% to 75% higher in CLL cells than in NBC.Overall, these findings strongly suggest that the mechanism of action of FD-895 and PLAD-B is mediated, at least in part, by IR/spliceosome modulation. The pro-apoptotic activity of FD-895 and PLAD-B was observed in all malignant cells tested (CLL, Ramos, Jurkat, Raji, A20 mouse lymphoma cell line), but it was much lower in normal T and B lymphocytes suggesting that malignant cells may be more dependent on the spliceosome system for their survival.This finding shows a therapeutic window that could facilitate future clinical development of this class of compounds. Not only did we observe IR induced by FD-895 and PLAD-B but we also found that these compounds have the ability to regulate alternative splicing.We demonstrated this by using apoptosis-related genes (MCL-1, BCL-X), which are known to be dependent on alternative splicing for their function. 32After incubation with FD-895 and PLAD-B we observed a change of isoform ratios that favor the expression of pro-apoptotic isoforms.This was not observed with F-ara-A or bendamustine.It is very likely that the modulation of alternative splicing induced by these compounds is broader and may involve other genes critical for cancer cell survival, proliferation, etc. Importantly, we did not observe alternative splicing induced by FD-895 or PLAD-B in normal lymphocytes suggesting that this is probably a very important pro-apoptotic mechanism of action that explains the differential sensitivity observed in CLL cells compared to NBC. In our experiments we used an in vitro model of stromal cell support that mimics conditions present in the tumor microenvironment.This is important, as the tumor microenvironment has been shown to provide anti-apoptotic protection to cancer cells, including CLL, and could be responsible for chemoresistance as well as persistence of minimal residual disease. 33Under these conditions, we showed that the pro-apoptotic activity of FD-895 and PLAD-B overcomes the protective effect of the microenvironment, suggesting that spliceosome modulators may have potential applications in refractory cancer and eradication of minimal residual disease. The pro-apoptotic activity of FD-895 and PLAD-B was independent of Del(17p) -TP53 or SF3B1 mutations found in CLL.Both of these compounds displayed com-parable activity in cells from CLL patients as well as cell lines with these genetic abnormalities and resistance to Fara-A (known for its p53-dependent cytotoxicity).The fact that the activity of FD-895 and PLAD-B is independent of p53 opens significant opportunities for the development of therapies for refractory malignancies in which the prevalence of p53 dysfunction is significantly high. 34n addition, the finding that the pro-apoptotic activity of these agents was independent of SF3B1 mutational status was somewhat unexpected, mainly because in vitro data have shown that SF3B1 mutations may confer resistance to this kind of compound. 15There are various potential explanations for this observation.First, all the mutations described in CLL patients (including the patients tested at UCSD) cluster in the heat repeat region 2-6 of the SF3b1 protein. 24,35On the other hand, the R1074H mutation that confers resistance to PLAD-B is located in a distant domain (9 th heat repeat region, Online Supplementary Figure S7).This highlights functional differences of these mutations and their impact on FD-895 and PLAD-B activity.Second, the activity of PLAD-B and FD-895 may be more complex than simply binding to SF3B1 as these compounds can also target other members of this protein family including SF3B1, SF3B3, SF3B4, and SF3B2 and potentially other proteins of the spliceosome system.Third, the fact that all CLL samples tested were sensitive to FD-895 and PLAD-B, regardless of SF3B1 mutation status, highlights the importance of this pathway in CLL and the potential relevance of a more global spliceosome deregulation in cancer. We found that the pro-apoptotic activity of FD-895 and PLAD-B in CLL cells was caspase-dependent (with activation of the intrinsic and extrinsic pathways) and correlated with changes in apoptosis-related proteins including poly (ADP-ribose) polymerase (PARP) cleavage and Mcl-1 (long isoform) down-regulation.It is possible that the IR and alternative splicing induced by FD-895 and PLAD-B tips over the strong anti-apoptotic protein balance that favors survival in CLL and other malignancies. 36This could have potential implications in terms of synergistic activity of these compounds in combination with other anti-cancer agents. Together, our studies provide a solid foundation for further therapeutic development of this class of compounds.We have shown for the first time that FD-895 and PLAD-B induce a global pattern of IR/spliceosome modulation and regulation of alternative RNA splicing at low nanomolar concentrations, and that the functional consequence of this process leads to apoptosis in vitro and in vivo preferentially in cancer cells.This constitutes evidence that the spliceosome system is a relevant target in leukemia and lymphoma and potentially other cancers. Our current efforts using key structure-activity relationships are focused on adapting this knowledge to further guide analog discovery and synthesis, 17 with the ultimate goal of developing novel spliceosome modulators for cancer therapy. ",
"section_name": "A B C D E F G H",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "TJK, and JEC, the UC San Diego Foundation Blood Cancer Research Fund (to TJK), the Bennett Family Foundation (to JEC), and support provided by Moores Cancer Center-UCSD for open access publication.",
"section_num": null
},
{
"section_content": "Targeting the spliceosome in leukemia and lymphoma haematologica | 2015; 100 (7) ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Targeting the spliceosome in leukemia and lymphoma haematologica | 2015; 100 (7) ",
"section_name": "The authors would like to thank the following organizations for their grant support: Lymphoma Research Foundation (LRF, grant #285871) to JEC and MDB, the American Cancer Society (RSG-06-011-01-CDD) to MDB, the National Institutes of Health (R01-GM086225) to MDB, the National Institutes of Health (PO1-CA081534)-CLL Research Consortium Grant to",
"section_num": null
},
{
"section_content": "",
"section_name": "Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.",
"section_num": null
}
] |
10.1038/s41598-017-12246-1
|
Identification and characterization of a novel Sso7d scaffold-based binder against Notch1
|
<jats:title>Abstract</jats:title><jats:p>Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11–13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.</jats:p>
|
[
{
"section_content": "Tenzin Gocha 1,3 , Balaji M. Rao 2 & Ramanuj DasGupta 1,3 Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers.Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials.However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds.Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest.Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold.N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes.We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1.Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3.Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies. Cell signaling constitutes a multitude of highly regulated protein-protein interactions (PPIs) through which cells communicate with each other.Misregulated cell signaling leads to disease, including cancer 1 .It is therefore important to develop novel molecular tools to disrupt specific PPIs that can help elucidate the nature and function of individual PPIs in modulating cell signaling, and subsequently cellular phenotypes.Furthermore, such tools have the potential to translate into efficient therapeutics.However, the ability to specifically inhibit a PPI in a given context remains challenging.Commonly used tools such as genetic knockouts or siRNA-mediated knockdown of protein expression cannot selectively target specific PPIs 2 .Additionally, the use of genetic manipulation to introduce targeted mutations or truncations may affect overall protein stability and confound studies on PPIs.Screening of small molecule libraries have resulted in identification of PPI-inhibitors.However, the use of these compounds is limited by their poor target specificity and toxicity 3 .Biologics (protein based drugs) on the other hand, exemplified by monoclonal antibodies (mAbs), have been very useful in perturbing PPIs and its success has been reflected in its recent adoption by the pharmaceutical companies in their drug portfolio [3][4][5][6] .Although mAbs have been very successful, antibodies have certain limitations based on their size (~150 kD), stability, and cost of production that restrict their large-scale adoption for various applications [7][8][9][10] .Due to these limitations, more recently alternative strategies have been adopted to develop affinity reagents 11 .This has led to a series of transitions from mAbs to Fabs (Fragment antigen binding) and then to scFvs (single chain fragment variable) 6,10,12 .ScFvs are approximately 7 times smaller than mAbs and have been shown to have added advantages of stability and productivity 10 . More recently, protein engineering technologies have enabled the use of smaller and synthesizable non-antibody scaffolds for generating binders with high affinity and specificity 4,10,[13][14][15] .Because of small size, robust protein scaffold, and the ease of recombinant expression in bacterial platforms, there is a wide-spread interest in binders based on non-antibody scaffolds for research as well as theranostic applications 7,14 .In this study we describe the identification of Notch1 specific binders by screening a combinatorial library obtained by mutagenesis of the Sso7d protein scaffold using yeast surface display (Fig. 1A) 16 .Sso7d is an ultra-stable 7 KDa protein from the hyperthermophilic archaeon Sulfolobus solfactaricus and has been shown to be a versatile scaffold for the generation of binders to a diverse range of target proteins 9,[16][17][18] .Notch is a receptor mediated signalling pathway, the dysregulation of which has been implicated in various diseases including cancer 19 .The binding of ligands, Jagged1/2 (JAG1/2) and Delta-like 4 (DLL4), to the Notch receptor is known to trigger a series of proteolytic cleavages that eventually results in the generation of the Notch Intracellular domain (NICD).NICD translocates to the nucleus, where it binds to Mastermind and CSL (CBF1, Suppressor of Hairless, Lag-1) to activate transcription of target genes [20][21][22] .The extracellular domain of Notch comprises of EGF Like Repeats (ELR) that have been shown to be critical for ligand binding 23,24 (Fig. 1B).Here we report the identification and functional characterization of an Sso7d variant, called Notch binder clone-9 or N9.We demonstrate that N9 binds to cell-surface expressed Notch1 and that binding of N9 to Notch1 inhibits its interactions with cognate ligand, such as JAG1 and DLL4.Consequently, N9 inhibits proliferation of Notch1-expressing breast cancer cell lines and downregulates expression of Notch target genes.Finally, N9 effectively reduces tumorsphere forming ability of breast and colorectal cancer stem-like cells, a property that has been shown to rely on active Notch signaling 25,26 .Our results highlight the potential use of Sso7d as a non-antibody scaffold for the modulation of cell signaling by perturbing specific ligand-receptor interactions. ",
"section_name": "Identification and characterization of a novel Sso7d scaffold-based binder against Notch1",
"section_num": null
},
{
"section_content": "Screen for sso7d binders to Notch1 ELR 11-15.Binders specific to Notch1 were isolated from a yeast display library of Sso7d mutants (~10 8 diversity), which was previously evaluated for its ability to generate high affinity ligands to different protein targets 13,16 .To identify binders that can interact with Notch1 and possibly modulate its ligand binding activity, we screened the Sso7d library against purified Notch1 ELR 11-15 region that constitutes the ligand binding domain 25,27 .Magnetic sorting and two rounds of FACS was used to isolate a pool of binders with the highest affinity for Notch1 (Supplementary Fig. S1, Fig. 1C). 10 clones were randomly picked from this pool; DNA sequencing identified 8 unique clones (Fig. 1D).Of these, we picked four individual clones for further characterization. N9 binds to Notch1 on the cell surface.Four individual binding clones were recombinantly expressed in E.coli and purified proteins were used to assess binding to NOTCH1 expressing cell lines.Because Notch1 has been shown to be overexpressed in breast cancer 28 , we used two well-studied breast cancer cell lines, MCF7 and MDA-MB-231 for the binding assay.Even though all candidate binders displayed some degree of binding to the two cell lines, clone Notch1-binder 9 (N9) exhibited the most robust binding (Fig. 2A), and was therefore chosen for further analysis. In order to confirm the specificity of N9 binding to endogenous Notch1, we performed siRNA-mediated knockdown of Notch1 in MCF7 cells.Knockdown of Notch1 significantly reduced binding of N9 to MCF7 cells (Fig. 2B, Supplementary Fig. S2A).Conversely, in gain-of-function studies, we observed increased N9 binding to 3T3 cells overexpressing Notch1 (3T3-hN1) compared to the control parental wild type line (3T3-WT) (Fig. 2C).We subsequently measured the binding affinity of N9 using the 3T3-hN1 cell line.N9 bound to 3T3-hN1 with an affinity (Kd) of 300 nM (Supplementary Fig. S2B-C).Notably, in ELISA-based binding assays in vitro, N9 displayed binding to purified ELR 11-15 with affinity of 110 ± 10 nM (Fig. 2D).Strikingly, although the yeast display screen was performed against Notch1 ELR 11-15, we observed cross-reactivity of N9 to other Notch receptors (Notch2/3) as well (Supplementary Fig. S3).Taken together, these results suggest that N9 can interact with Notch1 and that Notch1 expression is at least partly necessary as well as sufficient to confer N9 binding (Fig. 2B,C; Supplemetary Fig. S2A). N9 binds to ELR 11-13 of Notch1 and competes with ligand binding.Since N9 was screened against ELR 11-15, which constitutes the ligand binding domain of Notch1, we first tested the ability of N9 to compete for binding with one of its ligand, JAG1.In competitive ELISA, N9 inhibited Jagged1 binding to ELR 11-15 in a dose-dependent manner, Ki ~70 ± 20nM (Fig. 3A).It has previously been shown that ELR 11-13 is sufficient for interaction between Notch1 and its ligands, and that it constitutes the core recognition site [23][24][25]29 . Theefore, to map the binding domain of N9 on Notch1 we generated a shorter construct constituting ELR 11-13 and performed GST pull down assays.As expected, ELR 11-13, but not the GST control, pulled down N9 efficiently (Fig. 3B).Finally we tested whether N9 can inhibit Notch ligands JAG1 and DLL4 binding on cell surface.As a scaffold control, we evaluated N8, which didn't bind to the breast cancer cell lines as strongly as N9 (Fig. 2A).N9, but not N8, reduced binding of both JAG1 and DLL4 to HEK293-hN1 (Fig. 3C and Supplementary Fig. S4).Thus, our results confirm that N9 binds to EGF like repeat 11-13 of Notch1 and competes with binding of Notch1 to its cognate ligands. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "As N9 bound to ELR 11-13 and competed with ligand binding, we investigated whether it can modulate Notch1 pathway activity in vitro.Consistent with effective inhibition of JAG1 and DLL4 binding, addition of purified N9 to MCF7 cells significantly decreased expression of Notch target genes, including Hes1, Hey1 and HeyL (Fig. 4A).Since the Notch pathway has been reported to inhibit growth of breast cancer cell lines, we assessed whether N9 could affect proliferation of breast cancer cells.MCF7 cells were incubated with increasing concentrations of binders, and cell viability measured after 72 hours.Notably, N9 inhibited proliferation of MCF7 cells in a dose-dependent manner (Fig. 4B).Additionally, N9 treatment did not significantly affect the proliferation of control non-cancerous HEK293 cells (Supplementary Fig. S5).Importantly, N9-mediated inhibition of Notch target gene expression and reduced cell viability could be rescued by misexpression of an extracellular-domain truncated Notch (NEXT).These data suggest that the N9 phenotype is specifically dependent on its ability to modulate Notch signaling activity (Fig. 4C-D and Supplementary Fig. S6A-B).Notch pathway is implicated in cancer stem cell (CSC) maintenance and inhibition of Notch1 signaling has been reported to decrease growth of tumorspheres 25,26 .Therefore we investigated whether N9 could regulate the self-renewing capability of CSC-like cells.We cultured colorectal cancer (HCT116), breast cancer (MCF7) and patient-derived colorectal cancer cell lines in serum-free tumorsphere media in the presence or absence of binders.As shown in Figs 4E-F and 5, N9 significantly reduced tumorsphere formation to an extent similar to that of the known Notch1 inhibitor, DAPT.Altogether, these observations suggest that N9 can robustly target self-renewing ability of CSC-like cells, likely by blocking activity of the Notch pathway. We also evaluated whether N9 can be employed for immunofluorescent applications.Corroborating our previous data, N9 but not N8 effectively labeled MCF7 cells (Supplementary Fig. S7).Furthermore, when compared to a commercially available Notch1 antibody, we observed significant co-localization at the cell membrane with very little staining at free surfaces.(Fig. 6A).Finally, we transiently transfected HE293 cells with Notch1 and stained with either N9 or the commercially Notch1 antibody.As expected, strong colocalization was observed in cells expressing Notch1.Notably, no staining was observed in untransfected cells (Fig. 6C-C'). ",
"section_name": "Functional characterization of N9 in cell based assays.",
"section_num": null
},
{
"section_content": "The last few decades have witnessed a surge in FDA-approval for protein-based drugs, or biologics 5 .Despite the huge success of monoclonal antibodies, limitations based on size, post-translational modification, stability, productivity, and cost-effectiveness have restricted their applicability 10,11,30 .Considering these constraints, the use of small, non-antibody scaffolds, such as Sso7d have the potential to serve as complementary and robust alternative solutions in the domain of next-generation biologics.In this study we demonstrate the identification and functional characterization of an Sso7d variant, called N9 that binds to Notch1 with an equilibrium dissociation constant in the ~100 nM range.The dissociation constant for N9 obtained from direct ELISA and and JAG1 competition assay were similar, suggesting specific binding of N9 to the Notch1 receptor.Although binding site of JAG1 on Notch1 receptor is well studied and has been narrowed down to ELR 11-12 of Notch1, additional regions (ELR 6-15) have also been implicated in binding 31,32 .The fact that N9 bound to ELR 11-13 and competed with JAG1 suggests that ELR 11-13 is sufficient for binding and hence corroborates previous reports that ELR 11-12 contain the core recognition element of Notch/JAG1 interaction 24,29 . Despite binding of N9 to Notch1 on the of MCF7 cell lines, we noticed that knockdown of Notch2 also decreased N9 binding on cell surface.(SupplementaryFig. S8).Upon further investigation, we observed that knockdown of Notch receptors 1-3 generally decreased staining for N9 (Supplementary Fig. S3).Since ELR 11-13 of Notch1-3 exhibits ~70% sequence similarity, our data suggests that N9 binds to an epitope that is common to all these proteins.Previous studies have shown that Notch receptors can all bind JAG1, which suggests presence of common epitopes among Notch receptors 33,34 .Moreover, it has also been reported that monoclonal antibodies generated against one specific Notch receptor show cross-reactivity against other Notch receptors 35,36 .Knockdown of Notch4 did not reduce N9 staining (Supplementary Fig. S9).It is noteworthy that ELR 11-13 of Notch1 only has 50% sequence similarity with that of Notch4.If needed, paralog specificity for candidate binders can be attained by conducting negative selection screens against each paralog, prior to positive selection against the paralog of interest. Notably, the ability of N9 to downregulate Notch pathway activity, decrease proliferation of Notch-dependent cancer cells, and block the formation of tumorspheres highlights the promise of these binders to be developed as putative cancer therapeutic agents.It should be noted that these Notch1 binders were isolated from a naïve Sso7d library, without any further affinity maturation.Therefore it is conceivable that additional rounds of mutagenesis and screening may result in isolation of binders with significantly higher affinity.Our findings also confirm previous reports that blocking Notch1 function inhibits self-renewal capacity of CSC-like cells 25,26 .The importance of the findings in this study is underscored by the fact that antibodies against Notch pathway are being tested against different cancers in clinical trials as CSC-targeted therapy 37 .Therefore the discovery and characterization of small biologics-based Notch inhbitors could add to the rapidly evolving pipeline for Notch-targeted drugs. One of the additional potential application of small protein binders is in the field of imaging and diagnostics as illustrated by the success of radio-labelled affibodies 38 .The small size aids in enhanced biodistribution and faster clearance leading to increased signal-to-noise ratio 39 .In this report, we demonstrate that N9 can effectively label cells for immunostaining and flow-cytometry applications.These observations support previous reports that Notch1 mostly accumulates at cell-cell interface rather than free surfaces 40 .Future studies will be aimed at developing paralogue-specific Notch binders and optimizing them for therapeutic use and/or as molecular probes to study the function of Notch signaling in tumor progression and CSCs. Finally, this study exemplifies the qualities of Sso7d as a scaffold protein to develop binders against specific target proteins.We provide proof-of-concept for the use of Sso7d as a non-antibody scaffold for development of reagents that can modulate and/or monitor oncogenic signaling pathways.Sso7d as a fusion protein or binder has already been used for development of a variety of molecular tools, including the high fidelity DNA polymerase 41 and ELISA reagents.The fact that it could be used to perturb cell signaling networks by modulating PPIs opens a whole new era of application and opportunities. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Cell lines and medium.MDA-MB-231 and HCT116 cell lines were bought from ATCC and MCF7 cell lines was a gift from Michael Garabedian's laboratory (New York University School of medicine, New York, NY).3T3 and 3T3-hN1 cell lines were gifted by Ianis Aifantis's Laboratory (New York University School of medicine, New York, NY).HCT116 was cultured in McCoy's 5 A medium with 10% FBS and P/S.MCF7 and MDA-MB-231 was cultured in DMEM with 10% FBS and P/S.Plasmids, Cloning, protein expression and purification.Notch1 ELR 11-15 construct was a gift from Dighe Lab, Indian institute of Science.The plasmid was transformed into BL21 cells and purified using GST beads and s200 size exclusion chromatography.ELR 11-13 was made by mutating pGEX-4E ELR 11-15 construct by site-directed mutagenesis using Quikchange Site directed mutagenesis kit (Agilent) and confirmed by Sanger sequencing.hNotch1 construct was kindly provided by Professor Alison Banhan.NEXT construct was kindly provided by the Kopan Lab. Yeast display library Screening.The yeast display library of Sso7d mutants used for this study has been previously described 16 .The yeast display library was screened for binders to Notch1 using a combination of magnet-activated cell sorting and flow cytometry, as described 42 .Briefly, biotinylated Notch1 was incubated with 100μl of biotin-binder beads (4 × 10 8 beads per ml, Invitrogen) for 2 hours.The library was subjected to a negative selection step by incubation with with naked beads, and then with beads coated with GST control protein, for 2 hours each at 4 °C; yeast cells binding to the beads were discarded.Subsequently, the unbound yeast were incubated with magnetic beads coated with Notch1 for 1 hour at 4 °C, and bead-bound yeast cells were expanded.This pool of cells after the magnetic selection steps was further screened using two rounds of fluorescence activated cell sorting (FACS) to isolate high affinity binders..The first round of FACS was performed by incubating the library with 200 nM of biotinylated Notch1 for 1 hr at room temperature.For the second round of FACS, the pool of sorted cells from round 1 was labeled with 25 nM of biotinylated Notch1 and top population from 25 nM sort were collected.Collected populations were labelled with 200 nM of biotinylated Notch1 to validate binding. Following the last sort, the pool of yeast cells was plated on an SDCAA plate.Plasmids from individual colonies were recovered using Yeast Zymoprep kit and transformed into DH5alpha E. coli cells.Isolated plasmids from DH5alpha were then sequenced. Recombinant Sso7d protein production.Sso7d variants from the screen were cloned into pET28a and transformed in BL21 cells for protein expression.BL21 cells were grown in 1 L LB till OD reaches 0.6-0.8 and induced with 1 mM IPTG at 25 degree overnight in a shaker incubator.Cells were lysed in 50 mM Tris, 250 mM NaCL, 0.5 mM TCEP with EDTA free protease inhibitor and centrifuged at 14,000 rpm for 20 minutes.Supernatant was incubated with 2 ml Ni-NTA agarose beads (Pierce) for 1 hour and eluted with 250 mM Imidazole in lysis buffer.Sso7d containing fractions were then pooled and purified further using Size exclusion chromatograpy s75 column with 10 mM HEPES, 150 mM NaCL.For cell culture work, protein was then dialyzed in PBS buffer using Slide-a-lyzer cassette (Pierce). Flow cytometry binding and competition assay.MCF7, MDA-MB-231 cells were washed with PBS and harvested using 1 mM EDTA in PBS.Detached cells were passed through 25 G syringe to ensure single cell suspension.Cells were incubated with binders for 1 hour at room temperature and stained with anti Myc-488 (Millipore clone 4A6) antibody for 1 hour on ice.For JAG1 and DLL4 competition assay, Notch1 transfected HEK293 cells were preincubated with 20 μM of binders for 1 hour on ice. 2 nM of human recombinant JAG1-Fc or DLL4-Fc (R&D Systesms) was added and incubated for another 1 hour.Cells were stained with APC conjugated Mouse Anti-Fc antibody (H2, ab99768) for half hour on ice and analysed using Amnis ImageStream Imaging flow cytometer.siRNA mediated knockdown.MCF7 cells were plated in 6 well plate and after 12 hours, transfected with 5 nM siRNA (Ambion silencer select) using RNAimax (Thermofisher) in complete media without Penicillin and Streptomycin.48 hours after transfection, cells were harvested for flow cytometry or immunostaining. ELISA.50 ng of target protein was coated on Maxisorb plate overnight in carbonate buffer.After washing three times with PBS, plate was blocked with Pierce blocking buffer (Lot number) for 1 hour.Different concentrations of sso7d variants were added and incubated for 1 hour at room temperature.Plate was washed 3 times with PBST (PBS,0.05%Tween 20) and was incubated with 1:1000 dilution of Anti-myc antibody (Millipore clone 4A6) for one hour.After washing 5 times with PBST, wells were incubated with 1:5000 dilution of Goat anti-mouse HRP conjugate (Bethyl laboratories, A90-109P) or 1:50,000 dilution of protein G-HRP conjugate (Abcam, ab7460) for 1 hour.Wells were washed 5 times with PBST and 1-Step Ultra TMB (Thermofisher) ELISA substrate was added.After color development, the reaction was stopped using 2 M sulfuric acid and read at 450 using SpectraMax.Kd was determined using nonlinear regression, specific binding analysis in Graphpad Prism7.For JAG1 competition ELISA, wells were preincubated with serial dilution of N9 for 1 hour and 100 nM of human recombinant JAG1 Fc (R&D Systems) was added in each well.After one hour incubation, wells were incubated 1:50,000 dilution of protein G-HRP conjugate (Abcam, ab7460) for 1 hour.Wells were washed 5 times with PBST and 1-Step Ultra TMB (Thermofisher) ELISA substrate was added.After color development, the reaction was stopped using 2 M sulfuric acid and read at 450 using SpectraMax.Ki was determined using nonlinear regression, specific binding analysis in Graphpad Prism7. GST pull down.Equal amount of GST tagged proteins (~7 μg) were incubated with 20 μl GST beads for 1 hour at 4 degree.Bead-protein complex was incubated with the binders for another 1 hour at 4 degree and spun down.After several washes, protein was eluted either with 10 mM GSH or boiled with sample buffer (Biorad) and loaded on precast 4-15% gel (Biorad).Gel was transferred onto nictrocellulose membrane using Trans-Blot turbo transfer system (Biorad) and stained with Anti-GST (sc-138, Clone B14) or anti-Myc antibody (Millipore clone 4A6).Blots were detected with anti-mouse IR 800 secondary antibody using Odyssey imaging system (LI-COR). ",
"section_name": "Material and Methods",
"section_num": null
},
{
"section_content": "Primary colorectal cancer tissue from a patient undergoing surgical resection was obtained from National Cancer Center, Singapore, with patient's informed consent, as per the ethical guidelines and with approval from the Singhealth Centralized Institutional Review Board (CIRB).The tissue was aseptically minced and dissociated enzymatically using Collagenase and hyaluronidase at 37 °C into a cell suspension, which is then seeded onto plates pre-coated with Coating Matrix (Gibco).The cancer cells obtained are subsequently cultured in DMEM-F12 media supplemented with hEGF (20ng/mL), bFGF (10ng/mL) and 1% B27 without Vitamin A. Detailed protocol is available upon request. Immunostaining.HCT116, HEK293 or PDCRC cells were fixed with 4% Formaldehyde and permeablized with PBS + 0.5% Triton-X.Staining were performed over night at 4 degree in Odyssey blocking buffer (Licor).Antibodies used were Anti-Notch1, Cell signaling, D6F11 (1:200), Anti Myc-488, Millipore cl4A6 (1:1000), DAPI (1:1000) and imaged on Nikon TE2000-E microscope or PerkinElmer Operetta High-Content Imaging system. Cell proliferation and viability assay.MCF7, MDA-MB-231, HCT116, PDCRC or HEK293 cells were plated in 96 well plates at 5000 cells/well and binders were added after 12 hours at indicated concentrations.Cell viability was determined after 72 hours using Cell Titre Glo (Promega). qRT PCR.MCF7 or HCT116 cells were treated with 10 μM of binders for 72 hours.Cells were harvested and RNAs isolated from cell pellet using TRIzol reagent and purified using RNA cleanup kit (Qiagen).cDNA was prepared using High Capacity reverse transcription kit (Applied Biosystems) as per the manufacturer's protocol.Real time PCR were carried on using Brilliant II SYBR green masters mix (Applied Biosystems) using MxPro-Mx3005 P system (Stratagene).Gene expressions were normalized to GAPDH. ",
"section_name": "Collection and processing of patient derived colorectal cancer (PDCRC) cell line.",
"section_num": null
},
{
"section_content": "For NEXT (Extracellular-domain truncated Notch) mediated rescue experiment, HCT116 cells were grown in 6 well plate and transfected with 4ug of NEXT or vector control using lipofectamine 2000 (Thermofisher).Binders (10 uM) were added after 24 hrs and incubated for 72 hours.Gene expression and cell viability were measured as above. Tumorsphere assay.MCF7, HCT116 and PDCRC cells were washed once with PBS and harvested using 1 mM EDTA in PBS.Detached cells were passed through 25 G syringe three times to ensure single cell suspension.1000 cells/well were plated in ultra-low attachment 96 well plates in serum free DMEM F12 media supplemented with B27, rEGF and rFGF and 0.3% Low melting agarose.10 uM binders were added during cell seeding and spheres were imaged and counted after one week using Nikon TE000-E microscope, JOBS module (NIS Elements, Nikon). ",
"section_name": "Notch rescue experiment.",
"section_num": null
},
{
"section_content": "Significance was determined using Student's two-tailed T test based on at least three independent experiments, unless otherwise noted. ",
"section_name": "Statistical Analysis.",
"section_num": null
}
] |
[
{
"section_content": "We would like to thank Herbert Samuels, Lawrence Gardner and Stevan Hubbard for guidance, discussion and feedback for this project.We thank NYULMC Perlmutter Cancer Center and Department of Biochemistry and Molecular Pharmacology program for helpful discussion.We owe thanks to Sreeram at the Institute of Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, for helping with initial Flow cytometry screening experiments, and to Drs Robert Robinson, and Weilin Lee at IMCB, Singapore for help with protein purifications.Special thanks to Drs Shumei Chia, Jiamin Loo and Iain Tan, Genome Insitutute of Singapore, ASTAR for providing the patient derived colorectal cancer cell line.This work was supported by the National Institutes of Health (1R01CA155125-01) and GIS Institutional Funds (GIS/14-ARB3207) to RD. TG was partly supported by The Dalai Lama Trust. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-12246-1. 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": "",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-12246-1. ",
"section_name": "Additional Information",
"section_num": null
},
{
"section_content": "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": "Competing Interests:",
"section_num": null
}
] |
10.3390/curroncol30100643
|
Next-Generation Sequencing Analysis of Mutations in Circulating Tumor DNA from the Plasma of Patients with Head–Neck Cancer Undergoing Chemo-Radiotherapy Using a Pan-Cancer Cell-Free Assay
|
<jats:p>Using next-generation sequencing (NGS), we investigated DNA mutations in the plasma tumor cell-free circulating DNA (ctDNA) of 38 patients with inoperable squamous cell head neck cancer (SCHNC) before and after the completion of chemoradiotherapy (CRT). Baseline mutations of the TP53 were recorded in 10/38 (26.3%) and persisted in 4/10 patients after CRT. ΤP53 mutations were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline (overall rate 44.7%). Furthermore, 4/38 patients exhibited baseline mutations of the EGFR, AR, FGFR3, and FBXW3, and four new gene mutations were detected after CRT (MTOR, EGFR3, ALK, and SF3B1). Τ4 stage was related with a significantly higher rate of mutations (TP53 and overall). Mutations were observed in 8/30 (26.6%) responders (complete/partial response) vs. in 6/8 (75%) of the rest of the patients (p = 0.03). Significant poorer LRFS was noted for patients with mutations detected before and after CRT (p = 0.02). Patients who had detectable mutations either before or after CRT had significantly worse DMFS (p = 0.04 overall, and p = 0.02 for TP53 mutations). It was concluded that assessment of mutations before and after the end of CRT is essential to characterize patients with a high risk of locoregional recurrence or metastatic progression.</jats:p>
|
[
{
"section_content": "During disease progression, cancer cells and especially stem cells acquire genetic mutations that define clinical aggressiveness, invasion, metastasis, and resistance to radiotherapy (RT) and chemotherapy [1,2].Such mutations can appear even during therapy, either as a result of direct DNA damage and failure to properly repair the DNA strand breaks or as an accumulation and prevalence of existing resistant cancer cell clones with specific mutations [3,4]. The profile of genetic mutations of a tumor can be assessed with next-generation sequencing (NGS) based on tissue biopsy material.Indeed, this has been established as a routine test to identify molecular fingerprints that can guide therapy with molecular inhibitors or monoclonal antibodies, e.g., therapies targeting epidermal growth factor receptor (EGFR) or other gene mutations [5].Nevertheless, NGS can also be applied in cellfree DNA (cfDNA) extracted from the plasma or the saliva and other body fluids of patients. Fragmented DNA released by cancer cells through vesicles and exosomes or fragments from dying cancer cells that enter the circulation (circulating tumor DNA-ctDNA) can be isolated from the blood and body fluids.Testing ctDNA for tumor mutations in liquid biopsies has emerged as a convenient and reliable method for tumor profiling.In fact, Parkh et al. suggested that analysis of a single-lesion tumor biopsy alone is less effective than ctDNA analysis in identifying tumor genetic heterogeneity and alterations associated with resistance to therapy [6].Liquid biopsies, being non-invasive procedures, can be repeatedly obtained from patients without any discomfort during their therapy, conferring an important advantage over tissue biopsy analysis. Squamous cell head neck cancer (SCHNC) accounts for approximately 600,000 new cases annually, ranking 7th in prevalence among different cancer subtypes, with smoking and human papilloma virus (HPV) infections being major risk factors [7].Combination of surgery and adjuvant RT or chemoradiotherapy (CRT) and definitive/radical RT or CRT for inoperable cases are the established treatment modalities of this malignancy, offering high curability rates.While the incidence of distant metastases is less than 30%, locoregional recurrence eventually occurs in more than 50% of patients with a locally advanced disease [8].Specifically, the 2-year progression-free survival rates of locally advanced head and neck cancer patients treated with radical CRT range from 30% to 60% [9]. A number of studies investigating the cfDNA levels in the plasma or other body fluids of SCHNC patients before or after administration of RT or CRT have suggested that this method could potentially predict response to treatment and patient prognosis [10].In a prospective trial, we quantitatively assessed the cfDNA concentration in the plasma of a cohort of patients with locally advanced SCHNC treated with CRT [11].Increased levels were noted in 55% of patients and this was related to poorer response to therapy and worse prognosis.Beyond the quantity of cfDNA, gene mutation analysis could identify ctDNA that would ultimately prove to be of further prognostic and predictive relevance.In the current study, we report the analysis of DNA mutations in this cohort of patients.Detection of mutations was based on a panel of selected genes involved in the cell cycle, cell death pathways, cell signaling, and metabolism.These were assessed before and at the end of CRT, aiming to identify specific gene mutations involved in resistance to CRT and also assess an eventual prognostic role of persistent or newly emerging mutations after treatment. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "As previously reported [11], patients with histologically diagnosed inoperable SCHNC were prospectively treated with RT combined with chemotherapy (cisplatin and/or cetuximab).Only patients with a conventional type of squamous cell cancer of the head-neck area, as identified by the 5th edition of the World Health Organization Classification of Head and Neck tumors [12], were included.Thirty-eight patients were included in the current analysis.No patient selection was performed (patients sequential in time).Inclusion criteria were good performance status (0-1), no previous chemotherapy or RT treatment, normal blood and biochemical tests, and absence of major heart, kidney, lung, autoimmune, hematological or psychiatric disease.Pregnant women were also excluded.Supplemental Table S1 presents details regarding patient and disease characteristics.The median follow-up was 15 months (2-36 months), while for patients alive at the time of last follow-up, this was 18 months (6-36 months). ",
"section_name": "Patients",
"section_num": "2.1."
},
{
"section_content": "Patients were treated with image-guided RT (IGRT) and a Volumetric Modulated Arc Therapy (VMAT) technique as previously reported [11].Briefly, a simultaneous integrated boost (SIB) technique was applied to deliver 22 fractions, 5 fractions per week, within 30 days.Areas receiving prophylactic irradiation (e.g., neck) were treated with 2.15 Gy/fraction, while a daily booster dose of 0.40-0.55Gy was administered to the primary tumor.The dose to enlarged nodes was increased using a daily booster dose of 0.3-0.4Gy per fraction. The SIB regimen has been widely applied in our department for the treatment of SCHNC, as this provides an equivalent dose delivered in 2 Gy fractions (EQD2) of 62-66 Gy, using the linear quadratic formula for tumor α/β = 4-10 Gy.As this dose is delivered with a 15-day acceleration, the time-corrected (T) EQD2-T (for a λ-value = 0.4-0.8Gy/day) reaches an estimated biological dose of 68-78 Gy.This has been analyzed in detail in a previously reported study [11].A recent radiobiological study by Shuryak et al. has suggested that optimized hypofractionated and accelerated RT in the range of the above-reported regimen can be better tolerated and is highly effective [13]. Patients received concurrent chemotherapy with intravenous administration of cisplatin at a dose of 35-40 mg/m 2 per week, or cetuximab at a dose of 250 mg/m 2 /week, or a combination of both, as previously reported [14].Thirteen patients were treated with cisplatin, four patients received cetuximab and twenty-one patients were treated with both agents. ",
"section_name": "Treatment Technique",
"section_num": "2.2."
},
{
"section_content": "A CT or an MRI scan was performed two months after RT completion to assess tumor response, and these were repeated six-monthly after that during the follow-up of patients.The WHO criteria [15] were applied to assess response to CRT as follows: complete response (CR) was defined as a 95-100% reduction in 2D dimensions of all detectable and measurable lesions.Partial (PR) and minimal response (MR) refer to 50-95% and 25-49% reduction in tumor dimensions (2D), respectively.An increase in tumor dimensions by more than 25% was defined as progressive disease.All other cases were considered to correspond to stable disease. ",
"section_name": "Assessment of Response",
"section_num": "2.3."
},
{
"section_content": "Twelve ml of venous blood were collected in vacuum blood collection test tubes containing ethylenediaminetetraacetic acid (EDTA) vials.The first sampling was performed immediately before the administration of the first RT fraction and chemotherapy infusion.A second blood sample was obtained on the day of the last RT fraction.The technique of PBMC and plasma isolation and storage has been previously reported [11]. ",
"section_name": "Plasma Collection",
"section_num": "2.4."
},
{
"section_content": "Isolation of cell-free DNA was performed using the bead-based MagMAX™ Cell-free DNA extraction kit (catalog no.: A36716, Thermo Fisher Scientific, Waltham, MA, USA), specialized for high-quality isolation and specific enrichment of nucleic acids from liquid biopsies, as previously reported [11]. ",
"section_name": "Extraction and Quantification of Plasma cfDNA",
"section_num": "2.5."
},
{
"section_content": "NGS analysis was performed using the Oncomine Pan-cancer cell-free assay (Thermo Fisher Scientific, USA; https://www.thermofisher.com/order/catalog/product/A37664,accessed on 25 September 2023) according to the manufacturer's instructions.Library preparation was performed using cfDNA with concentrations ranging from 0.5 ng to 4 ng.Quantification of the isolated libraries was again performed via the Qubit system.The input range of each library used ranged from 10 ng to 20 ng.Sample analysis was performed using the Ion 540™ Kit-Chef system (Thermo Fisher Scientific).Automated preparation of Ion 540™ chips was performed, with each chip having six patient samples with different barcodes, and finally, sequencing of the samples was performed through the next-generation sequencing system using the Ion S5™ system.High-depth sequencing of the samples was performed using the Ion S5 sequencer, while the analysis of the samples was performed using Torrent Suite™ Software v. 5.12.3 and Ion Reporter™ version 5.20.2 (Thermo Fisher Scientific), using the Homo sapiens reference genome (hg19) as a reference library, according to the manufacturer's instructions, for the analysis of point mutations, deletions, insertions, fusions and CNV's in a panel of genes occurring in various cancer types, including AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4, SMO (Hotspot genes (SNVs) and short indels), ALK, BRAF, ERG, ETV1, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK3, RET, ROS1 (Gene fusions), MET (exon 14 skipping), CCND1, CCND2, CCND3, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET, MYC (CNVs) APC, FBXW7, PTEN, TP53 (Tumor suppressor genes), with a limit of detection (LOD) of a 0.1% allele frequency for SNVs and 1% for fusions.These genes are frequently mutated in multiple cancer types [16,17], including head and neck cancer [18]. ",
"section_name": "NGS Analysis",
"section_num": "2.6."
},
{
"section_content": "We used the GraphPad Prism 7.0 package for statistical analysis and graph presentation.The chi-square and Fisher's exact t-test were used to test associations between categorical variables, as appropriate.Kaplan-Meier locoregional relapse-free survival (LRFS), disease-specific overall survival (OS), and distant metastasis-free survival (DMFS) curves were plotted.For statistical significance, we considered a p-value < 0.05. ",
"section_name": "Statistical Analysis",
"section_num": "2.7."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The quality control of the analyzed DNA samples from patients before and after CRT showed a QC-test-limit-of-detection LoD % range from 0.1 to 1.3 (median 0.4) and from 0.1 to 1.2 (median 0.5), respectively.Table 1 reports the genes, mutations, and molecular frequencies recorded in patients before and after CRT. The baseline pre-CRT analysis showed a clear prevalence of mutations of the TP53, recorded in 10/38 (26.3%) of patients.Single mutation was recorded in 7/10 patients, while multiple mutations of the gene were recorded in 3/10 patients (two mutations in two patients and three in one).After CRT, TP53 mutations were undetectable in 5/10 of these patients, while baseline mutations persisted in 4/10.In one (1/10) additional patient, disappearance of the pre-existing TP53 mutation was noted, while a new mutation became detectable.Reduction in the number of TP53 mutations was observed in patients who presented with multiple mutations at baseline: patient no.22 had two mutations post CRT (at baseline, three mutations), and patient no.35, with two mutations at baseline, had only one post CRT.Both mutations in patient no.30 were undetectable post CRT.Of interest, mutations of the TP53 gene were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline.In this way, the rate of TP53 mutation detection (before and/or after CRT) was 17/38 (44.7%).Figure 1a shows the mutation rates of TP53 and changes after CRT, while Figure 1b shows the mutations of TP53 before, after CRT, and these persisted throughout therapy.Furthermore, 4/38 more patients exhibited mutations of EGFR, AR, FGFR3, and FBXW3 (one patient, respectively) before CRT.After CRT, the FGFR3 mutation remained detectable in the patient, while the rest were undetectable.Moreover, four additional mutations became detectable after CRT in four patients (one each) without pre-treatment detectable mutations.These concerned the MTOR, EGFR3, ALK, and SF3B1 genes, respectively (Table 2).We did not detect any CNVs or gene fusions in the current cohort of patients.Figure 1c shows the genes and mutation rates recorded before and after CRT.Appendix A reports the detected genes with mutations and their main biological functions in cancer biology.Analysis of TP53 mutations detected after CRT according to the chemotherapy regimen applied (cisplatin alone vs. cetuximab with or without cisplatin) did not reveal any statistically significant difference (mutation rate: 4/13 vs. 8/25 patients, respectively; p = 0.99). Furthermore, 4/38 more patients exhibited mutations of EGFR, AR, FGFR3, and FBXW3 (one patient, respectively) before CRT.After CRT, the FGFR3 mutation remained detectable in the patient, while the rest were undetectable.Moreover, four additional mutations became detectable after CRT in four patients (one each) without pre-treatment detectable mutations.These concerned the MTOR, EGFR3, ALK, and SF3B1 genes, respectively (Table 2).We did not detect any CNVs or gene fusions in the current cohort of patients.Figure 1c shows the genes and mutation rates recorded before and after CRT.Appendix A reports the detected genes with mutations and their main biological functions in cancer biology. Table 2. Univariate tables from Kaplan-Meier loco-regional relapse-free survival, disease-specific overall survival and distant metastasis-free survival analysis according to the presence of overall and TP53 mutations.The grouping of cases was performed using 4 variables: i mutations detected before (B) chemo-radiotherapy (CRT), ii.mutations detected after (A) CRT, iii.mutations detected before and/or after CRT, and iv.mutations detected before and after CRT.Abbreviations: LRFS = locoregional relapse-free survival, OS = disease-specific overall survival, DMFS = distant metastasis-free survival. ",
"section_name": "Gene Mutations",
"section_num": "3.1."
},
{
"section_content": "",
"section_name": "All Mutations",
"section_num": null
},
{
"section_content": "",
"section_name": "LRFS",
"section_num": null
},
{
"section_content": "Supplemental Table S2 reports the distribution of overall and TP53 mutations according to the age of patients and histopathological variables.For cases with the T4 stage, there was a significantly higher chance of detecting mutations before and after CRT (6/17 T4 patients vs. 1/21 T0-3 patients; p = 0.03).No other association with age, T stage, N stage, or histopathological grade was noted.Regarding TP53 mutations, these prevailed in the T4 stage compared to other stages, reaching a maximum significance for patients who had mutations both before and after CRT (5/17 T4 patients vs. 0/21 T0-3 patients; p = 0.007). ",
"section_name": "Associations with Histopathological Variables and Patient Age",
"section_num": "3.2."
},
{
"section_content": "Supplemental Table S3 reports the distribution of overall and TP53 mutations (recorded before and after CRT) in patients according to the response obtained after CRT.The only statistically significant association concerned the analysis of overall mutations assessed after the end of CRT.Mutations were observed in 8/30 (26.6%) responders (CR/PR) vs. in 6/8 (75%) of the rest of patients (p = 0.03).This difference showed a statistical trend after analysis for TP53 mutations (p = 0.08). ",
"section_name": "Associations with Response to CRT",
"section_num": "3.3."
},
{
"section_content": "Table 2 and Figure 2 report the univariate and Kaplan-Meier LRFS analysis, according to the presence of overall and TP53 mutations.Significantly poorer LRFS was noted for patients with persistent detection of mutations (mutations detected before and after CRT) (p = 0.02).A marginal association was observed for patients with detectable mutations after CRT (p = 0.08). Univariate tables from Kaplan-Meier loco-regional relapse-free survival, diseasespecific overall survival and distant metastasis-free survival analysis according to the presence of overall and TP53 mutations.The grouping of cases was performed using four variables: i. mutations detected before (B) CRT, ii.mutations detected after (A) CRT, iii.mutations detected before and/or after CRT, and iv.mutations detected before and after CRT.We found no association of mutations with the OS (Supplemental Figure S1).Analysis of DMFS showed that patients with mutations after CRT and patients with mutations after CRT had a marginally poorer outcome (p = 0.11 and 0.09, respectively), which reached significance for patients who had detectable mutations either before or after CRT (p = 0.04) (Table 2 and Figure 3).None of the patients without mutations (before or after CRT) developed metastasis during their follow-up.Analysis of TP53 mutations showed that patients with mutation after CRT and patients with mutations either before or after CRT had a significant association with poor prognosis (p = 0.05 and 0.02, respectively). We found no association of mutations with the OS (Supplemental Figure S1).Ana sis of DMFS showed that patients with mutations after CRT and patients with mutatio after CRT had a marginally poorer outcome (p = 0.11 and 0.09, respectively), wh reached significance for patients who had detectable mutations either before or after C (p = 0.04) (Table 2 and Figure 3).None of the patients without mutations (before or af CRT) developed metastasis during their follow-up.Analysis of TP53 mutations show that patients with mutation after CRT and patients with mutations either before or af CRT had a significant association with poor prognosis (p = 0.05 and 0.02, respectively). ",
"section_name": "Survival Aanalysis",
"section_num": "3.4."
},
{
"section_content": "Supplemental Tables S4 andS5 present the specific gene mutations recorded before and after CRT, respectively, in patients whose disease progressed or did not progress after CRT.Regarding TP53 mutations detected before CRT, p.R248W, p.V157F, p.Y220C, p.C238Y and p.C135S characterized patients who progressed after therapy.Regarding TP53 mutations detected after CRT, p.H179L, p.R213=, p.R248W, p.C238Y and of two newly detected mutations p.S241F, and p.V157F characterized patients who progressed after therapy. Among other gene mutations, the p.R505C mutation of the FBXW7, the p.E894K mutation of the AR, and the p.F384L mutation of the FGFR3 genes detected before CRT were recorded in patients with disease progression.In addition, detection of the p.F384L mutation of the FGFR3 and the p.R2217W mutation of the mTOR after CRT were found in two patients, respectively, with disease progression. ",
"section_name": "Specific Gene Mutations and Disease Progression",
"section_num": "3.5."
},
{
"section_content": "Gene mutations are frequently present in SCHNCs.These mutations concern genes involved in cell proliferation, survival, and death regulation pathways (e.g., p53 and EGFR signaling pathway), cellular differentiation (e.g., Wnt, NOTCH1, Hedgehog pathway), or regulation of the cell cycle (e.g., cyclins and related genes).TP53 mutations seem to have a dominant role in the biology of SCHNC [19].Huang et al. reported that mutations of the TP53 were noted in tissue samples of 55% of SCHNCs, and this rate was similar in HPV-positive and -negative tumors [20].Using NGS, TP53 mutations can also be detected in the plasma of SCHNC patients [21].Economopoulou et al. reported a 32.6%rate of TP53 mutations in a series of 45 SCHNC patients [22].Furthermore, in an investigation detecting gene alterations in the ctDNA from the saliva of SCHNC patients, mutations were recorded in 76% of cases [23].In the current study, we confirmed an evident prevalence of mutations of TP53 in SCHNCs, which concerned 26.3% of patients examined at baseline.In addition, we identified multiple TP53 mutations in a minority of patients, while mutations of other genes, like EGFR, AR, FGFR3, and FBXW3, were noted in 10% of patients.The rate of TP53 mutations reported herein are similar to the one reported by Economopoulou et al. [22], but certainly lower than the 50-80% rates reported in studies on tissue samples [19,20].Of interest, Porter et al. and Galot et al. recorded ctDNA TP53 mutations in 68% and 50% of patients with head and neck cancer, respectively [24,25].However, in both studies, blood samples were drawn from patients with recurrent or metastatic disease, a parameter that could potentially explain the higher rates of TP53 mutations. In this investigation, we also performed an analysis of gene mutations detected in the blood of SCHNC patients immediately after the end of CRT.In this way, we could identify the persistence, disappearance, or new mutations of genes after therapy.After CRT completion, TP53 mutations were undetectable in about half of patients with baseline detectable mutations.This may be a result of high intrinsic radiosensitivity and early elimination of cancer cell clones bearing these mutations during the course of CRT.Although TP53 mutations are involved in apoptosis inhibition and resistance to RT, this effect is not consistent as specific TP53 mutations have been linked with enhanced apoptotic tendency after irradiation [26].Additional molecular pathways may also counteract p53-mediated radioresistance and sustain radiosensitivity [27].For example, the FBXW7 gene, mutations of which were noted in one patient before CRT, has been shown to confer survival of cancer cells during RT by induction of p53 protein degradation and blockage of apoptosis [28]. Persistent detection of specific TP53 mutations, and, for one case, mutations of FGFR3, was noted in about 10% of patients after the end of CRT in our study.In addition, in 18% of the patients with undetectable TP53 mutations at baseline, new TP53 mutations could be detected post CRT.In this way, the total rate of TP53 mutations recorded in ctDNA was 44.7%.Moreover, new mutations of other genes, undetectable at baseline, were also recorded in a minority of patients, and these concerned MTOR, EGFR3, ALK, and SF3B1.Emerging mutations in patients with esophageal cancer progressing after CRT have also been noticed in a study by Azad et al. [29].Persistence of baseline mutations and the emergence of new detectable mutations could indicate radioresistance of the cancer cell compartment bearing these very gene mutations.Indeed, in the current study, patients with detectable mutations after CRT had a significantly lower tumor response rate.In this context, an interesting study in medulloblastoma suggested that the dominant clone at recurrence after RT emerges through selections of pre-existing minor clones [30], which may also apply to patients where new mutations were recorded after CRT. As far as prognosis is concerned, patients with persistent detection of mutations after CRT (detectable mutations at baseline) had significantly worse LRFS.A marginal association was also noted for patients with mutations detected after CRT.Although we found no significant association with OS, patients with detectable mutations after CRT, or mutations before and/or after CRT had a significantly higher rate of development of distant metastases.This finding was noted after taking into account all gene mutations and when analysis concerned TP53 mutations only.A retrospective mutation analysis of the ctDNA of 75 patients with SCHNC (stages I-IV, stage IV 62.7%) demonstrated that both overall ctDNA alterations and TP53 mutations significantly correlated with advanced tumor progression status and OS [31].In addition, it has been reported that the presence of ctDNA mutations either before or before and after treatment with CRT was linked with decreased survival [22].Taylor et al. published the results of a study in SCHNC patients treated with chemotherapy or immunotherapy, suggesting that, although baseline ctDNA abundance was not associated with OS, changes in the ctDNA variant allele frequency were predictive of progression-free survival [32].Two additional studies, although performed in squamous cell esophageal cancer, showed a significant association of ctDNA mutations with prognosis.Wang et al. reported that detectable ctDNA alterations one or several months after RT were linked with inferior progression-free survival of patients, while a better prognosis was recorded for patients whose ctDNA disappeared one month after therapy [33].Azad et al. also found an increased risk of disease progression in patients with squamous cell esophageal cancer when ctDNA mutations were recorded after CRT [29]. Beyond the well-known limitations related to the NGS procedure (quality of the isolated DNA, bioinformatic analysis variations, false negative results), other limitations of the study include the relatively low number of patients recruited in the prospective trial due to predefined funding and the high cost of NGS experiments.Moreover, although the study focused on SCHNC, this includes different primary tumor locations with eventual different pathogenesis, clinical behavior and prognosis.In addition, the HPV status was not studied in parallel with NGS.Longer follow-up could also have allowed the extraction of more robust conclusions.Nevertheless, the treatment was consistent for all patients and the inclusion of liquid biopsies after the end of therapy provided further insights of the biology behind the interplay of CRT with tumor biology. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Despite the aforementioned limitations, it is suggested that detection of TP53 and other gene mutations in the ctDNA from the plasma of patients with SCHNC treated with radical CRT can be achieved with NGS.Assessment of mutations before and after the end of CRT is, however, essential to characterize patients with high risk of locoregional recurrence or even metastatic progression.Persistent detection of mutations, pre-existing or new, appeared as the major identified parameter that predicted locoregional progression after CRT.Although TP53 mutations prevailed, detection of less frequently recorded mutations of other genes, like FGFR3, MTOR, EGFR3, ALK, and SF3B1, mutations after CRT seem also to contribute to the overall association of the mutational burden with disease progression.The genomic alterations post CRT described herein provide a platform for novel therapeutic approaches for SCHNC that test combined targeted therapies and CRT. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/curroncol30100643/s1, Figure S1: Kaplan-Meier disease specific overall survival curves according to the existence of overall mutations and TP53 mutations detected before chemo-radiotherapy, after CRT, before and/or after CRT and, finally, before and after CRT; Table S1: Patient and disease characteristics; Table S2: Distribution of overall and TP53 mutations, according to the age of patients and histopathological variables; Table S3: Distribution of overall and TP53 mutations; Table S4: Distribution of specific gene mutations before CRT in patients according to the progression status (after CRT); Table S5: Distribution of specific gene mutations at the end of CRT in patients according to the progression status (after CRT). ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "Data Availability Statement: All data are available in the files of the Department of Radiotherapy.and Oncology, Democritus University of Thrace.The data presented in this study are available on reasonable request from the corresponding author. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Author Contributions: Conceptualization, I.K. and C.N.B.; validation, M.I.K., C.N.B. and I.K.; formal analysis, M.I.K., E.X., S.P.F., C.K., I.M.K. and N.K.; investigation, M.I.K., C.N.B., E.X., S.P.F. and N.K.; writing-original draft preparation, I.M.K., writing-review and editing, M.I.K., E.X., S.P.F., C.K., I.K., C.N.B. and N.K.; supervision, M.I.K. and C.N.B.; funding acquisition, M.I.K. and C.N.B.All authors have read and agreed to the published version of the manuscript. Funding: This research has been co-financed by the European Regional Development Fund of the European Union and Greek National Funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T2EDK-03266, project acronym, and title: \"BIOKAKETRA-Identification of genomic and transcriptomic prognostic bio-signatures in head and neck cancer\"). The study has been approved by the local Ethics and Research Committee (ES1 23-01-2019 and ES2 22-02-2019) and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.Written informed consent to enter the trial was obtained from all patients before therapy. Written informed consent to enter the trial was obtained from all patients before therapy.Patients' consent included permission to publish their clinical and laboratory data for research and educational purposes anonymously. The authors declare no conflict of interest. Appendix A Table A1.Mutated genes and principal functions. A tumor suppressor gene.Encodes the tumor protein p53, a crucial regulator of apoptotic response, and guardian of the genome integrity.It also regulates DNA repair proteins and can induce cell cycle arrest at the G1/S cell cycle phase.Also involved in cellular senescence. The Epidermal Growth Factor Receptor or ErbB-1 gene encodes a transmembrane receptor that is activated by specific ligands like EGF and TGF-α, initiating a cascade of signaling events involved in proliferation, metabolism and resistance to chemotherapy and radiotherapy.Amplification and mutations of the gene promote aberrant activation lading to carcinogenesis and tumor progression. Androgen receptor gene encodes ARs, transcription factors that, following their binding to testosterone, enter the nuclei to activate several genes involved in tumor progression. Encodes a member of the fibroblast growth factor receptor family, a membrane protein that binds to the fibroblast growth factors of the tumor stroma, promoting proliferation and differentiation.Mutations of the FGFR3 have been detected in bladder cancer and glioblastomas and are involved in cell proliferation and resistance to anti-cancer therapy. The F-box and WD repeat domain containing 7 gene encodes a member of the F-box protein family with critical tumor suppressor functions.It controls the degradation of several oncoproteins (c-myc, mcl-2, mTOR, jun, cycline E) through the proteasome pathway.Its mutations promote carcinogenesis and tumor growth. The mammalian target of rapamycin gene regulates cell proliferation, autophagy, apoptosis and metabolism pathways including glycolysis.Its mutations promote carcinogenesis. Encodes a member of the EGFR family protein.Activating mutations lead to resistance to anti-cancer therapy. The anaplastic lymphoma kinase gene can be activated in a subgroup of solid tumors, driving cell growth and resistance to chemotherapy.Specific targeting drugs have been approved for the treatment of ALK-positive patients with lung cancer. It encodes subunit 1 of the splicing factor 3b protein complex.Mutations of the gene are linked with chronic lymphocytic leukemia, myelodysplastic syndromes, breast cancer, and orbital melanoma. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Author Contributions: Conceptualization, I.K. and C.N.B.; validation, M.I.K., C.N.B. and I.K.; formal analysis, M.I.K., E.X., S.P.F., C.K., I.M.K. and N.K.; investigation, M.I.K., C.N.B., E.X., S.P.F. and N.K.; writing-original draft preparation, I.M.K., writing-review and editing, M.I.K., E.X., S.P.F., C.K., I.K., C.N.B. and N.K.; supervision, M.I.K. and C.N.B.; funding acquisition, M.I.K. and C.N.B.All authors have read and agreed to the published version of the manuscript. Funding: This research has been co-financed by the European Regional Development Fund of the European Union and Greek National Funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T2EDK-03266, project acronym, and title: \"BIOKAKETRA-Identification of genomic and transcriptomic prognostic bio-signatures in head and neck cancer\"). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The study has been approved by the local Ethics and Research Committee (ES1 23-01-2019 and ES2 22-02-2019) and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.Written informed consent to enter the trial was obtained from all patients before therapy. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Written informed consent to enter the trial was obtained from all patients before therapy.Patients' consent included permission to publish their clinical and laboratory data for research and educational purposes anonymously. ",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. Appendix A Table A1.Mutated genes and principal functions. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "",
"section_name": "GENE FUNCTION",
"section_num": null
},
{
"section_content": "A tumor suppressor gene.Encodes the tumor protein p53, a crucial regulator of apoptotic response, and guardian of the genome integrity.It also regulates DNA repair proteins and can induce cell cycle arrest at the G1/S cell cycle phase.Also involved in cellular senescence. ",
"section_name": "TP53",
"section_num": null
},
{
"section_content": "The Epidermal Growth Factor Receptor or ErbB-1 gene encodes a transmembrane receptor that is activated by specific ligands like EGF and TGF-α, initiating a cascade of signaling events involved in proliferation, metabolism and resistance to chemotherapy and radiotherapy.Amplification and mutations of the gene promote aberrant activation lading to carcinogenesis and tumor progression. ",
"section_name": "EGFR/ErbB1",
"section_num": null
},
{
"section_content": "Androgen receptor gene encodes ARs, transcription factors that, following their binding to testosterone, enter the nuclei to activate several genes involved in tumor progression. ",
"section_name": "AR",
"section_num": null
},
{
"section_content": "Encodes a member of the fibroblast growth factor receptor family, a membrane protein that binds to the fibroblast growth factors of the tumor stroma, promoting proliferation and differentiation.Mutations of the FGFR3 have been detected in bladder cancer and glioblastomas and are involved in cell proliferation and resistance to anti-cancer therapy. ",
"section_name": "FGFR3",
"section_num": null
},
{
"section_content": "The F-box and WD repeat domain containing 7 gene encodes a member of the F-box protein family with critical tumor suppressor functions.It controls the degradation of several oncoproteins (c-myc, mcl-2, mTOR, jun, cycline E) through the proteasome pathway.Its mutations promote carcinogenesis and tumor growth. ",
"section_name": "FBXW7",
"section_num": null
},
{
"section_content": "The mammalian target of rapamycin gene regulates cell proliferation, autophagy, apoptosis and metabolism pathways including glycolysis.Its mutations promote carcinogenesis. ",
"section_name": "mTOR",
"section_num": null
},
{
"section_content": "Encodes a member of the EGFR family protein.Activating mutations lead to resistance to anti-cancer therapy. ",
"section_name": "ErbB3",
"section_num": null
},
{
"section_content": "The anaplastic lymphoma kinase gene can be activated in a subgroup of solid tumors, driving cell growth and resistance to chemotherapy.Specific targeting drugs have been approved for the treatment of ALK-positive patients with lung cancer. ",
"section_name": "ALK",
"section_num": null
},
{
"section_content": "It encodes subunit 1 of the splicing factor 3b protein complex.Mutations of the gene are linked with chronic lymphocytic leukemia, myelodysplastic syndromes, breast cancer, and orbital melanoma. ",
"section_name": "SF3B1",
"section_num": null
}
] |
10.1038/s41598-018-36853-8
|
BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo
|
<jats:title>Abstract</jats:title><jats:p>A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation <jats:italic>ex vivo</jats:italic> is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current <jats:italic>ex vivo</jats:italic> B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation <jats:italic>ex vivo</jats:italic>. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This <jats:italic>ex vivo</jats:italic> model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.</jats:p>
|
[
{
"section_content": "we previously described the specific transcriptional 15,16 and proteomic programs 17 which are induced in aggressive CLL cells following BCR ligation. Nevertheless, a sustained soluble stimulation of the BCR induces apoptosis in CLL cells 9,15,[18][19][20][21] and BCR-associated factors are mandatory in inducing CLL cells proliferation.Several factors, are known for their role in CLL cells survival or proliferation, among which IL-2, IL-4, IL-10, IL-15, IL-21 and CD40L are prominent [22][23][24][25] , but an exhaustive evaluation of their role as BCR-cofactors for CLL cells proliferation is still lacking.Difficulties to achieve robust CLL cell proliferation ex vivo led to the use of stromal cells 26,27 , activated T cells 22,[28][29][30][31] or fibroblast (eventually CD40L transfected) 21,22,30,[32][33][34] as feeder cells.However, feeder cells' interactions 35 and secretion of IL-6, IL-10 or TGF-β can also participate in CLL cells survival and proliferation 26 , which makes the identification of essential leukemogenic factors difficult and prevents the specific evaluation of BCR ligation in the proliferative response in these models. In this study, we aim to set-up culture conditions, primarily based on BCR ligation for patho-physiological relevance, inducing CLL cells proliferation.This study was conducted in two steps.We first aimed at establishing the optimal ex vivo model for CLL cells proliferation measured by carboxyfluorescein succinimidyl ester (CFSE) incorporation.For this, a selection of healthy and primary CLL cells were stimulated by anti-IgM ligation with or without co-stimulatory molecules (IL-2, IL-4, IL-10, IL-21, IL-15, sCD40L), at various concentration in different culture conditions.Next, using the optimized culture conditions, we analyzed the proliferative response of fresh negatively selected B cells isolated from a cohort of well characterized CLL patients, under informed consent, including clinical data, cell morphology, flow cytometry -including ZAP70 expression status-, FISH and IGHV mutational status, as these factors may impact the cell response to stimulation 22,28,30,31 .These culture conditions induced a proliferative response of a fraction of CLL cells, essentially ZAP70+, in soluble medium and a proliferation of nearly all CLL cells in 3D semi-solid medium, representing a valuable system for CLL functional studies. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Establishing culture conditions for CLL cells proliferation ex vivo.To establish culture conditions for CLL cells proliferation after ex vivo activation, we first evaluated CFSE labeling in a small series of patient samples (n = 8).This approach allows calculating the percentage of dividing cells and the number of cell generations (Fig. S1).We first confirmed data from previous studies showing that ex vivo BCR activation by means of anti-IgM ligation does not induce CLL cells proliferation when these cells are cultured in soluble medium (Figs 1A and S2A).Similarly, stimulation with IL-4, IL-21 or CD40L, used separately, in soluble medium, did not induce CLL cells proliferation either (Fig. 1A).We also confirmed that different combinations of cytokines, [CD40L + IL-4], [CD40L + IL-21] and [CD40L + IL-4 + IL-21] induced a weak (less than 40%) proliferation of CLL cells (Fig. 1A).Of note, IL-21, which has a pro-apoptotic effects on CLL cells 34 potentiates the proliferating effect of IL-4 when sequentially added after IL-4 23 and therefore IL-21 was added 24 h after all initial IL-4 stimulation.However, when we analyzed the proliferative effect of a combination of cytokines added after initial BCR stimulation (IgM ligation), we established that, even if BCR activation associated to [CD40L + IL-4] or [CD40L + IL-21] allowed a weak proliferation, the combination of anti-IgM with [CD40L + IL-4 + IL-21] induces a higher proliferation rate of CLL cells in soluble medium (Fig. 1A).Similar experiments confirmed the proliferative potential of these conditions on total B cells from healthy donors (Figs 1B andS2B).We analyzed the morphology of CLL cells submitted to these culture conditions.We observed the formation of clusters of proliferating cells in the culture medium (Fig. S1) and cytological analysis of these cells after cytocentrifugation at day 6 revealed in all cases a monomorphic evolution consisting in large cells with a high amount of basophilic cytoplasm, prominent nucleoli and a fine chromatin that were distinct from those of control unstimulated cells (Fig. S3A).Immunophenotyic analysis of proliferating CLL cells at day 6 after ex vivo stimulation showed a lower expression of CD5, an upregulation of CD138 but not of CD38 and no IgG expression on cell surface, as compared to the expression at day 0 before stimulation (Fig. S3B) which underlined the biological relevance of this model of ex vivo stimulation. Response of a cohort of CLL cells submitted to the selected culture conditions.Because of the clinical and biological heterogeneity of CLL patients, we analyzed the impact of these selected culture conditions on the proliferative response of fresh CLL cells harvested from sixty-five untreated patients referred in Strasbourg Hospitals, essentially Binet stage A (58/65) (Table 1).Among them, 25 harbored unmutated IGHV genes (UM-CLL) and expressed the ZAP70 protein (ZAP70+), 29 had mutated IGHV genes (M-CLL) and did not express the ZAP70 protein (ZAP70-), 10 were ZAP70 + M-CLL and one was ZAP70-UM-CLL.We also analyzed the proliferation of total B cells (20 healthy blood-donors) and naïve (CD19 + , CD27 -, IgM + ) B cells (16 healthy blood-donors).All of the total B-cells (20/20) and 12 out of 16 naïve B cells exhibited more than 25% of dividing cells at day 4 (Fig. 2A) (median 62%; confidence interval (CI) of median [56;82] with up to 4 cell generations for total B cells and median 60%; CI [24;71] for naïve B cells with up to 5 cell generations).In the same conditions, 24/59 (41%) of CLL cells samples proliferated (median 25%; CI [17;27]), showing up to four generations of proliferating cells at day 6 (Fig. 2A,B). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Then we analyzed the impact of a tri-dimensional environment using a semi-solid medium (methylcellulose) where the selected culture conditions also showed their proliferative action (Fig. S4).This culture condition enhanced the proliferation rates and number of cell generations of nearly all CLL cells (89%; 34/38 samples) (Fig. 3A), with up to six generations observed at day 6 (Fig. 3B,C).Of importance, CLL cells stimulation on feeding cells (CD40L-transfected 3T6 cells) neither drove a higher proliferation rate (compared to the soluble stimulation alone) nor increased the number of generations of proliferating cells (Fig. S5A,B). ",
"section_name": "3D semi-solid matrix increases CLL cells proliferation ex vivo.",
"section_num": null
},
{
"section_content": "Next, we tested the effects of CpG-ODN2006 (known to affect B cells proliferation by TLR9 activation) 31 in our culture conditions.While CpG-ODN2006 alone did not induce CLL cells proliferation (for 35 out of 39 CLL cells tested), we observed increased responses (p < 0.0001) when cells were stimulated with CpG-ODN2006 combined to BCR and cytokines in soluble and 3D semi-solid medium (Fig. 4A-F).In addition, CpG/DSP30 and IL-2 cocktails being routinely used as metaphases inductors for cytogenetic diagnosis, we then compared our stimulation conditions with CpG-ODN2006 + IL-2 or commercial premix DSP30/IL-2.The combination of CpG/DSP30 and IL-2 did not increase the proliferation rate, compared to BCR with our selected cytokine stimulation conditions, in soluble or semi-solid medium (Fig. S6A,B).Our work also confirmed previous reports 36 showing that the combination of CpG-ODN2006 and IL-15 is a modest inducer of CLL cells proliferation.Nevertheless, IL-15 addition did not increase the proliferation rate, compared to our soluble BCR stimulation (Fig. S7A,B). ",
"section_name": "TLR9 agonists further increases CLL cells proliferation.",
"section_num": null
},
{
"section_content": "Analyzing the rate of CLL cells proliferation after BCR and cytokine stimulation according to their biological characteristics, we observed that proliferating cells, in soluble medium, exhibit the highest ZAP70 expression levels, compared to non-proliferating cells (p = 0.0043) (Fig. 5).Accordingly, 15/30 (50%) ZAP70+ CLL cells responded to the stimulation with up to four generations (median: 2 generations, with a significant (p = 0.0198) Pearson's correlation coefficient between ZAP70 expression level and the number of cell generations), whereas only 8/28 ZAP70-CLL cells proliferate with a maximum of two generations.Of note, the percentage of IGHV gene identity and CD38 expression did not associate with the cell proliferation in this soluble model (not shown).In semi-solid medium, ZAP70+ CLL cells with mutated IGHV exhibit a proliferative advantage (Fig. 5). When BCR ligation and cytokines are associated with CpG-ODN2006 stimulation, all CLL-cells respond equally in soluble or semi-solid medium, irrespective of the level of ZAP70 and the IGHV status (Fig. 5). ",
"section_name": "Proliferative advantage of ZAP70+ CLL cells in soluble medium.",
"section_num": null
},
{
"section_content": "Given the heterogeneity of the proliferative response of ZAP70+ CLL-cells, we searched for signaling differences between responders (proliferating) and non-responders ZAP70+ cells.We performed western blots to analyze the main signaling pathways activated downstream of the BCR (e.g.ZAP70, pZAP/pSYK, pERK, pIkB) and the JAK/STAT pathway (e.g.pAKT and pSTAT6) in selected responders and non-responders amongst the ZAP70+ UM-CLL cells.Our results (Figs 6A and S8A,B) confirmed ZAP70 expression in all these CLL cells.They also suggested increased ZAP70 Tyr319 /SYK Tyr352 phosphorylation before stimulation (at the steady state) and a further increase upon stimulation in responding ZAP70+ CLL cells, compared to non-responding cells (p = 0.03).IkB phosphorylation was evidenced upon stimulation in both non-responding (p = 0.03) and responding (p = 0.05) CLL cells.The main signaling pathway downstream IL-21R (pSTAT3), revealed no difference between responders (proliferating) and non-responders ZAP70+ cells (Fig. S9A-C).However, we observed an increased STAT6 phosphorylation in responding CLL cells, compared to non-responders (p = 0.03).STAT6 being a major component of the IL-4 receptor signaling pathway, this result was corroborated by the significantly reduced CLL cells proliferation found in the absence of IL-4 in the stimulatory cocktails, or when a selective JAK3 inhibitor (PF-956980) was used (Fig. 6B). ",
"section_name": "Increased ZAP70/SYK and STAT6 phosphorylation in proliferating ZAP70+ CLL cells.",
"section_num": null
},
{
"section_content": "Engagement of the BCR is a crucial event in CLL leukemogenesis, but is not sufficient to induce cell proliferation ex vivo, and is even known to promote B cells apoptosis 15,18 .This caveat can be prevented with coated anti-IgM stimulation, which promotes CLL cells survival 9 but does not induce cell proliferation either.Therefore, BCR-induced CLL-cell proliferation in vivo likely requires additional co-stimulatory signals and proliferative properties of several soluble factors have been described in the literature.Here we performed an exhaustive study of the role of several co-stimulating factors, used solely or in combination, on top of anti-IgM stimulation, to identify mandatory factors sustaining CLL cells proliferation ex vivo.We evaluated CD40L (CD154), a TNF family member expressed on activated T-cells that activates the TNFRSF5 receptor on B cells and triggers several signaling pathways, including NF-kB and ERK, and participates in the survival, proliferation and differentiation of B cells 37 .IL-4, which is mainly secreted by T Follicular helper cell (TFH), activates STAT6 and participates in the activation and survival of B cells 38,39 , was also tested.Finally, we considered IL-21, which is produced by TFH, NKT and TH17 cells, for its role in the induction of JAK/STAT signaling in B cells 40 .IL-21 is known to induce apoptosis 34 , but plays also a role in CLL cells proliferation after priming by IL-4 and CD40L 22,23 , IL-21 was added at Day 1 in the culture medium.We also tested, isolated or in combination, IL-10, IL-2, IL-15, which did not show gain in BCR-induced proliferation effect (not shown) and were not considered further. Several groups have already used soluble CD40L, Il-2, IL-4, IL-10, IL15 or IL-21, isolated or in combination, to stimulate CLL cells [22][23][24]30,34,39 . However,to our knowledge, this is the first study to evaluate their role as BCR co-stimulating factors in soluble conditions without the support of feeder cells.Indeed, most ex vivo models of CLL cells proliferation described in the literature are not consistently defined, as they used co-cultures of fibroblasts expressing CD40L, sometimes in the presence of IL-21 22 , to favor CLL cells survival 32,33 .Co-cultures involving autologous activated T-cells have also been reported 22,28 , including in association with a fibroblast layer 30 .The nature of the cytokines used suggests a T cell dependent activation, possibly delivered by follicular helper T-cells found in the proliferative centers described in secondary lymphoid organs of CLL patients [41][42][43] . Our systematic analysis of BCR ligation associated to different co-stimulations enabled us to select the optimal condition combining BCR activation and co-stimulating factors [CD40L + IL-4 + IL-21] driving CLL cells proliferation.This cocktail was used to stimulate CLL cells harvested from a cohort of patients with different biological characteristics (IGHV UM/M, ZAP70+/-and CD38+/-) and control B cells from healthy donors.In these conditions, about 1/3 of CLL cells proliferate at day 6 after stimulation, among which ZAP70+ B cells appeared particularly responsive.The same stimulation, performed on a 3D semi-solid (methylcellulose) medium, induced the proliferation of nearly all (89%) CLL cells, with a high number of cell generation, representing an efficient model of CLL cells proliferation. A T-dependent help for CLL cells proliferation has not yet been proven in vivo.In our model, T-cells-derived cytokines (CD40L, IL-4 and IL-21) enable BCR-activated CLL proliferation which reinforces this hypothesis.Furthermore, it was shown in the literature that the proliferation of CLL cells xenografted in NOD-SCID mice required concomitant TFH graft in vivo 44 , which also sustains this model.Of note, our results showed the importance of IL-4 and IL-21 co-stimulation (in addition to anti-IgM and CD40L) for CLL cells proliferation, whereas the sole association of anti-IgM and CD40L was sufficient to induce healthy B cells proliferation.Finally, BCR and cytokine stimulation on a tri-dimensional matrix of methylcellulose allowed the proliferation of most of the CLL cells tested, irrespectively of their biological characteristics.This result, which may reflect the natural history of leukemogenesis of CLL cells within secondary lymphoid organs may further enhance the pathophysiological relevance of our ex vivo model.Furthermore, the individualization of clusters of proliferating cells, distributed in this 3D matrix, could allow studying the heterogeneity of intraclonal responsiveness to various drugs in future studies. High proliferation rate (up to 80% with 6-8 cell generations) was only observed in a subset of CLL cells.Such variability may reflect the clinical and biological heterogeneity of CLL patients, among whom those characterized by ZAP70+ CLL cells appeared particularly responsive, as also observed by others 32,45 .However, we noted that all ZAP70+ CLL cells do not equally respond to ex vivo stimulation, which prompted us to investigate in more details the molecular features of the responders and non-responders among this subpopulation.We first confirmed by Western blots the presence of ZAP70 protein in these cells.In CLL cells, ZAP70 activates and extends SYK Tyr352 phosphorylation, independently of ZAP70 phosphorylation 10,46 .This activation induces another SYK Tyr526 phosphorylation, leading to downstream BCR signaling.Our results, performed on a limited number of CLL cell samples (4R vs 3 NR) show that CLL cells that proliferate in soluble medium could achieved a higher initial ZAP70 Tyr319 /SYK Tyr352 phosphorylation, the mechanism of which remains to be investigated.We also show a concomitant increase in pSTAT6 in the proliferating ZAP70+ CLL cells, suggesting that signaling downstream of the IL-4R could participate in CLL proliferation, which reinforces the need to evaluate therapeutic agents inhibiting this pathway 39,47 .More generally, these results highlight the need to explore the functionality of multiple signaling pathways in relation to the heterogeneity of CLL patients.Indeed, as more kinases inhibitors are now available for therapeutic use, there appears to be a rational for further personalized use of these molecules. In conclusion, this study demonstrates the relevance of the BCR activation, combined with a defined set of cytokines, to recapitulate CLL cells proliferation ex vivo.In addition, it highlights the potential roles of T cells in this process.The soluble and 3D culture models established here represent valuable systems for further studies aimed at characterizing the initial steps of malignant evolution of the CLL, with the ultimate goal to identify novel targets for therapeutic purpose.Continued with relevant guidelines and regulations.CLL cells were negatively selected from fresh blood samples using the RosetteSep TM B cell enrichment cocktail (StemCell Technologies, Grenoble, France) and density gradient centrifugation (Ficoll ® Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France).IGHV gene mutation status and ZAP70 expression were evaluated for each patient following established protocols 48,49 .Cytogenetic abnormalities were identified by metaphase analysis and fluorescence in situ hybridization (FISH) using a panel of probes as previously reported 50 .Total (CD19+) or naïve (CD19+, CD27+, IgM+) B cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy blood donors using a negative selection kit (Human naïve B cell isolation kit, Human B cell isolation kit, Stemcell TM Technologies, Grenoble, France) after density gradient centrifugation (Ficoll ® Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France).The cell purity was then controlled by flow cytometry on a Cytomics FC500 System (Beckman-Coulter, Fullerton, CA) 52 .**<7 threshold of T-cells/CLL B cells ratio of ZAP70 mean fluorescence intensity expression for defining ZAP70 positive CLL cells 49 .***≥30% threshold for defining CD38 positive CLL.nd indicates non determined. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "using CD19 + or CD19 + /CD27 -staining (Beckman Coulter, Villepinte, France).CLL B cell purity was assessed after CD19 + /CD5 + staining (Beckman Coulter, Villepinte, France) and ranged from 90% to 99% (median 97%). Cell differentiation was studied after anti-CD38 and anti-CD138 stainings (Beckman Coulter, Villepinte, France) at days 0 and 6. Culture conditions.Cells were cultured in RPMI 1640 Medium (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS) (Dutscher, Brumath, France) and 1% penicillin/streptomycin (Gibco, New York, USA), with or without methylcellulose (MethoCultTM, Stemcell TM Technologies, Vancouver, Canada) at 37 °C, in an atmosphere enriched with 5% CO 2 .B cells at a density of 10 6 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab')2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R&D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).Il-21 was added 24 h after initial stimulation with anti-IgM, CD40L and IL-4 by up/down pipetting in soluble and methyl cellulose medium.In each well, 100 µl of fresh soluble medium was added at Day 3. Other culture conditions included CpG (ODN2006, 5 µg/ml, InvivoGen, San Diego, USA), IL-15 (15 ng/ml, R&D), IL-2 (10 ng/ml, R&D) and PremixAmpliB DSP30/IL-2 (50 or 100 µg/10 6 cells) (Amplitech, Compiegne, France).After 6 days, proliferation was assessed by flow cytometry.Control and CLL cells were co-cultured on fibroblasts (3T6 cells) stably transfected with either a plasmid encoding human CD40L (3T6-CD40L) or mock transfected (3T6).Fibroblasts were pre-cultured overnight in 48-well plates (Dutscher, Brumath, France) at 5.10 4 cells/well.At day 1, fibroblasts were X-ray-irradiated (30 Gy) and re-cultured overnight.At day 2, carboxyfluorescein succinimidyl ester (CFSE) labeled B cells (10 6 cells/ml) were added to the fibroblast layer (10 CLL cells/1 fibroblast).At day 6 and after CD19 staining, B cells proliferation was evaluated by flow cytometry. ",
"section_name": "Subjects and B cell isolation.",
"section_num": null
},
{
"section_content": "Freshly isolated B cells were labeled with 0.5 μM CellTrace TM CFSE (ThermoFisher, Waltham, MA, USA) and incubated for 10 min at 37 °C in the dark.Washed CFSE-labeled cells were stimulated and cultured at 37 °C/5% CO 2 .Four or six days later, B cell proliferation was evidenced by a cell division-dependent decrease in CFSE staining intensity as evaluated by flow cytometry (Fig. S1).Fluorescence data were analyzed with CXP (Beckman Coulter, Fullerton, CA) and FlowJo v.8.7 (TreeStar, Ashland, USA) softwares. ",
"section_name": "CFSE-based proliferation assays.",
"section_num": null
},
{
"section_content": "Cell apoptosis was evaluated using FITC annexin-V Apoptosis detection kit and propidium iodide (PI) (both from BD Pharmingen, BD Bioscience, San Jose, CA, USA).Cells (10 6 ) were washed in phosphate-buffered saline (PBS) and re-suspended in annexin buffer before the addition of FITC annexin-V Western blotting.After stimulation, B cells were centrifuged and cell pellets re-suspended in lysis buffer (1% Triton X-100, 20 mM Tris-HCl [pH 8], 130 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM PMSF, and protease inhibitors) for 20 minutes on ice.Lysates were centrifuged for 10 minutes at 300 g at 4 °C, and supernatants subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes.Membranes were then blocked using 5% milk in Tris-buffered saline (TBS; 20 mM Tris [pH 7.5], 150 mM NaCl) for 1 h at room temperature.The blots were then incubated with anti-ZAP70 (Clone E267) (Abcam, Paris, France), anti-phospho ZAP70 Tyr319 (Abcam, Paris, France) -which recognizes also SYK Tyr352 phosphorylation in CLL B cells 46 -, anti-phospho-SYK Tyr323 (Santa Cruz, Nanterre, France), anti-phospho-ERK1/2 Tyr204 (clone E-4) (Santa Cruz, Nanterre, France), for 2 h at room temperature, anti-phospho-STAT6 Tyr641 (Cell Signaling, France), anti-phospho-AKT Thr308 (clone D25E6) (Cell Signaling), anti-phospho-IkB Ser32/36 (clone 5A5) (Cell Signaling), anti-phospho-STAT3 Tyr705 (clone EP2147Y) (GeneTex), anti-STAT3 (clone 79D7) (Cell Signaling) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit monoclonal antibodies (1 h at 25 °C), and revealed by Electro Chemo Luminescence (ECL Plus Western blotting Detection Reagents (Amersham, Courtaboeuf, France) or SuperSignal West Femto Maximum Sensitivity substrate (Pierce, Courtaboeuf, France), according to manufacturers' instructions.To confirm the presence of equal amounts of loaded proteins, membranes were incubated with anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, clone 6C5) (Merck Millipore, Guyancourt, France).Signals were visualized by chemiluminescence and processed by the Image Lab TM software (BioRad, Marnes-la Coquette, France).The relative intensity of bands was measured and calculated using the Image J software (http://rsb.info.nih.gov/ij/index.html).The abundance of each protein has been normalized to GAPDH within the same sample on the same western blot. ",
"section_name": "Apoptosis assay.",
"section_num": null
},
{
"section_content": "Statistical analyses were performed using R 3.2.4(R Core Team, 2016, R Foundation for Statistical Computing, Vienna, Austria).Graphics were created using Graphpad Prism 7.0 (Graphpad Software; Inc, La Jolla, CA, USA).We used permutational ANOVA for repeated measurements (lmPerm R package, https://github.com/mtorchiano/lmPerm)to compare more than two groups.Posthoc tests were done using nonparametric multiple comparisons tests carried out with the nparcomp R package 51 .Differences between two groups were assessed using either nonparametric Wilcoxon matched-pairs signed rank test of nonparametric Mann-Whitney test (unpaired, when applicable).A p value of <0.05 was considered statistically significant.*p < 0.05; **p < 0.01; ***p < 0.001. ",
"section_name": "Statistical analyses.",
"section_num": null
}
] |
[
{
"section_content": "We would like to thank Drs Fanny Baran-Marszak, Frédéric Davi, Jean-Noël Freund, Delphine Rolland, Paolo Ghia and Robert Zeiser for critical reading of this manuscript.We are grateful to Drs Anne-Cécile Galoisy and Carine Gervais (Laboratoire d'Hématologie, Hôpitaux Universitaires de Strasbourg, France) for cytology analysis, Beatrice Uring-Lambert (Laboratoire d'Immunologie, Hôpitaux Universitaires de Strasbourg, France) for help with flow cytometry, Laurent Mailly (INSERM UMR_S 1110, Strasbourg, France) for access to the cell irradiaiton platform, Shanti Amé and Blandine Guffroy (Service d'Hématologie Adulte, Hôpitaux Universitaires de Strasbourg, France) for providing CLL samples, Manuela Tavian (INSERM UMR_S 949, Etablissement Français du Sang Grand Est, Strasbourg, France), Anne-Sophie Korganow (CNRS UPR9021, Strasbourg, France), Cendrine Seguin (CNRS UMR 7199, Illkirch, France) and Jozo Delic (Commissariat à l'Energie Atomique, Fontenay-aux-roses, France) for helpful suggestions and Ms Nathalie Perrusson (Laboratoire d'Hématologie, Hôpitaux Universitaires de Strasbourg, France) for technical help.This work was supported by grants from the Institut Thématique Multi-Organism (ITMO) cancer initiative within the framework plan cancer 2009-2013 \"GenPred project\", Initiative d'Excellence-CNRS, the Association pour la Recherche contre le Cancer (ARC), Alsace Cancer association, the Genomax, the Strasbourg School of Medicine Next Generation Sequencing center, the Institut Universitaire de France (IUF), the Ligue contre le Cancer and the LABEX Transplantex [ANR-11-LABX-0070_Transplantex] (French National Research Agency; ANR), INSERM UMR_S1109 and UMR_S1113. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "C.S., W.I. and O.T., S.F., Y.G. performed experiments, analyzed data and reviewed the manuscript.L.M., C.M.R. and L.M. performed C.L.L. cells biological characterization and reviewed the manuscript.L.M.F., E.T. and R.H. provided C.L.L. samples, clinical information and reviewed the manuscript.F.B. and M.M.B. performed statistical analysis.S.B. analyzed the data.T.M., P.G., S.B. wrote the manuscript.L.V. designed and supervised the study, analyzed data and wrote the manuscript.All authors read and concurred with the manuscript and its contents. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36853-8. 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": "C.S., W.I. and O.T., S.F., Y.G. performed experiments, analyzed data and reviewed the manuscript.L.M., C.M.R. and L.M. performed C.L.L. cells biological characterization and reviewed the manuscript.L.M.F., E.T. and R.H. provided C.L.L. samples, clinical information and reviewed the manuscript.F.B. and M.M.B. performed statistical analysis.S.B. analyzed the data.T.M., P.G., S.B. wrote the manuscript.L.V. designed and supervised the study, analyzed data and wrote the manuscript.All authors read and concurred with the manuscript and its contents. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36853-8. ",
"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
}
] |
10.1186/1756-0500-3-341
|
Evidences showing wide presence of small genomic aberrations in chronic lymphocytic leukemia
|
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western population. Although genetic factors are considered to contribute to CLL etiology, at present genomic aberrations identified in CLL are limited compared with those identified in other types of leukemia, which raises the question of the degree of genetic influence on CLL. We performed a high-resolution genome scanning study to address this issue.Using the restriction paired-end-based Ditag Genome Scanning technique, we analyzed three primary CLL samples at a kilobase resolution, and further validated the results in eight primary CLL samples including the two used for ditag collection. From 51,632 paired-end tags commonly detected in the three CLL samples representing 5% of the HindIII restriction fragments in the genomes, we identified 230 paired-end tags that were present in all three CLL genomes but not in multiple normal human genome reference sequences. Mapping the full-length sequences of the fragments detected by these unmapped tags in seven additional CLL samples confirmed that these are the genomic aberrations caused by small insertions and deletions, and base changes spreading across coding and non-coding regions.Our study identified hundreds of loci with insertion, deletion, base change, and restriction site polymorphism present in both coding and non-coding regions in CLL genomes, indicating the wide presence of small genomic aberrations in chronic lymphocytic leukemia. Our study supports the use of a whole genome sequencing approach for comprehensively decoding the CLL genome for better understanding of the genetic defects in CLL.
|
[
{
"section_content": "CLL (Chronic lymphocytic leukemia) is an incurable disease mainly affecting the B cell lineage in the western population, with a median age of diagnosis of 72 year old [1].Determining the cause of CLL is crucial for understanding the acquisition and for clinical diagnosis, treatment and prognosis of CLL.Genetic factors have been linked to the etiology of CLL.Cytogenetic analyses identified chromosomal abnormalities including del11q23 affecting the ATM gene, tri12, del 13q14, and del17p13 affecting TP53 gene [2].In addition, CGH studies found gains and losses in Xp11.2-p21 and Xq21qter [3].Molecular studies identified three genes: IgVH, CD38 and ZAP-70 that correlate with CLL prognosis [4][5][6].A CLL-specific microRNA signature was also identified, suggesting that microRNA deletion could be involved in CLL [7].SNP array studies identified 2q21.2, 6p22.1 and 18q21.1 abnormalities that follow a Mendelian inheritance pattern [8].Whole genome association studies also identified multiple loci at 2q37.3, 8q24.21,15q21.3 and 16q24.1 that appear to be associated with genetic susceptibility to CLL [9]. Although evidence supports the involvement of genetic factors in CLL, the frequency of genomic aberrations identified in CLL is relatively lower than those observed in the leukemias affecting other types of hematopoietic lineages [10].This information suggests that the CLL genome is relatively intact with fewer aberrations than other types of leukemia.Alternatively, more genomic aberrations may exist in CLL but these could mainly be small lesions in the CLL genome that are difficult to detect using conventional technologies due to their limited resolution.With the rapid progress of genome sequencing technologies, enthusiasm is increasing for pursuing comprehensive detection of genomic aberrations in cancer by sequencing cancer genomes.In the case of CLL, a critical issue is to know the degree of genomic aberrations in order to justify the use of whole genome sequencing approach to analyze CLL genome.We reasoned if we can scan certain CLL genomes at sufficient high resolution and at reasonable genome coverage, we should gain first-hand information to estimate the degree of genomic aberrations in CLL. We recently developed the DGS (Ditag Genome Scanning) technique that uses next-generation DNA sequencing technologies to collect paired-end sequences from restriction DNA fragments across a genome [11].Using this technique, we analyzed CLL genomes.Nine samples of peripheral blood from untreated CLL patients diagnosed in Northwestern University Lurie Cancer Center and University of Chicago Medical Center were used in this study, of which three were used for paired-end tag collection, and eight including two used in paired-end tag collection were used for full-length sequencing analysis (Additional file 1: Supplemental Table S1).Informed consent was made by the patients, and the use of clinical CLL samples was approved by the institutional review board of University of Chicago and Northwestern University following institutional guidelines.The detailed experimental process followed the published protocol [11] and outlined in Figure 1.Briefly, mononuclear cells were isolated from each CLL peripheral blood or bone marrow sample by using NycoPrep™ A solution (Axis-Shield).Human genomic DNA was extracted from mononuclear cells by using QIAamp DNA Blood Kit (QIAGEN) following the manufacturer's protocol.To generate the DGS library, genomic DNA was fractionated by HindIII restriction digestion.The restriction fragments were dephosphorylated by CIP and cloned into pDGS-HindIII vector that contains two MmeI sites next to the HindIII cloning site.The genomic library was digested by MmeI to release two tags from the cloned DNA fragments.The tag-vector-tag fragments were then gel-purified, and re-ligated to form a ditag library.Ditags were released from the vectors by HindIII digestion, gel-purified, and concatemerized by using T4 DNA ligase (Promega).The concatemers at 200 to 500 bps were agarose-gel-purified and used for ditag sequencing by using a 454 GS20 sequencer (454 Life Sciences).Ditags were extracted from the resulting sequences based on the HindIII sites.Same ditags were combined to generate a unique ditag with the corresponding copy numbers. To generate the reference ditag database, virtual Hin-dIII restriction fragments were generated from known human genomic sequences.Two 16-bp virtual tags were extracted from the 5' and the 3' ends of each virtual fragment, and connected to form a reference ditag representing the virtual DNA fragment.The following sequences were used to extract the reference ditags: 1. Human genome reference sequences (hg18 Initial ditag mapping was performed with perfect match between experimental ditags and hg18 reference ditags.For the unmapped experimental ditags, a singlebase mismatch in each single tag of the ditag was allowed to compensate for possible sequencing error or SNP.To identify the unmapped ditags related with homopolymer generated by 454 sequencing chemistry, the unmapped ditags with more than two homo-bases were stretched, e.g.AAA -> AAAA, or shortened, e.g.AAA -> AA, and mapped to reference ditags again.For the ditags remaining unmapped, they were mapped to the reference ditags of other sequence sources in the ditag reference database.The ditags remaining unmapped after these processes were defined as the unmapped ditags. Unmapped ditag sequences were used to design sense primers and antisense (reverse/complementary) primers, with four extra bases CAGC added to the 5' end of sense primer and CGCC to the 5' end of antisense primer.Genomic DNA digested by HindIII was used as the templates for PCR amplification.PCR was performed with 35 cycles at 95°C 30 sec, 57°C 60 sec, and 72°C 3 min, followed by extension at 72°C for 10 min.The amplified products in each reaction were cloned into Outline of the experimental process.Genomic DNA samples were digested by restriction enzymes.Ditags (paired-end tags) were collected from both ends of restriction fragments and sequenced.The ditag sequences were compared to known human reference genome sequences.The unmapped ditags were used as sense and antisense PCR primers to amplify their original DNA fragments to generate full-length sequences.The sequences were mapped to reference genome sequences to determine the type of genomic aberrations.pGEM-T vector (Promega), transformed into E. coli TOP10 (Invitrogen), and plated in a single well of the 48-well Qtrays (Genetix).Four clones from each transformation were amplified by colony-PCR using M13F and M13R primers, and sequenced by Big-Dye Terminator v3.1 Cycle Sequencing Kit (ABI) using M13F primer.For the sequences that did not reach the full-length, second sequencing reactions were performed using M13R primer.To determine the genomic aberrations, each full-length sequence was mapped to hg18 using BLAT at a minimum of 90% identity as the cut-off. The paired-end ditags were collected from three CLL samples.Genomic DNA from each sample was fractionated by HindIII digestion, which provides 3,561-bp resolution on average across the genome based on hg18 sequences [11].Unique paired-end ditags of 272,193, 320,283, and 307,547 was collected from each CLL sample, covering 32%, 34% and 38% HindIII fragments in each CLL genome respectively.Comparing the three ditag sets shows that between 87,968 and 108,579 ditags are present between two CLL samples, and 51,632 ditags are commonly present in all three CLL samples (Table 1A).The ditags present only in individual CLL sample could be the ditags representing individual genomic differences, the ditags potentially originating from experimental artifacts, or ditags detected in one but not in others due to unsaturated ditag collection in each CLL under the sequencing scale.The 51,632 ditags detected in all three CLL samples cover 5% of genomic DNA fragments commonly detected in the three CLL genomes.In order to provide high confidence for further downstream studies, we focused on the 51,632 common ditags for further mapping analysis.We compared the 51,632 common ditags with multiple known human genome sequences, including the human genome reference sequence hg18, human SNP, human GM15510 genome sequences, chimpanzee genome sequences that are highly homologous to the humans, Watson genome sequences, and Venter genome sequences.Of the 51,632 ditags used for the mapping, 98.3% (50,799) map to hg18 that represent normal genomic fragments in the CLL genomes, 0.4% (230) are unmapped ditags that represent potential genomic aberrations commonly present in all three CLL genomes, and the remaining ditags map to other genomes that represent normal genome variations (Table 1B). To determine the types of genomic aberrations for the unmapped ditags, we generated full-length sequence for the restriction DNA fragment detected by the unmapped ditags by using the \"ditag-PCR\" method, in which the ditag sequences were used as PCR sense and antisense primers to amplify the original DNA fragment that derived the unmapped ditag.We performed 192 reactions in eight CLL samples including two used in ditag collection and six additional CLL samples.Under the conditions that a full-length sequence must be longer than 50 bases and detected at least in the CLL used in ditag collection or at least in two additional CLL samples, 220 full-length sequences were generated from 100 unmapped ditags.Mapping the full-length sequences to hg18 identified different types of genomic aberrations caused by insertion, deletion and base change.Many of these aberrations created new HindIII restriction site that leads to the release of unmapped ditag, or the change of ditag sequence composition that prevents ditag mapping.These aberrations were observed in both coding and non-coding regions in CLL genome.For example, aberrations were detected in exons of NEK8, RUNX1 and MUC2 genes, and introns of 20 other genes (Table 2A, Additional file 2: Supplementary table S2).NEK8 encodes a member of the serine/threonine protein kinase family, which plays a role in cell cycle progression from G2 to M phase and is over-expressed in breast cancer [12].A 353-base sequence converted from the unmapped ditag AAGCT-TACCCTCTGGACGCCTGTATGAAGCTT maps to the last exon (Exon 15) coding for the 3' UTR of NEK8.Two HindIII restriction sites were inserted in the sequence that are not present in the wild-type NEK8 gene.RUNX1 is a gene involved in AML through its involvement in the t(8;21) [13].A 434-base full-length sequence from a ditag AAGCTTCGGCCTATAG/ ACAACCTAACAAGCTT was detected in all eight CLL samples, and maps to intron 3 and exon 4 of RUNX1. Analyzing the mapped region shows a T to C singlebase change between the sequence and exon 4 of RUNX1 gene.Searching dbSNP reveals that this is a SNP (rs1235270).Due to the uncertainty of RUNX1 protein coding sequence itself, it is not certain if this germline SNP causes a coding amino acid change.Several bases are also changed in the mapped intron 3 of RUNX1 gene.These base changes raise an interesting question whether RUNX1 could be involved in CLL. MUC2 is a member of the MUCIN family, which codes for high molecular weight glycoproteins.The abnormalities of MUC2 is linked with colorectal and pancreas cancer [14].A 410-base sequence derived from an unmapped ditag AAGCTTCCGGTCGGCTTCGCAG-TAGAAAGCTT covers intron 29, exon 30 and intron 30 of MUC2 gene.This sequence also contains two Hin-dIII restriction sites AAGCTT inserted at both its ends that do not exist in the wild-type MUC2 gene.Only three aberrations were detected in the exon of three known genes.This could be attributed to the limited genome coverage of the study and the low percentage of the exon-coding sequences in the genome.With increased genome coverage, it would be possible to identify the aberrations affecting more exons.Aberrations also affect the introns of multiple genes.FHIT encodes diadenosine 5',5'''-P1,P3-triphosphate hydrolase involved in purine metabolism [15].It is located in the common fragile site FRA3B on chromosome 3, where carcinogen-induced damage can lead to translocations in several cancers.A 283-base sequence maps to intron 8 of FHIT gene but its tag 1 contains GA to TG change.HYDIN encodes an axonemal protein; mutation of HYDIN is related to congenital hydrocephalus [16].Two full-length sequences of 605-bp and 614-bp from two different unmapped ditags were obtained from seven CLL samples.Both sequences map to 21st intron of HYDIN.The 605-bp sequence contains CCTACGGCG in its tag 2 converted from wild-type gCcACaGCa (lowercase refers to the changed base), and the 614-bp sequence contains CGCC converted from wild-type tGCt in its tag 1 and an internal insertion.NCOR2 is a transcriptional regulator that recruits histone deacetylases to promoters [17].A 582-base sequence maps to intron 1 of NCOR2, but its tag 1 contains an AAGC insertion, and tag 2 contains a C to T change, an AG deletion, and a T insertion.TYK2 is a member of the JAK family involving in IFN-g, IL-6, IL-10 and IL-12 signaling.Mutation in this gene is associated with hyperimmunoglobulin E syndrome [18].A 268-base sequence maps to intron 14 of TYK2 but its tag 1 contains an AAGCTTA insertion and its tag 2 contains a TGAAGCTT insertion.Both insertions create HindIII restriction sites that lead to the generation of the unmapped ditag.A 197-base sequence was detected in seven CLL samples and two different sequences of 112-base and 170-base were generated from the CLL used in ditag collection.All three sequences map to UBAP2 located at 9p13.3, a gene involved in the ubiquitination pathway [19].For the 197-base sequence, its 178 bases map to intron 6 of UBAP2 gene and the remaining 18 bases have no map, whereas the 112-base and 170-base sequences contain different insertions.Although the aberrations in many of these genes have been correlated with different types of cancer, most have not been linked with CLL. Non-coding regions contribute to the majority of the genome, and contain important functional elements involving DNA replication, genome stability, regulation of gene expression, and coding for non-coding transcripts etc. Extensive characterization of non-coding region could provide rich candidate markers for clinical applications and identify the hotspots of genomic aberrations involving cancer development.A total of 37 sequences generated from 30 unmapped ditags mapped to the non-coding regions in the genome with various types of abnormalities (Table 3, Additional file 3: Supplemental Table S3).Although these loci are not directly located in the coding regions, many genes are located nearby the mapped locations.Of the 26 loci specifically mapped by the sequences, 15 have genes located either upstream, downstream or both within 100 kb distance.For example, a 614 base sequence maps to 5q35.One hundred and forty seven full-length sequences converted from 57 unmapped tags map to the highly repetitive sequences in the non-coding regions.Of these sequences, 110 sequences map to the ALR/Alpha satellite sequences of the centromere, and chromosome 2, S4): 23 sequences converted from 13 unmapped tags map to the centromere of chromosome 2 at 2p11.1, 41 sequences converted from 16 ditags map to the centromere of chromosome 10 at 10q11.1, and 22 sequences converted from 6 unmapped ditags map to the centromere of chromosome 17 at 17p11.1.The presence of highly frequent aberrations in ALR/Alpha satellite sequences in these three chromosomes suggests that these could be the hot spot of genomic aberrations in CLL.Aberrations in repetitive sequences have been shown to contribute to cancer development [20].However, it is difficult to analyze the aberrations in these highly repetitive regions using the hybridization-based approach due to the difficulty to designing specific probes.Our results show that restriction sequencing-based approach provides a useful tool to study the aberrations in these regions. Ten full-length sequences generated from eight unmapped ditags did not map to known human genome sequences (Table 2B.Additional file 5: Supplementary table S5).For example, a 107-base full-length sequence converted from an unmapped ditag AAGCTTAGATAGAGCG-CAGTCAACTGAAGCTT was detected in all eight CLL samples.However, it does not map to the reference genome sequences.These sequences represent the DNA contents present in CLL genomes but not in normal genomes. Through high-resolution scanning of three CLL genomes and verifying the results using full-length sequences and additional CLL genomes, our study provides evidence showing the wide presence of genomic aberrations in CLL, of which most are small lesions.Studies with increased number of CLL samples and at high genome coverage will be required to better understand the genetic aberrations in CLL.Although the study used multiple genomics databases to eliminate the changes from normal genomic polymorphism, further studies with normal DNA from the same patient will be required to fully distinguish somatic mutations from germline variations in CLL. ",
"section_name": "Findings",
"section_num": null
}
] |
[
{
"section_content": "We wish to thank Dr. Janet Rowley for comments on the study.The study was supported by Mazza Foundation and Guglielmi Fidelity Charitable Fund (SW). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Additional file 1: Supplementary table S1.CLL samples used for the study. Additional file 2: Supplementary table S2.Aberrations in exon and intron. Additional file 3: Supplementary table S3.Aberrations in the intergenic region. Additional file 4: Supplementary table S4.Aberrations in the repetitive region. Authors' contributions YK, YCJ, JC, AHA, PK performed laboratory work.YZ, SM, SR provided clinical samples and data analysis, SR, SW designed the experiment.SW wrote the paper.All authors read and approved the final manuscript. The authors declare that they have no competing interests. ",
"section_name": "Additional material",
"section_num": null
},
{
"section_content": "Additional file 1: Supplementary table S1.CLL samples used for the study. Additional file 2: Supplementary table S2.Aberrations in exon and intron. Additional file 3: Supplementary table S3.Aberrations in the intergenic region. Additional file 4: Supplementary table S4.Aberrations in the repetitive region. ",
"section_name": "Additional material",
"section_num": null
},
{
"section_content": "Authors' contributions YK, YCJ, JC, AHA, PK performed laboratory work.YZ, SM, SR provided clinical samples and data analysis, SR, SW designed the experiment.SW wrote the paper.All authors read and approved the final manuscript. ",
"section_name": "Additional file 5: Supplementary table S5. Aberrations only present in CLL genomes.",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1007/s00259-019-04417-1
|
Targeting B-cell malignancies with the beta-emitting anti-CD37 radioimmunoconjugate 177Lu-NNV003
|
The aim of this study was to explore the β-emitting lutetium-177 labelled anti-CD37 antibody NNV003 (177Lu-NNV003, Humalutin®) for the treatment of non-Hodgkin's lymphoma in in vitro studies and in animal models.Cytotoxicity of 177Lu-NNV003 was measured in REC-1 (mantle cell lymphoma) and DOHH-2 (diffuse large B cell lymphoma) cell lines. Biodistribution was studied in mice bearing subcutaneous DOHH-2 or MEC-2 (chronic lymphocytic leukaemia) xenografts. The therapeutic effect of a single injection of 177Lu-NNV003 was measured in mice intravenously or subcutaneously injected with REC-1 cells. Haematological and histopathological assessments were used to evaluate the toxic effect of 177Lu-NNV003. The immunotherapeutic effect of NNV003 was assessed by measuring binding to Fcγ receptors, activation of ADCC and ADCP. NNV003's immunogenicity potential was assessed using in silico immunogenicity prediction tools.177Lu-NNV003 showed an activity dependent antiproliferative effect in all cell lines. Maximum tumour uptake in vivo was 45% of injected activity/g in MEC-2 tumours and 15% injected activity/g in DOHH-2 tumours. In mice injected intravenously with REC-1 cells, 177Lu-NNV003 (50-100 MBq/kg) improved survival compared to control groups (p < 0.02). In mice with subcutaneous REC-1 xenografts, 500 MBq/kg 177Lu-NNV003 extended survival compared to the control treatments (p < 0.005). Transient haematological toxicity was observed in all mice treated with radioactivity. NNV003 induced ADCC and ADCP and was predicted to have a lower immunogenicity potential than its murine counterpart.177Lu-NNV003 had a significant anti-tumour effect and a favourable toxicity profile. These results warrant further clinical testing in patients with CD37-expressing B cell malignancies.
|
[
{
"section_content": "Non-Hodgkin's lymphoma (NHL) accounts for 2.7% of all cancers and 2.4% of all cancer deaths worldwide [1].Among NHL, diffuse large B cell lymphoma (DLBCL) is the most common subtype, whereas mantle cell lymphoma (MCL) is relatively rare [2].Chronic lymphocytic leukaemia (CLL), another B cell malignancy, is the most common leukaemia.Chemotherapy is still the cornerstone in the treatment of these diseases, often accompanied by monoclonal antibodies against the CD20 antigen [3][4][5].However, targeted therapies like Bruton's tyrosine kinase (BTK) inhibitors and BCL-2 inhibitors are emerging as novel treatment options, particularly in MCL and CLL [3,4,6]. Survival rates for these haematological diseases have improved over recent decades [7][8][9].However, many patients will still relapse and become refractory to chemotherapy, anti-CD20 therapy and even the novel targeted treatments [10][11][12].Consequently, there is a medical need for new approaches and other targets to overcome resistance.In this study we evaluate a new compound, a next generation radioimmunoconjugate (RIC), for treatment of B cell malignancies.The RIC, 177 Lu-NNV003, consists of a chimeric mouse-human anti-CD37 antibody (NNV003), conjugated with p-SCN-Bn-DOTA (DOTA) that chelates the β-emitting radionuclide lutetium-177. Two β-emitting anti-CD20 radioimmunotherapies were approved by the FDA for NHL treatment in the early 2000's: 90 Yibritumomab tiuxetan (Zevalin®) and 131 I-tositumomab (Bexxar®).Despite their strong anti-tumour activity these radioimmunoconjugates have not been implemented in clinical practice, as testified by the withdrawal of I 131 -tositumomab in 2014 from the market for business reasons [13]. CD37 is a highly glycosylated transmembrane protein selectively expressed by normal B cells, as well by many B cell malignancies [14].CD37 has been used as target for naked antibodies and two antibody drug conjugates [15][16][17][18][19]. Furthermore, the next generation RIC ( 177 Lu-lilotomab satetraxetan, Betalutin®), which is the murine version of 177 Lu-NNV003, is currently in clinical trials for relapsed/ refractory lymphomas (NCT01796171, NCT02658968) [20,21] and, based on preclinical data [22], in combination with rituximab for previously treated follicular lymphoma (NCT03806179).In the present study, we have explored the efficacy of unlabelled and lutetium-177 labelled NNV003 chimeric antibody in CLL, MCL and DLBCL models. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Labelling and quality control of antibodies with 177 Lu NNV003 (IgG1, mouse variable regions, κ, and human constant region, κ) and cetuximab (Merck KGaA, mouse-human chimeric IgG1, κ) were conjugated with p-SCN-Bn-DOTA (Macrocyclics, Texas, USA) and labelled with 177 Lu as previously described (see Online Resource for details) [20]. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "MEC-2 (CLL, DSMZ GmbH, Braunschweig, Germany) was cultured in IMDM medium; REC-1 (MCL, ATCC, USA) and DOHH-2 (DLBCL, DSMZ GmbH, Braunschweig, Germany) were cultured in RPMI medium.The media were supplemented with 10% heat inactivated fetal bovine serum and 1% penicillinstreptomycin (Thermo Fisher Scientific, Massachusetts, USA). ",
"section_name": "Cell lines",
"section_num": null
},
{
"section_content": "The binding properties of NNV003 and lilotomab to human and mouse Fcγ receptors (hFcγRs and mFcγRs) and to the neonatal Fc receptor (FcRn) were evaluated by ELISA as previously described [23]. ",
"section_name": "Binding of NNV003 and lilotomab to effector molecules",
"section_num": null
},
{
"section_content": "Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) induction in MEC-2, REC-1 and DOHH-2 cell lines were measured using an ADCP-and ADCC Reporter Bioassay kit (Promega, USA) according to the manufacturer's instructions (see Online Resource for details). ",
"section_name": "ADCP and ADCC assays",
"section_num": null
},
{
"section_content": "Cell proliferation after treatment with unlabelled or 177 Lu labelled NNV003, or unspecific isotype antibody ( 177 Lucetuximab) was measured in REC-1 and DOHH-2 using CyQUANT™ NF Cell Proliferation Assay Kit (Thermo Fisher Scientific, USA) (see Online Resource for details). ",
"section_name": "Cell cytotoxicity of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "Animal studies were approved by the National Animal Research Authorities and carried out according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes regulations.All mice were housed under pathogen free condition and had ad libitum access to food and water.In all animal models health and body weight conditions were monitored using a score system to track symptoms severity.Animals were euthanized when a humane endpoint was reached. ",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "",
"section_name": "Biodistribution of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "The biodistribution data from the DOHH-2 s.c.model was used to calculate the absorbed radiation doses from 177 Lu-NNV003 in different organs as previously described [24] (see Online Resource for details). ",
"section_name": "Radiation dosimetry of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "To assess the anti-tumour effect of the unlabelled NNV003 compared to lilotomab in vivo, female CB17 SCID mice (C.B-Igh-1 b /IcrTac-Prkdc scid , Taconic, Denmark, 8 weeks old, average weight 18.5 g) were injected intravenously (i.v.) with 10 × 10 6 REC-1 cells one day prior to treatment; 100 μg antibodies were injected i.v.twice a week for four weeks (n = 10 mice per group).Humane end-points were > 10% weight loss/ gain over a period of one week, palpable tumour > 12 mm, or signs of substantial discomfort.The remaining mice were then euthanized and necropsied 166 days after cell injection. Anti-tumour effect of a single injection 177 Lu-NNV003 was investigated in female CB17 SCID mice (CB-17/Icr-Prkdc scid/scid /Rj, Janvier Labs, France, 9-10 weeks old, average weight 20 g), who were injected i.v. with 20 × 10 6 REC-1 cells two days prior to therapy injections.Six groups (n = 8) were treated with 0.9% NaCl, 0.167 mg/kg unlabelled isotype control (cetuximab), 100 MBq/kg 177 Lu labelled cetuximab, 0.167 mg/kg NNV003, 50 MBq/kg or 100 MBq/kg 177 Lu-NNV003.Mouse IgG2a-κ was injected i.p. the day before therapy injection.Humane end-points were hind leg paralysis, weight loss > 15%, palpable tumour > 15 mm or any signs of severe sickness or discomfort.The remaining mice were then euthanized and necropsied 169 days after cell injection. To compare the anti-tumour effect of 177 Lu-NNV003 and NNV003, therapies were tested in a s.c.model where 10 × 10 6 REC-1 cells were injected s.c. in each flank of female nude mice (Hsd:Athymic Nude-Foxn1 nu , Envigo, France, 8 weeks old, average weight 22.0 g).To ensure tumour growth, 80 μl Anti-asialo (diluted in 2 ml, Wako Chemicals, USA) was injected i.p. at day -1, 4 and 8 from cell inoculation.When the average tumour size was 173 mm 3 13 days after cell inoculation, mice were treated with 0.9% NaCl, 10 mg/kg unlabelled isotype control (cetuximab), 500 MBq/kg 177 Lu labelled isotype control ( 177 Lu-cetuximab), 1.6 or 10 mg/kg NNV003, or 500 MBq/kg 177 Lu-NNV003 (n = 10).Mouse IgG2a-κ was injected i.p. the day before therapy injection.Humane end-point was reached when total tumour volume was 2500 mm 3 (tumour burden within 10% of body weight) or ulcer on the tumour surface. GraphPad Prism 7.00 (GraphPad Software, La Jolla California, USA) was used to create Kaplan-Meier survival curves and to perform comparisons of the groups using Logrank test and Holm-Sidak method with α = 0.05. ",
"section_name": "Therapeutic effect of NNV003 and 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "Female Rag2 deficient mice (129S6/SvEvTac-Rag2 tm1Fwa , Taconic, USA, 9-13 weeks old, average weight 20.5 g) were injected i.v. with 10 × 10 6 DOHH-2 cells three days prior to therapy injections.Mice (n = 9 or 10) were treated with 200, 300 or 400 MBq/kg 177 Lu-NNV003, 2 or 30 mg/kg unlabelled NNV003, 0.9% NaCl, 2 mg/kg unlabelled or 300 MBq/kg 177 Lu labelled cetuximab.Mouse IgG2a-κ was injected i.p. the day before therapy injection.Blood was sampled in three to five mice in each group 2.5 weeks before therapy injection and 1.5, 3, 4.5, and 6 weeks after.Red blood cell, white blood cell and platelet concentrations were measured using a haematology analyser (MS4 analyser, Melet Schloeing Laboratories, France).Serum was collected when the animals were euthanized and alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea and creatinine were measured on a VetScan VS2 (Abaxis, USA).One-way ANOVA was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla California, USA) to test for significant differences.Histopathological examinations were performed on selected organs. ",
"section_name": "Toxicity of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The binding properties of NNV003 and its mouse counterpart lilotomab to the classical human and murine forms of the FcγRs are shown in Figs.1a andb.Generally, NNV003 bound more strongly than lilotomab to most of the activating hFcγRs, while lilotomab did not bind to hFcγRI, hFcγRIIIa and hFcγRIIIb.Both antibodies bound weakly to human C1q.Lilotomab bound more strongly than NNV003 to mFcγRIIb and mFcγRIII.However, NNV003, but not lilotomab, bound to mFcγRI and mFcγRIV.Only NNV003 bound pH dependently to human FcRn while lilotomab did not bind (Fig. 1c), which is in line with the fact that mouse IgG subclasses do not bind significantly to the human receptor, and as such NNV003 and not lilotomab is expected to be rescued from degradation via FcRn in vivo [25,26], indicating that the biological halflife of NNV003 will be longer than for lilotomab. Both ADCC and ADCP induction was highly dependent on target cell line (Fig. 1d ande).The level of induction by NNV003 was comparable to that of rituximab (positive control) in MEC-2 cells, but generally lower in other target cells.Cell lysis by ADCC by both NNV003 and lilotomab was low compared with rituximab and comparable to the no antibody control in most of the target cell lines (Fig. 1f). No cytotoxic effect of NNV003 alone was observed in vitro (Fig. 1g); however, treatment with 100 μg NNV003 twice a week for four weeks resulted in 100% survival of mice i.v.injected with REC-1 cells with more than 150 days observation time (Fig. 1h). The predicted immunogenicity potential of NNV003 was lower than for lilotomab (Table 1, Fig. S1 in Online Resource). Anti-proliferative effect, binding and internalisation of 177 Lu-NNV003 in vitro 177 Lu-NNV003 showed an anti-proliferative effect against the cell lines tested (Fig. 2).However, only in DOHH-2 cells did 177 Lu-NNV003 significantly inhibit proliferation more than the 177 Lu-IgG1 control, indicating that the radiation from the unbound activity in the serum was important for the antiproliferative effect.Binding studies with 177 Lu-NNV003 showed that the antibody bound to REC-1, MEC-2 and DOHH-2 with an affinity of approximately 1 nM and that the cell lines expressed 60 000 (REC-1), 80 300 (MEC-2) and 104 000 CD37 antigens per cell (Table S1 in Online Resource).After 18 h of incubation, REC-1 had internalised 37% of the bound 177 Lu-NNV003, MEC-2 had internalised 14% and DOHH-2 6% (Fig. S2 in Online Resource). ",
"section_name": "Characterisation of NNV003",
"section_num": null
},
{
"section_content": "The biodistribution of 177 Lu-NNV003 was performed in NSG mice with s.c.MEC-2 xenografts and in CB17 SCID mice with s.c.DOHH-2 xenografts (Fig. 3).The day prior to RIC injection, 200 μg mouse IgG2a-κ was administered i.p. to inhibit the binding of 177 Lu-NNV003 to murine Fc receptors and thus to prevent clearance of antibodies in the spleen and liver.Mouse IgG2a binds with a similar affinity to murine Fc receptors as human IgG1 [27][28][29]. 177Lu-NNV003 reached a maximum uptake of 45% of injected activity/g in MEC-2 tumours after three days and 15% in DOHH-2 tumours after two days.The retention of 177 Lu-NNV003 in tumour was more stable for DOHH-2 tumours than for MEC-2; the injected activity/g was approximately 12% for DOHH-2 and 10% for MEC-2 7 days after injection.After initial distribution to normal organs, no redistribution of the radionuclide was observed.The absorbed radiation dose after injection of 100 MBq/kg 177 Lu-NNV003 in DOHH-2 tumours was 4.6 Gy and lower than 1.2 Gy in all of the normal tissues, except in blood (Fig. 4). ",
"section_name": "Biodistribution and radiation dosimetry of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "In CB17 SCID mice i.v.injected with REC-1 cells two days prior to therapy, improved survival was observed in animals treated with 177 Lu-NNV003 (50 and 100 MBq/kg) compared to the IgG1 and the NaCl control groups (p < 0.02) (Fig. 5a).NNV003 had a modest anti-tumour effect, but survival was not significantly prolonged compared to the NaCl and IgG1 control groups (p > 0.15).At the end of the study, 38% of the mice treated with NNV003 survived, while 63% and 50% in the 50 and 100 MBq/kg 177 Lu-NNV003 groups survived, respectively.There was no statistically significant difference between the groups treated with NNV003 or 177 Lu-NNV003 (p > 0.55).Histopathological examination of normal organs from surviving mice showed no signs of tumour infiltration (data not shown), while tumour infiltration in the pelvic region, abdomen, ovaries and skull was confirmed by autopsy in the non-responding mice euthanized the end of the study. Two additional therapy studies in i.v.models were performed (Fig. S3 in Online Resource).In a MEC-2 i.v.model, only the treatment with 177 Lu-NNV003 increased survival compared with control groups, but in a DOHH-2 i.v.model, NNV003 and 177 Lu-NNV003 treatments were equally efficient in extending survival of the mice. Since the range of β particles is more suited to treat bulky tumours, a therapy study in nude mice with REC-1 s.c.tumours was performed.A dose of 500 MBq/kg 177 Lu-NNV003 significantly increased survival and slowed down tumour growth, compared to control treatment and to NNV003 treatment (p < 0.005), even at high doses of 10 mg/kg of NNV003 (Fig. 5b andc). ",
"section_name": "Therapeutic effect of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "Female Rag2 mice were injected i.v. with DOHH-2 cells three days prior to therapy with 200-400 MBq/kg 177 Lu-NNV003 or control treatments.A modest and temporary decrease in white blood cell count was observed in the groups treated with 177 Lu-NNV003 or 177 Lu-IgG1 (Fig. 6), while platelet and red blood cell counts did not significantly change. Mice treated with 300 and 400 MBq/kg 177 Lu-NNV003 showed a modest increase in both ALP (significant difference) and ALT (non-significant difference) compared to control mice, indicating slight hepatotoxicity (Fig. 7).Levels urea and creatinine indicated no signs of renal toxicity.All mice survived until end of the study without clinical signs of disease, and no histopathological abnormalities were found in any of the potentially affected organs.Atrophy, interstitial cell hyperplasia and a low incidence of cysts were observed in the ovaries in 177 Lu-NNV003 treated mice (data not shown). ",
"section_name": "Toxicity of 177 Lu-NNV003",
"section_num": null
},
{
"section_content": "Despite the advances in NHL treatments in recent years, there is still an unmet medical need for patients resistant to chemotherapy, CD20 immunotherapies and kinase inhibitors.The mouse-human IgG1 chimeric anti-CD37 RIC 177 Lu-NNV003 represents a new treatment option with a different target and mechanism of action.Here we show that 177 Lu-NNV003 increased survival of mice with xenografts of human CLL, MCL and DLBCL.The chimeric NNV003 antibody targets CD37 positive cells both in vitro and in vivo. The mouse monoclonal antibody lilotomab has been shown to bind CD37 on normal B cells and B cell malignancies with a high level of specificity [20].NNV003 is a mousehuman chimeric IgG1 version of lilotomab and binds to the same epitope on CD37.The higher anti-tumour effect of NNV003 than of lilotomab in the mouse models is likely due to stronger binding of NNV003 to the mFcγRs.Stronger binding of mouse-human chimeric IgG1 antibodies than of mouse antibodies to the mFcγRs is consistent with other studies [27,28]. The anti-proliferative effect of 177 Lu-NNV003 on the cell lines in vitro was most pronounced for DOHH-2, which may be due to the higher CD37 expression on DOHH-2 compared to REC-1.In REC-1 the cytotoxic effect of 177 Lu-NNV003 differed only slightly from that of lutetium-177 labelled unspecific antibody.This unspecific effect was probably due to cross-irradiation from unbound conjugate during the incubation period before washing the cells [30]. the in vivo studies, however, 177 Lu-IgG1 did not improve survival. A high uptake of 177 Lu-NNV003 in CD37-expressing tumours compared to normal organs and was observed.The higher uptake in MEC-2 tumours than in DOHH-2 tumours, even though the CD37 expression was similar, might be explained by higher degree of 1 7 7 Lu-NNV003 internalisation in MEC-2 cells (Online Resource, Table S1).Biodistribution and dosimetry were not performed for REC-1, but it is reasonable to expect similar results for this animal model. In an i.v.REC-1 xenograft model, CB17 SCID mice treated with 177 Lu-NNV003 showed prolonged survival compared to controls.Some efficacy was also observed with NNV003, probably due to an immunotherapeutic effect of the antibody.In another i.v.model using DOHH-2 cells, the anti-tumour [43] from haematology measurements from 39 Rag2 mice effect of NNV003 was equal to the effect of Lu-NNV003 (Online Resource, Fig. S3b).However, in a s.c.model mimicking a bulkier disease, there was no effect of NNV003 alone, even at a clinically relevant dose of 10 mg/kg.In this model, the immunotherapeutic effect was not potent enough, while 177 Lu-NNV003 delayed tumour growth and prolonged survival significantly.All animal models utilized mouse strains with functional NK-cells and macrophages, capable of ADCC and ADCP activity [31,32]. We observed a modest and temporary reduction in white blood cell counts after 1 7 7 Lu-NNV003 treatment.Additionally, while some biochemical indication of hepatotoxicity was seen, no abnormal findings of the liver were observed by histopathological examination of organs.In the present study, treatment related changes were mainly seen in the ovaries, consistent with pre-clinical studies with 177 Lulilotomab satetraxetan [33].No treatment related changes were identified in the bone marrow, possibly attributed to bone marrow regeneration due to the long study duration.In a phase 1/2a clinical trial of 177 Lu-lilotomab satetraxetan for treatment of patients with relapsed indolent NHL, reversible thrombocytopenia and neutropenia were the most common adverse events observed [21]. The favourable efficacy and toxicity profile of 177 Lu-NNV003 suggests a beneficial therapeutic window.Moreover, this compound binds to a different antigen than CD20, which is the most common target in the treatment of B cell malignancies.Accordingly, most NHL patients with recurrent disease will become refractory to treatments targeting CD20 [12].By pursuing a different target, like CD37, 177 Lu-NNV003 may overcome this resistance and become a valid option for patients with recurrent disease.Other novel treatments like BTK inhibitors and BCL2-inhibitors have offered effective treatment options, particularly for patients with CLL and MCL [34][35][36][37][38][39][40].However, most patients will eventually relapse again and 177 Lu-NNV003 might then represent a tolerable and effective treatment option. The absorbed doses to tumours (4.6 Gy in DOHH-2) and normal tissues are consistent with that of 177 Lu labelled lilotomab in in vivo models [24].The range of the tumour doses (0.75-7.94 Gy) reported clinically with 177 Lu-lilotomab satetraxetan, resulted in an overall response rate of 61% and 28% complete response [21,41], indicating that the absorbed radiation doses were high enough to achieve a clinical response.This is in line with a previous report showing that 4 Gy of external beam radiotherapy is very effective for local control in patients with indolent NHL [42]. In conclusion, the next generation chimeric anti-CD37 RIC 177 Lu-NNV003 was shown to improve survival of mice with CLL, MCL and DLBCL xenografts and to have a favourable toxicity profile.These encouraging results warrant further investigation of 177 Lu-NNV003 in clinical trials to determine whether the additional immunotherapeutic component, longer biological half-life and potential reduced risk of immunogenicity offers any clinical advantage to patients with relapsed B cell malignancies. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgments We are grateful to the Norwegian Research Council for the funding provided to this project. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Funding This study was partially funded by the Norwegian Research Council, grant number 256790, and by Nordic Nanovector ASA. ",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Funding This study was partially funded by the Norwegian Research Council, grant number 256790, and by Nordic Nanovector ASA. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Ethical approval All applicable international, national, and institutional guidelines for the care and use of animals were followed.Animal studies were approved by the National Animal Research Authorities.This article does not contain any studies with human participants performed by any of the authors. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "",
"section_num": ""
}
] |
10.1186/2047-1440-2-s1-s5
|
Alemtuzumab induction therapy in solid organ transplantation
|
Alemtuzumab (Campath) is a monoclonal antibody that has a profound lymphocyte-depleting effect, targeting the CD52 antigen that is present on all lymphocytes. Alemtuzumab has been used for the treatment of chronic lymphocytic leukaemia and various autoimmune disorders, and has also shown potential as an induction agent in the prevention of rejection following solid organ transplantation. Alemtuzumab has been studied in randomised controlled trials and has demonstrated low levels of rejection in renal transplant recipients compared with other induction agents, albeit mainly in the early months following transplantation. Studies have shown that alemtuzumab enables the use of lower calcineurin inhibitor (CNI) maintenance drugs; however, this reduction in nephrotoxic immunosuppression has not consistently been matched by an improvement in renal function. The hypothesis has been suggested that alemtuzumab might allow the development of immunosuppressive regimens that avoid CNIs completely; studies have investigated the combination of alemtuzumab with mammalian target of rapamycin-inhibitor maintenance therapy, and, in particular, sirolimus. Initial studies with this combination showed that regimens of sirolimus alone and of sirolimus with mycophenolate mofetil were unsuccessful, with a high rate of rejection and complications. Subsequent studies have targeted the combination of alemtuzumab induction with a short course of a CNI, before switching to maintenance therapy with sirolimus. This regimen might combine good protection from acute cellular rejection and chronic nephrotoxicity. A randomised controlled trial has been established to study this regimen, with results pending.
|
[
{
"section_content": "Th e design of an immunosuppressive strategy must take into account the three major causes of morbidity following organ transplantation: ischaemia-reperfusion injury (particularly in marginal donor organs); rejection; and complications of drug treatment (toxicity, infection and cancer).Induction immunosuppression is now used in the majority of transplant programmes in solid organ transplantation. Induction therapy is intended to protect the graft from the immediate postoperative period of high rejection risk.However, there is increasing interest in the eff ects of induction agents in mitigating the eff ects of ischaemiareperfusion injury.With respect to mainte nance therapy, the priority in the early post operative weeks is to prevent rejection and minimise postsurgical infections and other surgical complications.Subse quently, drug toxicity, opportunistic infection and cancer risk become the primary concerns. Despite several decades of experience, there is no consensus regarding many aspects of induction therapy.Both polyclonal antibodies and monoclonal antibodies are used with no clear evidence as to which is preferable.Similarly, there is little agreement as to whether depleting or nondepleting antibodies are preferable.Th ere is a range of targets from the highly selective anti-CD25 antibodies (basiliximab) that target an antigen (the interleukin-2 receptor) only present on activated lymphocytes to antibodies that recognise all peripheral blood lymphocytes (for example, antithymocyte globulin (ATG), alemtu zumab).Although anti-CD25 antibodies are very widely used globally, there is now increasing interest in the potential benefi ts of depleting agents with a broader spectrum.Th e advantages that have been hypothesised for these agents include reduction of ischaemia-reperfusion injury, a more profound protection from early acute rejection and, fi nally, a long-lasting eff ect in relation to donor-specifi c hyporesponsiveness. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Alemtuzumab (Campath) is a monoclonal antibody that recognises the CD52 antigen, present on all peripheral blood lymphocytes.Campath antibodies have been tested experimentally and clinically for 30 years [1]. Initially an IgM rat-derived antibody (Campath-1M) was used for the removal of T cells from bone marrow for transplantation; this was carried out as a means to prevent graft versus host disease.It was noted at the time that an antibody of profound lymphocyte-depleting capabilities would have potential application in solid organ transplantation.Campath-1M was used in clinical renal transplant trials and was found to have a profound eff ect on peripheral lymphocyte numbers, although with rapid recovery between doses [2], and was also shown to reduce the incidence of acute rejection [3].Smaller studies with the IgG2b isotype (Campath-1G) demonstrated a more profound and long-lasting depletion of lymphocytes [4]. Th e humanised antibody (Campath-1H) was developed with the potential advantages of greater effi cacy and, second, a lower risk of sensitisation.Th is antibody was used for (and subsequently licensed for) the treatment of chronic lymphocytic leukaemia [5].Campath-1H was also used in a number of autoimmune conditions [6][7][8] and shown to be eff ective in treating multiple sclerosis [9].Th e fi rst report of the use of the humanised antibody in solid organ transplantation was in the treatment of acute cellular rejection of kidney transplants [10], which was associated with a high incidence of infections. ",
"section_name": "Alemtuzumab",
"section_num": null
},
{
"section_content": "A number of randomised trials of alemtuzumab in solid organ transplantation have been published.In 2008 Margreiter and colleagues studied 131 patients randomised to receive induction therapy with alemtuzumab (2 × 20 mg) versus no induction [11].All patients were subsequently treated with tacrolimus and patients who had not received induction therapy also received mycophenolate.Th is trial demonstrated a signifi cant reduction in the incidence of acute rejection at 6 months (29% vs. 15%, P = 0.05).However, by 12 months the incidence of rejection was no longer signifi cantly diff er ent (32% vs. 30%).Th ere was no diff erence in renal function between the two groups and, apart from an increase in the incidence of cytomegalovirus infection, there was no other adverse consequence of induction therapy. In 2011 Chan and colleagues reported a randomised controlled trial of 82 alemtuzumab-treated patients (with tacrolimus monotherapy) versus 42 controls (receiving dacluzimab, tacrolimus, mycophenolate), all patients having a rapid steroid taper [12].Th is study showed a lower incidence of rejection in the alemtuzumab-treated group at 6 months but not 12 months and no diff erence in graft survival or function.However, the study confi rmed that alemtu zu mab therapy does enable simple and eff ective tacrolimus monotherapy with a very low rate of rejection.Also in 2011, Hanaway and colleagues carried out a randomised trial involving 474 kidney transplant recipients [13].Th ese were divided into high-risk patients (defi ned as panel reactive antibody >20%, black race, retransplant; n = 139) and low-risk patients (all others).Th e high-risk group was treated with either ATG (1.5 mg/ kg × 4, n = 70) or alemtuzumab (30 mg × 1, n = 69).Th e low-risk group of patients was treated with basiliximab (20 mg × 2, n = 171) or alemtuzumab (30 mg × 1, n = 164).All patients received tacrolimus 8 to 14 ng/ml, mycophenolate 2 g/day, and steroids for 5 days.Patients were followed up for 3 years.Th is trial demonstrated that patients treated with alemtuzumab had a signifi cantly lower incidence of cellular rejection at 6, 12 and 36 months (3% vs. 15%, 5% vs. 17% and 13% vs. 20% respectively).When divided into the high-risk and lowrisk subgroups, it was clear that the reduction in rejection was entirely within the group of patients at low risk of rejection (alemtuzumab vs. basiliximab) and that the incidence of rejection in the high-risk patients was similar between the alemtuzumab and ATG groups.Th ere was no signifi cant diff erence in graft or patient survival.Analysis of lymphocyte populations demonstrated that alemtuzumab and rabbit ATG had similar eff ects with respect to lymphocyte depletion and the rate of recovery of lymphocyte populations.Th is was contrasted in the low-risk group, in which the lymphocyte population was profoundly suppressed by alemtuzumab but very little altered by treatment with basiliximab.Th ere was no eff ect on renal function.Th is trial did show that alemtuzumab-treated patients are more liable to late rejection episodes (after 12 months), a fi nding also noted by others including Watson and colleagues [14].What is also notable from this and other large series of alemtuzumab-treated transplant patients is the relative absence of autoimmune complications, a problem that has been seen more commonly in patients treated for multiple sclerosis [15]. When the potency and duration of lymphocyte depletion induced by alemtuzumab was recognised, a number of investigators considered whether this may lead to the induction of donor-specifi c hyporesponsive ness.Th is followed the important observations of Knechtle and colleagues in demonstrating that profound lymphocyte depletion, using an anti-CD3 monoclonal antibody conjugated to diphtheria toxin, can lead to long-lasting donor-specifi c hyporesponsiveness in a non human primate experimental model of kidney trans plan tation [16].Calne and colleagues tested the use of alemtuzumab induction (2 × 20 mg) followed by low-dose cyclosporin monotherapy (75 to 125 mg/ml) [17].Th is study, although without a control group, demonstrated the capability of a low-dose mono therapy immunosup pression regimen to achieve low levels of rejection and good levels of graft survival and function.Th e same group of patients was reviewed 5 years after transplantation [14] and was compared with a group of matched controls treated with conventional therapy (cyclosporin, prednisolone, azathioprine).Lympho cyte counts were signifi cantly lower in the alemtuzumab group only in the fi rst 3 months post operatively.Cyclosporin levels were significantly lower in the alemtuzumab group for approximately 2 years.However, despite lower cyclosporin levels, there was no signifi cant diff erence in renal function.An early benefi t with respect to acute rejection in the alemtuzumab-treated patients was matched by an increased level of rejection after 6 months, culminating in a similar overall level of rejection between the two groups.Th ere was no signifi cant diff erence in patient or graft survival or in the incidence of infection or other serious adverse events. ",
"section_name": "Randomised trials of alemtuzumab in solid organ transplantation",
"section_num": null
},
{
"section_content": "Th e ability to lower calcineurin inhibitor (CNI) doses was regarded as one of the primary advantages of alemtu zumab therapy, particularly because of the desire to avoid chronic nephrotoxicity [18].However, despite the ability of alemtuzumab to enable lower levels of CNI therapy, none of the above studies demonstrated improvement of renal function in patients treated with alemtuzumab.Th e profound lympho-depletion caused by alemtuzumab led Kirk and colleagues to question whether the antibody alone would enable tolerance and avoid the need for main tenance therapy.Seven living donor recipients were treated with three or four doses of alemtuzumab preoperatively; despite profound depletion of lymphocytes in the peripheral blood and lymph nodes, all patients developed rejection (reversible) and required conversion to maintenance sirolimus [19].Th e availability of mammalian target of rapamycin inhibitors, however, provided the opportunity for long-term maintenance of patients without the use of nephrotoxic immunosuppression; the combination of alemtuzumab induction with sirolimus monotherapy was a theoretically attractive proposition. In the fi rst study of an immunosuppression strategy of this design, 29 patients were treated with alemtuzumab (20 mg × 2) followed by low-dose maintenance sirolimus therapy [20].Eight patients required treatment for rejection and one graft was lost.Th e investigators concluded that sirolimus monotherapy was inadequate in this context.Other investigators tested the combination of sirolimus with mycophenolate following alemtuzumab induction [21].Twenty-two patients were treated with alemtuzumab induction (30 mg × 2) and maintenance therapy sirolimus (8 to 12 mg/ml) and mycophenolate (500 mg/twice daily).Th ere was an acute rejection incidence of 36%, leuko paenia in 27% of patients and acute respiratory distress syndrome in two patients.Th e investigators concluded that the combination of alem tuzumab, sirolimus and mycophenolate, when started at the time of trans plan tation, was associated with a high rejection rate and a high incidence of other complications. Attention then moved to the use of alemtuzumab induction followed by a short course of CNI therapy, before switching to sirolimus maintenance therapy for the longer term.In a study of 30 patients, alemtuzumab (30 mg × 2) induction was followed by mycophenolate (500 mg/twice daily for 12 months), tacrolimus (5 to 8 ng/ml for 6 months) and, after tacrolimus withdrawal, sirolimus (5 to 8 ng/ml) (data submitted for publication).A number of rejection episodes occurred following mycophenolate withdrawal at 12 months (all reversible with steroids) and the protocol was therefore amended to continue mycopheno late 250 mg twice daily thereafter.Th is protocol was associated with a low incidence of rejection (6.6% at 12 months).Following the change of protocol to maintain low-dose mycophenolate after 12 months, there were no acute rejection episodes after 12 months.Two patients were withdrawn from the protocol because of respiratory side eff ects of sirolimus.Eighty-fi ve per cent of patients remained steroid and CNI free 5 years postoperatively. Th is trial demonstrated the potential utility of alemtuzumab in enabling patients to be established safely on CNI-free therapy.Th e real benefi t of this strategy is in the potential absence of chronic nephrotoxicity and the consequent long-term improvement in graft half-life.Clearly this hypothesis required formal testing in an adequately powered randomised control clinical trial.Such a trial was established in 2010 with the intention of testing the eff ects of alemtuzumab versus basiliximab as an induction agent and testing the eff ect of switching to sirolimus from tacrolimus at 6 months.Th e end points for this study will fi rst be the incidence of acute cellular rejection and then medium-term graft function.Th e results of this study (Campath, calcineurin inhibitor reduction, chronic allograft nephropathy) are awaited. ",
"section_name": "Alemtuzumab and mammalian target of rapamycin inhibitors",
"section_num": null
}
] |
[
{
"section_content": "Abbreviations ATG, antithymocyte globulin; CNI, calcineurin inhibitor. PJF has received honoraria from Pfi zer for lecturing. This article has been published as part of Transplantation Research Volume 2 Suppl 1, 2013: Proceedings of the 12th International Symposium on Transplantation.The full contents of the supplement are available at http://www.transplantationresearch.com/supplements/2/S1.The supplement is based on the 12th International Transplantation Symposium held on 16-17 April 2012, in Istanbul, Turkey.The symposium and publication based on these proceedings were supported by an unrestricted educational grant from Pfi zer Inc., who had no infl uence on the editorial content.Editorial support was provided by Synergy, Richmond, UK and was funded by Pfi zer Inc.Rapamune (sirolimus) is a product marketed by Pfi zer Inc.The articles have been through the journal's standard peer review process and the Supplement Editors declare that they have no competing interests. Published: 20 November 2013 ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations ATG, antithymocyte globulin; CNI, calcineurin inhibitor. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "PJF has received honoraria from Pfi zer for lecturing. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "This article has been published as part of Transplantation Research Volume 2 Suppl 1, 2013: Proceedings of the 12th International Symposium on Transplantation.The full contents of the supplement are available at http://www.transplantationresearch.com/supplements/2/S1.The supplement is based on the 12th International Transplantation Symposium held on 16-17 April 2012, in Istanbul, Turkey.The symposium and publication based on these proceedings were supported by an unrestricted educational grant from Pfi zer Inc., who had no infl uence on the editorial content.Editorial support was provided by Synergy, Richmond, UK and was funded by Pfi zer Inc.Rapamune (sirolimus) is a product marketed by Pfi zer Inc.The articles have been through the journal's standard peer review process and the Supplement Editors declare that they have no competing interests. Published: 20 November 2013 ",
"section_name": "Declaration",
"section_num": null
}
] |
10.1523/jneurosci.1348-08.2008
|
Notch1 Signaling in Pyramidal Neurons Regulates Synaptic Connectivity and Experience-Dependent Modifications of Acuity in the Visual Cortex
|
<jats:p>How the visual cortex responds to specific stimuli is strongly influenced by visual experience during development. Monocular deprivation, for example, changes the likelihood of neurons in the visual cortex to respond to input from the deprived eye and reduces its visual acuity. Because these functional changes are accompanied by extensive reorganization of neurite morphology and dendritic spine turnover, genes regulating neuronal morphology are likely to be involved in visual plasticity. In recent years, Notch1 has been shown to mediate contact inhibition of neurite outgrowth in postmitotic neurons and implicated in the pathogenesis of various degenerative diseases of the CNS. Here, we provide the first evidence for the involvement of neuronal Notch1 signaling in synaptic morphology and plasticity in the visual cortex. By making use of the Cre/Lox system, we expressed an active form of Notch1 in cortical pyramidal neurons several weeks after birth. We show that neuronal Notch1 signals reduce dendritic spine and filopodia densities in a cell-autonomous manner and limit long-term potentiation in the visual cortex. After monocular deprivation, these effects of Notch1 activity predominantly affect responses to visual stimuli with higher spatial frequencies. This results in an enhanced effect of monocular deprivation on visual acuity.</jats:p>
|
[
{
"section_content": "The primary visual cortex (V1) responds selectively to various features of visual stimuli such as orientation, direction, and spatial frequency (Hu ¨bener, 2003).Moreover, most neurons in V1 are more responsive to stimuli from one eye than from the other.How neurons respond in adulthood is strongly influenced by visual experience during postnatal development.When during a critical period of development one eye is deprived, for example, responsiveness of V1 to the deprived eye decreases, whereas that of the nondeprived eye increases, resulting in a shift of ocular dominance (OD) (Wiesel and Hubel, 1963;Gordon and Stryker, 1996).Monocular deprivation (MD) also counteracts the development of responsiveness to stimuli with high spatial frequencies that occurs during the critical period, thus reducing visual acuity of the deprived eye (Giffin and Mitchell, 1978;Fagiolini et al., 1994;Prusky and Douglas, 2003).These functional changes are accompanied by extensive structural changes involving growth and retraction of thalamocortical and intracortical axons and turnover of dendritic spines, suggesting that signaling cascades regulating neuronal morphology play an important role in visual plasticity (Shatz and Stryker, 1978;Antonini et al., 1999;Majewska and Sur, 2003;Mataga et al., 2004;Oray et al., 2004;Pizzorusso et al., 2006). One of these cascades is signaling through the transmembrane receptor Notch1.Although Notch1 (Bray, 2006;Weinmaster and Kopan, 2006) is mostly known for its role in cell-fate decisions during development, it has in recent years been shown to also regulate structural changes in developing neurons.Notch1 is activated after interacting with ligands on neighboring cells, resulting in release of its intracellular domain (NICD) (De Strooper et al., 1999), which together with CBF1/RBP-J activates transcription of target genes (Lu and Lux, 1996).This cascade stimulates neurite branching but inhibits neurite growth in postmitotic cortical neurons (Berezovska et al., 1999;Sestan et al., 1999;Redmond et al., 2000), neuroblastoma cells (Franklin et al., 1999) and newborn neurons in the dentate gyrus (Breunig et al., 2007).Also the primary transcriptional target of Notch1, hairy and enhancer of split 1 (Hes1), has been shown to restrict neurite outgrowth (Jessen et al., 2003).Because Notch1 signaling is upregulated with increasing contacts between neurons in the developing brain (Sestan et al., 1999) and downregulated during a hippocampusdependent learning task (Conboy et al., 2007), Notch1 has been suggested to provide a negative feedback signal controlling neuronal growth and connectivity. In this study, we used V1 to investigate whether neuronal Notch1 signaling plays a role in plasticity of the mammalian brain.Several studies suggest that it may (Costa et al., 2003;Wang et al., 2004;Costa et al., 2005) but do not exclude the possibility that the observed effects are caused by developmental deficits and/or altered Notch1 signals in non-neuronal cell types.To circumvent these issues, we used the Cre/Lox system to target expression of constitutively active Notch1 to individual or all pyramidal neurons of the cortex, starting in the fourth postnatal week just before the beginning of the critical period of OD plasticity.Using this approach, we show for the first time that neuronal Notch1 signaling cell-autonomously regulates synaptic connectivity of cortical neurons and affects experience-dependent plasticity in V1. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "DNA constructs and transgenic mice.A construct for Cre-dependent expression of NICD and green fluorescent protein (GFP) was created by cloning cDNA encoding amino acids 1741-2531 of mouse Notch1 into pCDNA3.1 containing the poliovirus internal ribosome entry site (IRES) followed by GFP.The fragment encoding NICD-IRES-GFP was cloned into pThy-lox-stop-lox (LSL) (Chakravarthy et al., 2006), rendering pThyLSL-NICD-IRES-GFP.Transgenic mice were created by pronuclear injections of linearized DNA into fertilized C57BL/6 oocytes.New founders were crossed to the G35-3-Cre line (Sawtell et al., 2003), which had been kept on a C57BL/6 background for at least six generations.Transgenic mice expressing membrane-associated GFP (mGFP) in a Cre-dependent manner have been described previously [line TLG498 (Chakravarthy et al., 2006(Chakravarthy et al., , 2008))].All experiments involving mice were approved by the Institutional Animal Care and Use Committee of the Royal Netherlands Academy of Arts and Sciences. RNA isolation and quantitative PCR.Male mice were anesthetized by intraperitoneal injection of 0.1 ml/10 g bodyweight pentobarbital (Nembutal; Ceva Sante Animale).Occipital cortex was dissected, snap frozen in liquid nitrogen and stored at Ϫ80°C until RNA isolation using Trizol (Invitrogen).RNA yield was quantified on a Nanodrop (Isogen Life Science) and quality assessed using an Agilent Bioanalyser.cDNA was synthesized using the SuperScript III First-Strand cDNA Synthesis Kit (Invitrogen).Oligonucleotide primers were designed with Primer Express 2.0 software and purchased from Eurogentec or Biolegio. Forward and reverse primer sequences for quantitative PCR (qPCR) were as follows: ACTG1-forward (fw), CATTGCTGACAGGATGCA-GAA; ACTG1-reverse (rv), ACATCTGCTGGAAGGTGGACA; EF1␣fw, AAGAAGATCGGCTACAACCCAG; EF1␣-rv, TTACGCTC-TACTTTCCAGCCCT; GAPDH-fw, ATGTGTCCGTCGTGGATCTGA; GAPDH-rv, ATGCCTGCTTCACCACCTTCT; G6PDX-fw, GTCCA-GAATCTCATGGTGCTGA; G6PDX-rv, GCAATGTTGTCTCGATTC-CAGA; HPRT-fw, GCAAACTTTGCTTTCCCTGG; HPRT-rv, TTC- Polr2a-rv, TGCCCCTTAGATTTGGCCA; Rs27a-fw, GGCCAAGATCCAGGATAAGGA; Rs27a-rv, CCATCTTCCAGCT-GCTTACCA; TBP-fw, CACGGACAACTGCGTTGATTT; TBP-rv, GC-CCAACTTCTGCACAACTCT; NICD-fw, CGTACTCCGTTACATG-CAGCA; NICD-rv, AGGATCAGTGGAGTTGTGCCA; Hes1-fw, TCAACACGACACCGGACAAA; Hes1-rv, CCTTCGCCTCTT-CTCCATGAT. qPCRs were run on the 1/20 diluted cDNA for genes of interest and candidate reference genes.SYBR Green technology was used on a 7300 Real-Time PCR System (Applied Biosystems).Using GeNorm software (http://medgen.ugent.be/ϳjvdesomp/genorm/),an optimal set of reference genes was chosen based on similar expression patterns across sam-ples (Vandesompele et al., 2002).A geometrical mean of the set was used to normalize results of the genes of interest. Lentiviral injections.NICD ϩ mGFP ϩ or mGFP ϩ transgenic males were injected with a lentiviral vector-mediating expression of a CreGFP fusion protein (LV-CreGFP) at postnatal day 22 (P22)-P25.Mice were anesthetized with isoflurane (Abbott).The eyes were protected using artificial tears (Methocel; Novartis Pharma) and patches. Holes were drilled 2.8 mm caudal to bregma and 2.5 mm lateral to the midline.Two hundred nanoliters of LV-CreGFP were injected bilaterally into V1 250 -300 m below the cortical surface using a Nanoject II pump (Drummond Scientific).Mice were analyzed at P38. Immunohistochemistry. Mice were anesthetized by intraperitoneal injection with pentobarbital and perfused with 4% paraformaldehyde (Sigma-Aldrich) in PBS.After 2 h postfixation, 50 m coronal sections of V1 were made using a Leica VT1000S vibratome (Leica).To enhance the EGFP signal and allow long-term storage, free-floating sections were stained using monoclonal mouse anti-GFP (1:500; Millipore Bioscience Research Reagents), followed by goat anti-mouse Cy3 (1:500; Jackson ImmunoResearch).Sections were embedded in Mowiol [10% w/v Mowiol 4-88 (Merck); 25% glycerol; and 0.1 M Tris HCl; pH 8.5] and stored at 4°C in the dark until imaging. For analyzing parvalbumin (PV)-containing boutons surrounding pyramidal neurons, sections were stained using rabbit anti-PV (1:1000; Swant) and mouse anti-NeuN (1:500; Millipore Bioscience Research Reagents), followed by goat anti-rabbit Alexa568 and goat anti-mouse Al-exa488 (both 1:500; Invitrogen). Confocal microscopy and morphological analysis.Cy3-stained mGFPexpressing layer 2/3 pyramidal neurons in V1 were imaged on a Zeiss LSM510 Meta confocal microscope (Zeiss) using a He/Ne (543 nm) laser.Dendritic protrusions on basal dendrites distal to the first branch point were imaged if the distance to either the next branch point or the end of the dendrite was at least 20 m.Z-Stacks were made with 200 nm Z-intervals (63ϫ oil objective; numerical aperture 1.4; optical zoom was adjusted for each dendrite).For each neuron of which a dendrite segment was imaged, a single-plane image of the largest cross-sectional area of its soma was acquired (63ϫ objective, 2.5ϫ optical zoom).An 80 m pinhole was used.Laser intensity, detector gain, and amplifier offset were adjusted for each individual image acquisition to allow use of the entire detector range and avoid saturation.Three-dimensional reconstruction of image stacks was done to facilitate analysis. Morphological analysis was performed using Zeiss LSM Image Browser (http://www.embl-heidelberg.de/eamnet/html/body_image_browser.html).Dendritic protrusions of V1 pyramidal neurons expressing mGFP (473 protrusions, 29 dendrites, 26 neurons, 8 mice) or NICD and mGFP (518 protrusions, 39 dendrites, 32 neurons, 10 mice) were analyzed.Spine neck length and the longest straight line in the spine head were determined.Statistical significance of differences in protrusion density, spine neck length, head size, soma size, and densities of filopodia (longer than 2 m, no head), mushroom spines (clear neck and head), stubby spines (wider than 0.30 m, no neck), and thin spines (no head, shorter than 2 m and a width Ͻ0.30 m) was determined by Student's t test.At all stages, the investigator was blind to the experimental group. For analysis of perisomatic inhibitory boutons, PV-containing puncta around NeuN-positive neurons of layer 2/3 cells from V1 were imaged using a 488 nm argon laser and a 543 nm He/Ne laser.The same settings were used for imaging sections of control and NICD ϩ Cre ϩ transgenic animals.Single-plane confocal images were converted into TIFF images and analyzed in ImagePro 6 (Media Cybernetics).NeuN-positive cells were marked with a free-drawing tool.Using customized macros, a 2 m ring was drawn around NeuN-positive cells.The number, intensity, and size of PV-puncta within the ring were counted automatically.Analysis was performed blind to genotype.A total of 10 mice were used for the analysis, with five NICD ϩ Cre ϩ mice (64 cells; 221 puncta) and five littermate controls (55 cells; 181 puncta). Slice electrophysiology.Coronal slices (325 m thickness) of V1 were prepared from 29-to 34-d-old NICD ϩ Cre ϩ male mice (10 slices; 6 animals) or littermate controls (24 slices; 12 animals).Slices were cut in ice-cold slicing artificial CSF (ACSF) containing the following (in mM): 125 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 10 glucose, 3 MgSO 4 , and1 CaCl 2 (0.300 mOsm) and carboxygenated with 95% O 2 /5% CO 2 .Slices were allowed to equilibrate for 1 h at room temperature in carboxygenated ACSF containing the following (in mM): 125 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 10 glucose, 1 MgSO 4 , 3 CaCl 2 , and 0.01 glycine (300 mOsm). Slices were put onto a 64-channel multielectrode recording probe (MED64; Alpha MED Sciences) with the electrodes covering layers 1-5 and superfused with 4 ml/min carboxygenated ACSF at 32°C.Layer 4 was stimulated with a bipolar extracellular electrode (0.15 ms; current was adjusted per experiment to evoke half-maximum response), and synaptic response was quantified as the maximum amplitude of the field EPSP (fEPSP) response.After 20 min baseline recording at 0.1 Hz, long-term potentiation (LTP) was induced by theta burst stimulation (TBS; eight trains of four pulses at 100 Hz, delivered at 200 ms intervals).TBS was performed three times at 10 s intervals.After LTP induction, fEPSPs were recorded for 40 min at 0.1 Hz.The number of electrodes in layer 2/3 recording a clear synaptic response was not different between transgenic and control animals (data not shown). Synaptic responses were analyzed off-line using the recording software Performer 2.0, Igor Pro (WaveMetrics), Excel, and SPSS.Only recordings showing baselines stable within 1.5% were included.For statistical comparisons, the magnitude of LTP was taken as the average of the 10 -40 min post-TBS time frame.Normality of the distribution was assessed using Kolmogorov-Smirnov testing, followed by two-tailed Student's t test or Kruskal-Wallis analysis.A p value Ͻ0.05 indicates statistically significant differences.At all stages, the investigator was blind to experimental conditions. For input/output (I/O) curves, the smallest stimulus size yielding a detectable synaptic response was designated threshold stimulus.Multiples of this stimulus size (generally 10 -15 A) were then used to characterize the input-output relationship.For each stimulus amplitude, six repetitions were performed, yielding an average response size per slice. In vivo intrinsic signal imaging.Intrinsic signal was imaged transcranially in 62 NICD ϩ Cre ϩ or control mice (NICD ϩ Cre Ϫ , NICD Ϫ Cre ϩ , or NICD Ϫ Cre Ϫ littermates) as previously described (Heimel et al., 2007).Mice were anesthetized by intraperitoneal injection of urethane (20% in saline, 2 g/kg; Sigma-Aldrich).Heads were fixed and scalps resected.Atropine sulfate (0.05 mg/ml in saline, 0.1 mg/kg; Pharmachemie) was injected subcutaneously to reduce mucous excretions.A monitor covered the mice's visual fields from Ϫ15 to 75°horizontally and from Ϫ45 to 45°vertically.The screen was divided into 2 ϫ 2 patches, and drifting gratings were used to map the retinotopic representation of V1.The representation of the upper nasal screen patch was used to calculate responses to subsequent tests.For OD measurements, computer-controlled shutters alternated visual stimulation of the eyes using drifting square wave gratings [0.05 cycles per degree (cpd)].An imaged ocular dominance index (iODI) was defined as iODI ϭ (contra response Ϫ ipsi response)/(contra response ϩ ipsi response).A response to the contralateral eye only corresponds to an iODI of 1, and iODI ϭ Ϫ1 indicates only ipsilateral response.Acuity was determined with 90% contrast sinusoidal gratings of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.7 cpd, phase reversing at 2 Hz and changing orientation every 0.75 s.A spatial frequency tuning curve was fit to a linear-threshold function using a least-mean-squares procedure.The intersection with zero response strength was our measure of spatial acuity.Student's t tests were used to compute significance levels. Eyelid suturing.Right eyelids were sutured at P28 under isoflurane anesthesia (Abbott) as previously described (Heimel et al., 2007).At P35 the eyelid was reopened at the start of the imaging session. Golgi impregnation.Adult (P72-P88), male mice (NICD ϩ Cre ϩ or control) were anesthetized by intraperitoneal injection of a mixture of 0.36 mg/kg fentanyl citrate, 11.25 mg/kg fluanisone (sold together as Hypnorm; VetaPharma) and 2.8 mg/kg midazolam (Dormicum; Roche).The brain was rapidly removed, cut midsagittally, and placed in Golgi-Cox solution (1% potassium dichromate, 1% mercuric chloride, and 0.8% potassium chromate in water).The solution was refreshed once.After 3-4 weeks the tissue was dehydrated and embedded in celloidin (Merck). Coronal sections (200 m) were cut (Reichert Jung Polycut S microtome; Lame ´ris Laboratorium), developed, fixed, dehydrated, and mounted.Slides were coded to render the analyzer blind to experimental condition. Sholl analysis.Analysis of dendritic branching was done on layer 2/3 pyramidal neurons (controls 42 neurons, 4 mice; NICD ϩ Cre ϩ 17 neurons, 4 mice).Camera lucida drawings were made using a Zeiss binocular microscope, with a 40ϫ oil-immersion objective and a camera lucida drawing tube.Selected neurons showed a fully impregnated dendritic tree and spines and no obscuring of branches by blood vessels, precipitate, or other artifacts. Sholl analysis was performed using an overlay of concentric circles centered on the soma (Sholl, 1953).The number of dendritic intersections crossing each circle (diameters of 25,50,75,100,125,150,200,250, and 400 m) was counted. ",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To activate Notch1 signaling in excitatory neurons of mouse V1, we produced a mouse line carrying a transgene driving expression of NICD, a constitutively active form of Notch1 (missing the extracellular and transmembrane domains) followed by an IRES and GFP.The transgene contained the Thy1promoter, restricting expression to neuronal cell types, followed by a \"stop-cassette\" flanked by loxP sites, rendering it Cre dependent (Fig. 1).Founders were crossed with Cre transgenic line G35-3, expressing Cre-recombinase specifically in all excitatory neurons of cortex and hippocampus (Sawtell et al., 2003).One founder (TLNic403) showed broad and detectable GFP expression in the visual cortex and will be referred to as NICD transgenic mice.In NICD ϩ Cre ϩ offspring, the transgene was expressed in Ͼ80% of pyramidal neurons of cortical layers 2/3 and 5/6 (Fig. 2 A), as well as in the dentate gyrus and areas CA1 and CA3.No transgene expression was detected in layer 4, probably because of the lack of Thy1promoter activity in this layer (Fig. 2 A).To confirm that NICD was expressed and functionally active, we used qPCR to analyze expression of NICD and Hes1 (a classical Notch target) at different ages.By P26, expression of Notch1 was twice as high in the cerebral cortex of NICD ϩ Cre ϩ animals as in control littermates, 4 -5 times as high by P29 and 8 times as high in adults (2-3 months old) (Fig. 2 B).As expected, Credependent transgene expression occurred in cortex but not in the cerebellum or retina (Fig. 2 B).Hes1 expression was approximately doubled in the cortex of NICD ϩ Cre ϩ mice, confirming that the NICD transgene was functional (Fig. 2C). ",
"section_name": "Production of transgenic mice expressing NICD in V1 pyramidal neurons",
"section_num": null
},
{
"section_content": "Because Notch1 has been shown to restrict neuronal growth with increasing neuronal contacts, we asked whether Notch1 also restricts neuronal connectivity at the synaptic level.To study cellautonomous effects of Notch1 on morphology in postmitotic neurons in V1 in vivo, we activated the Cre-dependent NICD transgene by lentivirus-mediated CreGFP expression (LV-CreGFP (Ahmed et al., 2004)).Because the level of GFP expression from the NICD(-IRES-GFP) transgene alone was not sufficient for visualizing dendritic protrusions and the GFP of the CreGFP fusion protein was localized to the nucleus, we crossed NICD transgenic animals with mice expressing a Cre-dependent, membrane-associated form of GFP (line TLG498, referred to as mGFP transgenic mice) (Chakravarthy et al., 2006).Bilateral, intracranial LV-CreGFP injections were made in NICD ϩ mGFP ϩ mice or in mGFP ϩ single transgenic littermates at P22-P25.The resulting Golgi-staining-like transgene expression pattern allowed detailed morphological analyses (Fig. 3A) of dendritic protrusions from basal dendrites of layer 2/3 pyramidal neurons (Fig. 3 B,C). NICD expression until P38 resulted in an 18.6% reduction in total protrusion density (16.31Ϯ 1.08 in controls vs 13.28 Ϯ 0.93 in transgene expressing cells; p ϭ 0.037) (Fig. 3D), caused mostly by a 22.0% reduction in mushroom spine density (12.83 Ϯ 0.97 in controls vs 10.00 Ϯ 0.82 in transgene-expressing cells; p ϭ 0.024) (Fig. 3D) and a 59.1% reduction in filopodial density (0.69 Ϯ 0.17 per 15 m in controls vs 0.28 Ϯ 0.07 in NICD transgene expressing cells; p ϭ 0.016) (Fig. 3D).The cross section of the soma was reduced by 8.1% in NICD-expressing neurons (177.46 m 2 Ϯ 3.42, vs 193.02 m 2 Ϯ 4.36 in controls; p ϭ 0.006) (Fig. 3E). No changes were observed in the spine head size between neurons expressing NICD and mGFP (0.57 Ϯ 0.01 m) and neurons expressing mGFP only (0.56 Ϯ 0.01 m; p ϭ 0.37) (Fig. 3F ), nor did NICD expression significantly affect spine neck length (0.80 Ϯ 0.03 m, vs 0.87 Ϯ 0.03 m in controls; p ϭ 0.096) (Fig. 3F ).Dendrite thickness was not affected by transgene expression (data not shown). Next, we addressed the question whether increased Notch1 activity in pyramidal neurons also affected the number or size of boutons providing perisomatic inhibitory input.This form of inhibitory input is considered a crucial determinant of plasticity during the critical period, and is provided predominantly by PVexpressing basket cells (Fagiolini et al., 2004).To this end, PVpuncta surrounding layer 2/3 pyramidal neuron somata were analyzed.Sections of broad-expressing 30-d-old transgenic animals (Fig. 2 A) and littermate controls were stained for the neuronal nucleus marker NeuN and PV, after which PV-puncta in a ring surrounding the pyramidal neurons were quantified. The number of PV-puncta per pyramidal cell was not affected by transgene expression (3.29 Ϯ 0.27 in controls versus 3.45 Ϯ 0.31 in transgenics; p ϭ 0.93) (Fig. 3G), nor was their size, as determined by the percentage of the ring area covered by PVpuncta (controls 3.89 Ϯ 0.43%; transgenics 3.95 Ϯ 0.37%; p ϭ 0.87) (Fig. 3G).Punctum intensity was the same between controls and transgenics. ",
"section_name": "Notch1 activity cell-autonomously reduces dendritic spine and filopodium density but leaves perisomatic inhibitory boutons unaffected",
"section_num": null
},
{
"section_content": "The finding that NICD expression reduced spine and filopodia density prompted the question whether NICD expression would also affect LTP induction, which is associated with formation of new spines, probably through filopodia growth and stabilization (Engert and Bonhoeffer, 1999;Maletic-Savatic et al., 1999).LTP induction in connections from layer 4 to layer 2/3 of V1 was analyzed in NICD ϩ Cre ϩ mice and control animals. Synaptic transmission from layer 4 to layer 2/3 was recorded in coronal V1 slices of 29-to 34-d-old mice, before and after induction of LTP by theta-burst stimulation (TBS).In NICD ϩ Cre ϩ mice, TBS resulted in a smaller increase in synaptic responses (104.9Ϯ 0.9%) than in littermate controls (109.3Ϯ 1.3%; p ϭ 0.045) (Fig. 4C, example traces in Fig. 4 B).No differences in basic physiology were observed.Neither the I/O curve nor the stimulus strength required for obtaining a halfmaximal response was affected by transgene expression (Fig. 4 A).Because in NICD ϩ Cre ϩ mice, transgenic NICD was expressed in layer 2/3 but not in layer 4 (Fig. 2 A), these data indicate that restriction of LTP is caused by increased Notch1 signaling in the postsynaptic neurons. ",
"section_name": "Postsynaptic Notch1 activity reduces LTP",
"section_num": null
},
{
"section_content": "The reduction in LTP, filopodia formation and total spine numbers suggested that NICD expression in pyramidal neurons reduces their capacity to form new connections.To test whether this had any consequences for OD plasticity in NICD transgenic mice, they were subjected to monocular eyelid suture at the peak of the critical period (P28) and analyzed at P35. Seven days of MD in control animals resulted in an OD shift, measured by optical imaging of intrinsic signal in the visual cortex using moving square wave grating at 0.05 cpd as a visual stimulus.Examples of ocular dominance maps under the different experimental conditions are shown in Figure 5A.After 7 d of MD, the iODI was reduced in monocularly deprived animals (0.07 Ϯ 0.05; 6 animals) compared with nondeprived littermate controls (0.35 Ϯ 0.02; n ϭ 8 animals; p Ͻ 0.0001) (Fig. 5B).This was not different in NICD ϩ Cre ϩ transgenic animals [0.31 Ϯ 0.03 in nondeprived transgenics (n ϭ 11), 0.04 Ϯ 0.06 after MD (n ϭ 5); p ϭ 0.001] (Fig. 5B).Interestingly, we noticed that in control animals, the OD shift was caused by both a decrease in deprived eye responses [0.74 Ϯ 0.17 (value normalized to nondeprived control)] and an increase in open eye responses (1.47 Ϯ 0.37) (Fig. 5C) [consistent with previous imaging literature (Hofer et al., 2006;Heimel et al., 2008)], whereas in NICD ϩ Cre ϩ animals the shift seemed to be caused predominantly by decreased responses of the deprived eye (0.62 Ϯ 0.10), whereas open eye responses remained unchanged (1.05 Ϯ 0.19) (Fig. 5C).Although the differences between the genotypes were not significant, the observation suggested that more subtle differences in visual responsiveness could be occurring between control and NICD ϩ Cre ϩ animals after MD. ",
"section_name": "Notch1 activity does not affect the magnitude of ocular dominance plasticity determined using a low spatial frequency stimulus",
"section_num": null
},
{
"section_content": "Apart from changing OD, MD also counteracts the development of high visual acuity in the deprived eye that normally occurs during the critical period [from 0.3 cpd at P25 to 0.5 cpd at P35 (Heimel et al., 2007)].The findings that changes in visual acuity could still be induced in adult rats despite the absence of OD plasticity at this age (Iny et al., 2006) and do not always mirror OD in kittens (Murphy and Mitchell, 1986;Faulkner et al., 2006) suggested to us that acuity may be the more sensitive measure for changes in visual responsiveness.We therefore assessed visual acuity in control and NICD ϩ Cre ϩ animals 7 d after MD at P35 using optical imaging of intrinsic signals (Heimel et al., 2007).We determined that nondeprived control mice had a spatial acuity of 0.52 Ϯ 0.02 cpd in the contralateral eye (n ϭ 3 animals) (Fig. 6 A).MD from P28 -P35 resulted in a lower acuity (0.41 Ϯ 0.02 cpd; n ϭ 8; p ϭ 0.03).In NICD ϩ Cre ϩ mice, the acuity under control conditions was unaffected (0.51 Ϯ 0.05 cpd; n ϭ 9).However, after MD, the acuity was strongly reduced (0.30 Ϯ 0.02 cpd; n ϭ 7; p ϭ 0.001), significantly lower than in deprived control mice ( p ϭ 0.004) (Fig. 6 A).We noticed that although after MD, NICD ϩ Cre ϩ mice showed especially weak responses to visual stimuli with the highest spatial frequencies (0.3-0.4 cpd), responses to lower spatial frequency stimuli (0.1-0.2 cpd) were also more affected than in deprived control animals (Fig. 6 B).Thus, Notch1 activity caused MD to have a greater impact on responses to visual stimuli with increasing spatial frequencies. ",
"section_name": "Notch1 activity increases effects of MD on visual acuity",
"section_num": null
},
{
"section_content": "We next analyzed whether Notch1 activity had similar effects in the adult visual cortex.Like in juvenile animals, we did not see any reduction of acuity in nondeprived adult (2-6 months of age) NICD ϩ Cre ϩ mice (0.52 Ϯ 0.07; n ϭ 8) compared with controls (0.53 Ϯ 0.03; n ϭ 11).However, in contrast to the situation during the critical period, we did not observe differences in the effects of adult MD on visual acuity in NICD ϩ Cre ϩ mice (0.39 Ϯ 0.03; n ϭ 5 animals) or control animals (0.40 Ϯ 0.04; n ϭ 6) (Fig. 7A). ",
"section_name": "NICD expression does not affect acuity in adult animals",
"section_num": null
},
{
"section_content": "Because it has been proposed that Notch1 activity could cause neuronal degeneration in the adult brain, we assessed whether prolonged NICD expression resulted in any changes in dendritic morphology of pyramidal neurons in adult NICD ϩ Cre ϩ mice.Brains of 2.5-month-old animals were Golgi-impregnated, after which Sholl analysis was performed on layer 2/3 pyramidal neurons, using coronal slices through V1.The number of intersections of dendrites with respective concentric circles drawn at different distances around the soma was not significantly reduced in NICD ϩ Cre ϩ mice compared with controls (Fig. 7B).Also, when all intersections were added up per neuron, the difference between controls (75.38 Ϯ 2.84) and NICD ϩ Cre ϩ (66.71Ϯ 4.87) was not significant ( p ϭ 0.116). ",
"section_name": "Notch1 signaling does not cause dendritic degeneration in adult V1",
"section_num": null
},
{
"section_content": "Plasticity in the visual cortex is accompanied by structural rearrangements, including the loss and gain of dendritic spines (Fifkova ´, 1968;Majewska and Sur, 2003;Mataga et al., 2004;Oray et al., 2004).Signaling pathways regulating neuronal morphology are therefore likely to be involved in this form of plasticity.In this respect, the Notch1 signaling pathway is of particular interest.Apart from its well known function in cell-fate decisions and regulation of proliferation and apoptosis, it has also been implicated in limiting neurite outgrowth of postmitotic neurons in the first weeks after birth (Berezovska et al., 1999;Sestan et al., 1999;Redmond et al., 2000).Moreover, various interactions between Notch1 and amyloid precursor protein processing have been observed (Song et al., 1999;Fassa et al., 2005;Fischer et al., 2005), and its expression is increased in Alzheimer's disease (Selkoe, 2001) and other brain disorders (Berezovska et al., 1998;Fischer et al., 2005;Ishikura et al., 2005;Nagarsheth et al., 2006), suggesting that it may play a role in neurodegeneration.Whether neuronal Notch1 signaling regulates synaptic plasticity (Costa et al., 2005) has remained an unanswered question with important clinical implications.In this study, we addressed this question by analyzing the consequences of increased Notch1 activity in cortical neurons on ocular dominance plasticity. We first wanted to establish whether Notch1 activity affected spine density and morphology during the critical period.We found that cell-autonomous activation of Notch1 activity of neurons in the juvenile visual cortex (P23-P38) resulted in a reduction in mushroom spine densities (by 22%), whereas spine head sizes and neck lengths remained unchanged.In addition, there was a strong reduction in the number of filopodia (by 59%) after Notch1 activation.We observed a small reduction in the soma size of neurons expressing active Notch1, which we believe reflects the reduction of total synapse numbers.Because perisomatic inhibitory innervation by PVexpressing basket cells is an important determinant of critical period plasticity, we also determined the size and numbers of PV-positive boutons surrounding pyramidal neurons but detected no differences between NICD transgenic and control mice. These results show that Notch1 not only restricts dendritic growth during early development, but that it also limits synaptic connectivity at later stages of cortical maturation.Our spine morphological analyses do not allow us to distinguish whether the reduced spine numbers are caused by reduced spine formation or increased spine loss.However, the concurrent reduction in the density of filopodia, which are believed to be precursors of dendritic spines, seems more consistent with the idea that Notch1 activity reduces the potential of neurons to form new excitatory synapses. We next addressed the question of whether the effect of Notch1 on dendritic spine densities had any consequences for cortical plasticity.Because the induction of LTP has been shown to result in the induction of filopodia (Maletic-Savatic et al., 1999) and spine formation (Engert and Bonhoeffer, 1999), we analyzed whether activation of Notch1 signaling in layer 2/3 pyramidal neurons in V1 reduced the level of LTP that can be induced in the connections between layers 4 and 2/3.We indeed found that postsynaptic NICD expression reduced LTP induction, confirming a functional correlate for the effects of Notch1 on neuronal morphology and providing for the first time direct evidence for a role of neuronal Notch1 signaling in synaptic plasticity. In apparent contrast to our observation, a previous report has shown that ubiquitous transgenic expression of Notch1 antisense RNA (NAS) decreased LTP in the hippocampus, whereas addition of the Notch1 ligand Jagged-1 had the opposite effect (Wang et al., 2004).In that report, however, Notch1 signaling was not only altered in pyramidal neurons but in many cell types, and already during development.Because Notch1 signaling in oligodendrocytes interferes with their differentiation (Wang et al., 1998) and affects myelination (Givogri et al., 2002) it seems plau-sible that other (or additional) mechanisms are affected in NAS transgenic animals, explaining the phenotypic difference. Having confirmed the influence of Notch1 on neuronal morphology and plasticity, we analyzed whether Notch1 would also affect OD plasticity.To our initial surprise, overexpression of NICD in V1 did not appear to affect the size of the OD shift that is induced by MD.However, further experiments revealed that although visual acuity developed normally in NICD transgenic animals, after MD it was significantly lower than in deprived control animals.This seemed to be caused predominantly by the more strongly reduced responsiveness to the deprived eye in NICD transgenic mice, which became especially prominent with increasing spatial frequency, resulting in a more pronounced reduction of visual acuity after MD.Only because the increase in responsiveness to the nondeprived eye was more apparent in control than in NICD transgenic mice, the relative change in OD was the same in both types of animals. Thus, neuronal Notch1 activity causes the development or stability of visual acuity to be more dependent on visual experience.What causes this increased vulnerability of the responses to high spatial frequencies in NICD transgenic animals?Currently we can only speculate about this, but the most parsimonious explanation seems to be that although responsiveness to low spatial frequencies is already high before the critical period [and develops even in the absence of experience (Fagiolini et al., 1994)], responsiveness to higher spatial frequencies increases strongly during the critical period (Heimel et al., 2007).The reduced capacity for synaptic strengthening as a result of Notch1 activity could well cause the formation or stability of these developing responses to be more dependent on visual input.This idea is supported by our observation that also an increase in responsiveness to the nondeprived eye does not occur in NICD transgenic animals after MD.This explanation opens the interesting possibility that a relationship between LTP and visual plasticity (Daw et al., 2004) may be found more readily in the development of visual acuity than in altered OD plasticity assessed using optimal visual stimuli. Our data provide clear evidence for the involvement of neuronal Notch1 signaling in experience-dependent synaptic plasticity during the critical period of OD plasticity, and support the idea that Notch1 signals limit neuronal connectivity.At the same time, we did not obtain convincing evidence for a possible role of Notch1 in dendritic degeneration.Dendritic morphology as assessed with Sholl analysis was not different between adult NICD transgenic animals and controls.We did not find any evidence for a loss in visual performance in adult NICD transgenic mice up to 6 months of age, nor did monocular deprivation during adulthood affect visual acuity more in NICD transgenic animals than in control mice (Fig. 7).Of course, this does not rule out the possibility that Notch1 may have effects on neurite morphology or neuronal maintenance when neurons are at the same time otherwise affected by CNS pathology. Apart from providing insight into the role of Notch1 in cortical plasticity, our data exemplify how apparently small changes in OD plasticity can have important implications for the development of visual acuity.This stresses the need for studying the mechanisms that underpin plasticity of visual acuity (Giffin and Mitchell, 1978;Murphy and Mitchell, 1986;Fagiolini et al., 1994;Faulkner et al., 2006;Iny et al., 2006;Fischer et al., 2007), espe-cially when taking into consideration that loss of acuity is the major problem for people with amblyopia. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by a research support grant and a Vidi Grant from the Netherlands Organization for Health Research and Development (ZonMW).H.D.M. and C.N.L. are supported by the Netherlands Academy of Arts and Sciences and ZonMW.J.M.H., J.A.H., L.H.V.W., and C.N.L. are supported by a \"Bsik\" grant from SenterNovem.We thank Sean Perry, Hans Lodder, Svetlana S ˇkulj, and Sitha Scheltinga for technical assistance, Dr. Susumu Tonegawa for supplying the G35-3-Cre mice, and Dr. Chris de Zeeuw for critical reading of this manuscript. ",
"section_name": "",
"section_num": ""
}
] |
10.1186/s13059-021-02453-5
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Chromatin interaction neural network (ChINN): a machine learning-based method for predicting chromatin interactions from DNA sequences
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<jats:title>Abstract</jats:title><jats:p>Chromatin interactions play important roles in regulating gene expression. However, the availability of genome-wide chromatin interaction data is limited. We develop a computational method, chromatin interaction neural network (ChINN), to predict chromatin interactions between open chromatin regions using only DNA sequences. ChINN predicts CTCF- and RNA polymerase II-associated and Hi-C chromatin interactions. ChINN shows good across-sample performances and captures various sequence features for chromatin interaction prediction. We apply ChINN to 6 chronic lymphocytic leukemia (CLL) patient samples and a published cohort of 84 CLL open chromatin samples. Our results demonstrate extensive heterogeneity in chromatin interactions among CLL patient samples.</jats:p>
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[
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"section_content": "Chromatin interactions play important roles in regulating gene expression [1][2][3].They bridge enhancers to genes [4][5][6] and create insulated domains to constrain the reach of enhancers [7].High-throughput experimental techniques such as high-throughput chromosome conformation capture (Hi-C) [8] and chromatin interaction analysis with paired-end tags (ChIA-PET) [9] have been developed to detect genome-wide chromatin interactions.These techniques greatly advanced the understanding of genome organization and its roles in transcription regulation [4,[10][11][12].However, due to costs and technical challenges, these methods have not been widely applied to large cohorts of cell lines or clinical samples.Hence, our understanding of how common or rare chromatin interactions are in different patient samples is limited. A predictor that uses DNA sequences to predict chromatin interactions could potentially expand our understanding of genome organization.Sophisticated computational methods such as DeepSea [13] and DeepBind [14] have demonstrated that many transcription factors binding sites in open chromatin regions could be predicted from DNA sequences.Additionally, various computational methods have been developed to predict chromatin interactions to complement the experimental techniques [15][16][17][18][19][20][21].Many of these methods rely on using various functional genomics data, meaning the use of chromatin immunoprecipitation sequencing (ChIP-seq) data of transcription factors and histone modifications, open chromatin data, and transcription data [15,17,19,21]. Methods such as RIPPLE [17], TargetFinder [19], and JEME [15] reported high performances in predicting enhancer-promoter interactions using supervised machine learning approaches.However, the reported performances were exaggerated by using cross-validation with random splitting of samples [22,23].The lack of effective machine learning approaches has motivated the field to develop new methods. Recently, the convolutional neural network framework was adapted to predict Hi-C contact matrices from 1-dimentional sequence data in a method called \"Akita\" [24].There are other methods that predict Hi-C-like data and chromatin interactions, namely DeepTACT [25], SEPT [26], and DeepC [27].A detailed comparison and description of these methods is discussed in Additional file 1: Table S1. CTCF-associated genome folding patterns can be observed in the prediction results of Akita, suggesting the importance of CTCF in regulating chromatin interactions.In addition, prediction results can recapture the differences in genome folding between a normal and genetically altered cell lines, indicating that machine learning framework can predict different genome folding profiles given different input DNA sequences. However, there are several limitations to these methods.First, Akita and DeepC only performs predictions with limited sequence regions (in the case of Akita, this is 1 Mb), thus long-range chromatin interactions cannot be predicted and genome-wide chromatin interactions cannot be obtained with these methods.Second, it is unclear whether ChIA-PET data can be predicted, as DeepTACT predicts promoter capture Hi-C data [25], and Akita, DeepC, and SEPT are restricted to Hi-C data.Third, none of these methods have been tested for their abilities to predict chromatin interactions de novo in patient cancer samples. To overcome these challenges, in this study, we investigated the possibility of utilizing DNA sequence features to predict chromatin interactions between open chromatin regions, regardless of distance between them.Our study has several advantages.First, we demonstrated that open chromatin interactions can be predicted accurately from functional genomic data at the resolutions of the experimental techniques. Second, we then developed a novel method, called chromatin interaction neural network (ChINN) to predict open chromatin interactions from DNA sequences.This model has been developed for RNA Polymerase II (RNA Pol II) ChIA-PET interactions, CTCF ChIA-PET interactions, and Hi-C interactions, overcoming previous limitations in terms of data input.Moreover, ChINN is able to identify open chromatin interactions in a genome-wide manner, overcoming the limitations of previous methods which were restricted to specific genomic regions. Third, we extensively validated our method.ChINN was able to identify convergent CTCF motifs, AP-1 transcription family member motifs such as FOS, and other transcription factors such as MYC as being important in predicting chromatin interactions.Moreover, we further applied our model to a set of 6 newly generated chronic lymphocytic leukemia samples, which showed patient-specific chromatin interactions.We were able to validate predicted interactions by Hi-C and 4C.The models were then applied to a cohort of previously published 84 chronic lymphocytic leukemia (CLL) samples [28].Thus, we demonstrated the prediction power of our method in practice. Fourth, we used ChINN to characterize the levels of open chromatin interaction heterogeneity in patient samples.While we found that many chromatin interactions are ubiquitous, we also found widespread evidence for patient-specific open chromatin interactions, and open chromatin interactions that were different in different subtypes of CLL. Taken together, our results indicate both functional genomics models and ChINN can predict open chromatin interactions, and application of ChINN to cancer patient samples demonstrates widespread patient heterogeneity in chromatin interactions. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In light of Xi et al. [22] and our previous study [23] showing that the existing prediction methods have exaggerated performances, we first tried to demonstrate that chromatin interactions could be predicted from functional genomic data.Many previous studies focused on enhancer-promoter interactions that were annotated using chromatin interactions derived from Hi-C or ChIA-PET [15,17,19].The enhancers used were typically hundreds of base pairs, while the chromatin interaction anchors were much larger in size.For example, Hi-C anchors are normally 5 to 100 kb long (while only in rare case with extremely deep sequencing are the anchors down to 1 kb size) [29,30], and ChIA-PET is normally several kilobase pairs long [31,32].The resolution discrepancy could lead to the introduction of a lot of noises to the training datasets (Fig. 1a).Thus, we used the chromatin interaction anchors directly.Positive samples were constructed from ChIA-PET datasets separately and the corresponding distance-matched negative datasets were generated (Additional file 1: Fig. S1).The resulting distance-matched datasets have positive-to-negative ratios of approximately 1:5 and all chromatin interactions were between open chromatin regions in the corresponding cell types (Additional file 1: Table S2).We used ChIP-seq data of transcription factors and histone modifications commonly available to GM12878, K562, and HelaS3 and DNase-seq data from ENCODE [33] to annotate the anchors and build the feature vectors (Additional file 1: Table S3).For each chromatin interaction, the average signal of each transcription factor, histone modification, and open chromatin were calculated for both anchors.The distance between two anchors was also used as a feature. Gradient boosted trees [34] were used to build models for each dataset.We tested three feature sets: (1) all common functional genomics data and distance, (2) distance only, and (3) common functional genomics data only.A precision-recall curve (PR) curve shows the trade-off between precision and recall across different decision thresholds.The auPRC is calculated as the area under the PR curve.The models trained on all features achieved auPRC ranging from 0.62 to 0.77 (Fig. 1b), while models trained on distance are mostly at baseline (Fig. 1d), showing that distance is properly controlled between positive and negative samples.The models trained on functional genomics features achieved auPRCs ranging from 0.58 to 0.69 (Fig. 1c), lower than models trained on all features.These results showed that although distance alone cannot predict chromatin interactions, combining distance feature with other features together can help to distinguish the positive and negative chromatin interaction considering the working mechanisms of the GB model (Additional file 1: Text S1). The across-sample performances were lower than within-sample performances (Fig. 1e).Using peak counts instead of signal values produced better acrosssample performances but lower within-sample performances (Fig. 1f).Models trained on RNA Pol II datasets generalize well to each other.Models trained on CTCF ChIA-PET datasets, however, did not generalize well to each other.Models trained on CTCF ChIA-PET data perform poorly on RNA Pol II ChIA-PET datasets and vice versa. ",
"section_name": "Open chromatin interactions can be predicted from functional genomic features",
"section_num": null
},
{
"section_content": "In our previous section, we showed that open chromatin interactions can be predicted from functional genomics data, which consists of transcription factor data.As transcription factor binding can be predicted from sequences as shown by methods such as DeepSea (Zhou et al, Nature Methods, 2015) and DeepBind (Alipanahi et al, Nature Biotech, 2015), we reasoned that open chromatin interactions can be predicted from DNA sequences.Consequently, we went on to explore whether open chromatin interactions can be predicted from DNA sequences. We built a convolutional neural network, ChINN, to predict chromatin interactions between open chromatin regions using DNA sequences (Fig. 2a).These models are called the ChINN sequence-based models.The models were trained on GM12878 CTCF, GM12878 RNA Pol II, HelaS3 CTCF, K562 RNA Pol II, and MCF-7 RNA Pol II datasets separately. Compared to using functional genomics data for prediction, using sequences produced better within-sample performances for CTCF ChIA-PET datasets with auPRCs of 0.77 for GM12878 CTCF and 0.75 for HelaS3 CTCF (Fig. 2b), but worse withinsample performances for RNA Pol II ChIA-PET datasets with auPRC of 0.51 for GM12878 RNA Pol II, 0.6 for K562 RNA Pol II, and 0.47 for MCF-7 RNA Pol II.Including distance as a feature to classifier only slightly improved the performances for The \"Pol2\" in the figure represents \"RNA Pol II\". a The architecture of the sequence-based models using to train on distance-matched datasets.Precision-recall curves of the sequence-based models on distancematched datasets using b only sequence features or c sequence features with distance.The numbers in the brackets indicates the area-under precision-recall curves.Across-sample performances as measured by areaunder precision-recall curve (auPRC) of the models on distance-matched datasets using d only sequence features or e sequence features with distance.Precision-recall curves of the sequence-based models on distance-matched Hi-C datasets using f only sequence features or g sequence features with distance.The numbers in the brackets indicates the area-under precision-recall curves.Across-sample performances as measured by area-under precision-recall curve (auPRC) of the models on distance-matched Hi-C datasets using h only sequence features or i sequence features with distance the distance-matched datasets (Fig. 2c).The across-sample performances of CTCF models showed well generalizability to each other (Fig. 2d).RNA Pol II models can also generalize to each other.Models trained on CTCF ChIA-PET datasets perform poorly on RNA Pol II ChIA-PET datasets and vice versa (Fig. 2d,e).The inability to generalize between CTCF chromatin interactions and RNA Pol II chromatin interactions could be attributed to the different sequence contexts. For each model, we obtained and matched the position-weight matrices for all kernels on the first convolutional layer to known transcription factor binding motifs (Additional file 1: Fig. S2).As expected, CTCF motif was captured by both CTCF models.Other than the CTCF motif, the remaining known transcription factor binding motifs learned by the two models were different, indicating the possible cell-typespecific motifs.Our findings of the cell-type-specific motifs were supported by other pieces of evidence: studies show that cell-type-specific CTCF-mediated interactions are important in gene regulation [35,36] and CTCF binding sites vary extensively across cell types [37,38].The patterns learned by RNA Pol II models showed more diversity and no matching transcription factor binding motif was shared among the three models.Interestingly, some of the transcription factors identified, such as ZNF143 in K562 and GATA3 in MCF-7, play important roles in the relevant cancer types [39,40]. Besides, we also trained ChINN model on GM12878, HeLaS3, HMEC, HUVEC, IMR90, K562, KBM7, and NHEK Hi-C data, respectively.The auPRCs of within-sample performances using only sequences range from 0.52 to 0.77 for the above eight Hi-C models (Fig. 2f).Including distance as a feature to classifier only slightly improved the performances for the GM12878, HeLaS3, and NHEK Hi-C models (Fig. 2g).The across-sample performances of all eight Hi-C models showed well generalizability to each other (Fig. 2h,i). Similarly, we obtained and matched the position-weight matrices for all kernels on the first convolutional layer to known transcription factor binding motifs for eight Hi-C datasets (Additional file 1: Table S4) and counted how many times each motif was detected (Additional file 1: Table S5).The CTCF motif was captured by all Hi-C models.The known transcription factor binding motifs learned by different Hi-C models were different.Some motifs, such as FOS, were learned by all models, but other motifs showed diversity, for example, ZN436 is detected by all other models except for HMEC, and ZIC3 is only detected by HeLaS3 (Additional file 1: Table S5).We noticed that the motifs detected in all cell lines exhibit smaller p-values than the cell-typespecific motifs, indicating that these \"general\" motifs are very important in predicting chromatin interactions.We speculate a model of chromatin interactions whereby there are general chromatin interactions facilitated by general transcription factors and common across different cell types, as well as cell-type specific chromatin interactions facilitated by cell-type specific transcription factors which can control cell-type specific transcription. ",
"section_name": "Open chromatin interactions can be predicted from DNA sequences",
"section_num": null
},
{
"section_content": "After extracting the sequence features from both the forward and reverse complement sequences of the anchors, the sequence features were fed into the classifier to obtain a probability score that indicated how likely the pair of anchors were involved in a chromatin interaction.We obtained the feature importance scores of the gradient boosted trees trained and validated using a set of extended datasets that includes more negative samples than the distance-matched datasets (Methods, Additional file 1: Fig. S3a-d).We noted that the PR curves of the datasets that used sequence features and distance (Additional file 1: Fig. S3a) were better than that of sequence features alone (Additional file 1: Fig. S3b).However, distance alone was uninformative by itself in predicting chromatin interactions (Additional file 1: Fig. S3c), suggesting that it is the combination of distance as a property in addition to sequence features that provide predictive power. The sequence and distance-trained datasets were able to predict chromatin interactions across different cell types (Additional file 1: Fig. S3d).Consequently, we focused on the sequence features that were important for the prediction.As convergent CTCF motif has been observed in the anchor regions of CTCF loops [41][42][43], this suggests that the other sequence features or binding motifs at CTCF ChIA-PET anchors may also have such convergent orientation.Interestingly, in CTCF models the important sequence features were on different strands of the two anchors in a convergent manner (Fig. 3a, Additional file 1: Fig. S3e), while RNA Pol II models did not show such pattern (Fig. 3b, Additional file 1: Fig. S3f-g).For the CTCF models, importance scores of features on different strands of the two anchors showed good correlation, while importance scores of features on the same strand of the two anchors did not show much 3c).In contrast, the importance scores of features of RNA Pol II models were generally highly correlated regardless of the strand.These results are consistent with the previously observed convergent CTCF motifs at CTCF ChIA-PET and further suggest that other transcription factors also binds to CTCF loops in a similar manner. The kernels on the last convolutional layer that generated the most important features in the extended CTCF models captured the CTCF motif (Additional file 1: Fig. S3h), suggesting that convergent CTCF motifs were important for the prediction of CTCF-associated chromatin interactions.However, using only CTCF motif information for the prediction of CTCF-associated open chromatin interactions could not recapitulate the performance achieved by the convolutional neural network (Additional file 1: Fig. S3i), indicating that CTCF was not the sole determining factor of chromatin interactions.We also showed the results when training with NN model (same NN structure as ChINN sequence-based models) on the same datasets using sequence and distance feature to illustrate the superiority of GB model here (Additional file 1: Fig. S3j). Similarly, we trained gradient boosted trees with the corresponding extended datasets for eight Hi-C datasets.Distance was the largest contributor (in terms of feature importance score) when it was used together with sequence features (Additional file 1: Fig. S4a-d).But on its own, it was not very informative.This suggests its interaction with the sequence features is informative.When we visualized the sequence feature importance, although not as obvious as that of the CTCF models, we observed that the important sequence features were on different strands of the two anchors according to the corresponding mean values (Fig. 3d, Additional file 1: Fig. S4e).However, the importance scores of features did not show high correlation on Hi-C datasets (Fig. 3e).All the extended Hi-C models captured the CTCF motif via the kernels of the most important feature on the last convolutional layer (Additional file 1: Fig. S4f), indicating that convergent CTCF motifs were important for the prediction of Hi-C data chromatin interactions.The results trained with NN model using sequence and distance feature were also shown for reference (Additional file 1: Fig. S4g). ",
"section_name": "Convergent CTCF motifs are important for prediction of CTCF-associated open chromatin interactions",
"section_num": null
},
{
"section_content": "The above models were trained and evaluated on known chromatin interactions.Without knowledge of chromatin interactions, as is the case for many clinical samples and cell types, the locations of the anchors would not be known.To be able to predict chromatin interactions between open chromatin regions, the models need to be able to predict chromatin interactions between paired genomic regions (anchors) of open chromatin regions. We tested different combinations of merging distances and extension sizes (Fig. 4a) based on validation datasets and determined that the merging distance of 3000 bp and extension size of 1000 bp for the construction of anchors in GM12878 cells (Additional file 1: Fig. S5a). The pairs generated between anchors constructed from open chromatin regions in GM12878 were used to train gradient boosted trees for both CTCF and RNA Pol II models (see Methods).The positive-to-negative ratios were about 1:122 for CTCF chromatin interaction labeled samples and 1:186 for RNA Pol II chromatin interaction labeled samples.The CTCF model achieved within-sample auPRC of 0.514 and the RNA Pol II model achieved auPRC of 0.347 (Fig. 4b).In cross-sample evaluation, the CTCF model achieved auPRC of 0.359 on HelaS3 dataset and the RNA Pol II model achieved auPRCs of 0.232 and 0.164 on K562 and MCF-7 datasets, respectively (Fig. 4b).We were able to validate some of the predicted chromatin interactions in MCF-7 cells using 4C-seq (Additional file 1: Fig. S5b-d).Some of the validated chromatin interactions were not captured by the MCF-7 RNA Pol II ChIA-PET dataset, thus ChINN is able to identify bona fide chromatin interactions that might have been previously missed out due to insufficient sequence coverage. We also generated pairs between anchors constructed from open chromatin regions in GM12878 and K562 Hi-C datasets with different combinations of merging distances and extension sizes (Additional file 1: Fig. S6a).We kept to the same parameters as the CTCF model, i.e., merging size of 3000 and extension size of 1000, to train gradient boosted trees due to the insignificant difference in auROC achieved by different parameters.The GM12878 and K562 Hi-C model had relatively low auPRC in the withinsample and cross-sample evaluation (Fig. 4c).However, we found that the auPRC of our ChINN method showed at least 4 times improvement over that of the random classifier.In cell line IMR90 tested by K562 model, ChINN showed as high as 57 times improvement.As there are a lot of data in the datasets (for example, the IMR90 dataset has 979,699 samples), these improvements in the auPRC could lead to many chromatin interactions being predicted correctly.Moreover, some of the predicted chromatin interactions in MCF-7 cells using 4C-seq were able to be validated by our Hi-C models (Additional file 1: Fig. S6b-d). Other methods that predict Hi-C-like data and chromatin interactions are available, namely DeepTACT [25], SEPT [26], Akita [24], and DeepC [27].However, except for SEPT, the other three machine learning methods are very different from ChINN in terms of the data.For example, DeepTACT uses promoter capture Hi-C input data, which is quite different from the use of Hi-C and ChIA-PET input data.For Akita and DeepC, their output consists of Hi-C contact matrices on lists of user-specified genomic regions, while our output is chromatin loops and probabilities of interaction across the whole genome.As a consequence, we cannot call loops from these partial Hi-C matrices of Akita and DeepC, because we would not know the background genomic interaction distribution.Therefore, direct comparison between ChINN output and Akita/DeepC output is not possible. As for the SEPT, following its pipeline, we extended the input sequences or cut to 3 or 2 kb flanking regions from the center.But SEPT performs worse as compared with ChINN on our dataset (AUPRC = 0.0016 evaluated on K562 Hi-C test datasets with HeLaS3 as source data), as the sequences in our dataset are longer than these input sequences.Therefore, cutting the sequence to 3 or 2 kb according to what SEPT does would not let the model learn much useful information. We concluded that each method is designed to investing ate different questions, and in Additional file 1: Fig. S7, we summarized the decision making process for researchers who wish to use the different methods.For example, if the researcher is interested in promoter-promoter or promoter-enhancer interactions identified by PCHi-C (Promoter Capture Hi-C), they should use DeepTACT.If the researcher is interested in an output that is shown as a Hi-C heatmap, they should use Akita.If the researcher is interested to look at general chromatin interactions predicted from Hi-C data, or RNA Pol II and CTCF chromatin interactions predicted from ChIA-PET data, they should use ChINN.ChINN is the only machine learning method currently available for predicting Hi-C and ChIA-PET chromatin interactions from sequences with outputs specified as open chromatin associated chromatin interactions instead of Hi-C matrices. ",
"section_name": "Predicting chromatin interactions from open chromatin regions",
"section_num": null
},
{
"section_content": "Next, we wished to apply our machine learning methods to patient samples to understand if our method could predict chromatin interactions in a completely new dataset.We obtained 6 chronic lymphocytic leukemia (CLL) patient samples.The clinical characteristics are described in Additional file 1: Table S6. We prepared integrated Hi-C, ATAC-Seq, and RNA-Seq libraries from these 6 samples.We used Juicer to call topologically associated domains and loops from these patient samples.Our CLL samples showed many TADs and loops (Additional file 1: Table S7), thus indicating that we were able to perform Hi-C in these patient samples. Next, we applied GM12878 and K562 Hi-C models to six new CLL samples.We used GM12878 and K562 Hi-C models for this prediction because CLL, GM12878, and K562 all come from hematopoietic lineages, and therefore GM12878 and K562 predictions would be likely to have captured both general chromatin interaction mechanisms and tissue-specific mechanisms that are relevant to hematopoietic cells. The auPRC achieved by GM12878 Hi-C model range from 0.2772 to 0.4362, which are a bit higher than that of K562 Hi-C model, whose auPRC range from 0.2607 to 0.3996 (Fig. 5a).We calculated the F-score with different thresholds and finally determined the threshold of 0.025 for GM12878 model and 0.016 for K562 model to make the prediction on new CLL samples (Additional file 1: Fig. S8a-b), where the corresponding confusion matrix was shown as Fig. 5b andc. One question we asked was whether there is patient heterogeneity in Hi-C data.We use \"heterogeneity\" to indicate that the chromatin interactions are different across patients. Clinical samples differ from each other due to a wide variety of factors including different driver mutations and different underlying genetics and epigenetics of each patient. Here, we asked whether the subtype of the CLL samples could be one factor giving rise to patient heterogeneity.The CLL samples could be divided into two subtypes based on IGHV mutation status.In our data, two samples (102 and 344) are IGHVunmutated CLL (uCLL) type and 4 samples (312, 324, 401, and 484) are IGHVmutated CLL (mCLL) type.IGHV mutation status is an important prognostic biomarker in CLL, with mCLL being less aggressive [44]. Genomic sequences are almost identical across different patient samples, except for regions of patient-specific cancer structural variations and single nucleotide variations.Thus, if two anchors are identical in a different cell type, the probability that they are interacting given by the model will be the same.In this study, we have limited the scope of chromatin interaction prediction to only open chromatin regions, and call the predicted chromatin interactions \"open chromatin interactions\".As a first step to investigate this question, we applied our ChINN framework on the six new CLL samples and built models using Hi-C and ATAC-seq data from each CLL sample.Models built using CLL samples would have captured general chromatin interaction mechanisms and tissue-specific mechanisms relevant to hematopoietic cells, as well as CLL-specific mechanisms. Figure 5d showed the predicted chromatin interactions in 6 new CLL samples and the differences between uCLL and mCLL samples.With the selected threshold, a total of 152,202 Hi-C-associated open chromatin interactions were predicted (Fig. 5d) by GM12878 Hi-C model.We found extensive patient heterogeneity (Fig. 5e,f), as observed from the lack of similarity of chromatin interactions across the new CLL samples and the overlapping peaks between new CLL samples and GM12878 Hi-C peaks.For example, Fig. 5e indicates that 37% of Hi-C identified chromatin interactions can be found in only one sample, but not the other five samples.This indicates that many open chromatin interactions can only be found in one sample, and is an illustration of the level of heterogeneity in terms of the presence and absence of open chromatin regions and their associated chromatin interactions. In addition, we also applied our ChINN framework on the six new CLL samples and built models using Hi-C and ATAC-seq data from each CLL sample.Our Hi-C libraries identified 1795 open chromatin interactions unique to uCLL samples and 10663 open chromatin interactions unique to mCLL samples (Fig. 6a).Moreover, uniqueness analysis of the Hi-C interactions from these six CLL samples similarly showed high patient heterogeneity (Fig. 6b).Thus, both predicted open chromatin interactions and Hi-C identified interactions indicate high patient heterogeneity. These models have auPRC range from 0.37 to 0.58 (Fig. 6c).In addition, acrosssample testing of these CLL models on other datasets from other CLL sample suggests a comparable performance (Fig. 6d).Inclusion of distance did not result in dramatic increase of the model performance (Additional file 1: Fig. S9a-9b).Moreover, the first convolutional layers of all CLL models were able to capture the CTCF and AP-1 transcription family member (FOS, JUN, JUNB, JUND) binding motif (Additional file 1: Fig. S9c), similar to the Hi-C models we showed earlier (Additional file 1: Fig. S4e; Additional file 1: Table S4-5). After that, we trained gradient boosted trees with the corresponding extended datasets of the CLL samples.We observed that similar correlation of the important sequence features on different strands of the two anchors (Fig. 6e; Additional file 1: Fig. S9d-9e), although the within-sample and cross-sample auPRC were decreased (Additional file 1: Fig. S9f-9g). We also generated open chromatin pairs using ATAC-seq to train the gradient boosted trees (merging size, 3000 bp; extension size, 1000 bp).Although the performances decreased compared with using Hi-C anchor region pairs as input, they were still higher than the random auPRC values (Additional file 1: Fig. S9h-9k).We further used the 401 CLL sample model to predict open chromatin interactions in MCF7 cells, as the 401 CLL model has the highest within-sample and across-sample performance.The predicted interactions correlate quite well with the real 4C-seq interactions (Fig. 6f, g, Additional file 1: Fig. S9l-9o, threshold = 0.016). One question we asked was whether there is patient heterogeneity in Hi-C data.We first tried to associate the real and predicted Hi-C interactions with differentially expressed genes identified from RNA-seq data.The results showed that although the trend of different IFC scores (the fold change of the average number of open chromatin interactions observed at the gene promoter in uCLL samples over that in mCLL samples) could be observed, these differences were not significant (Additional file 1: Fig. S9p-9q).We also observed that the Hi-C interactions and ATAC-seq peaks in the new CLL samples showed high patient heterogeneity (Additional file 1: Fig. S9r).These patient heterogeneities may be a reason for the limited sample size in the IFC score analysis after we collapsed all six samples into mutated and unmutated categories (Additional file 1: Fig. S9p-9q). Taken together, our results demonstrate across-sample prediction capability for the ChINN model.In addition, we observed high patient heterogeneity in the new CLL samples from the predicted open chromatin interactions as well as the Hi-C identified chromatin interactions. ",
"section_name": "Exploring chromatin interactions in patient samples",
"section_num": null
},
{
"section_content": "Next, we used our machine learning method to predict open chromatin interactions in a cohort of patient samples and then analyzed the data.We applied the above models to 84 chronic lymphocytic leukemia (CLL) samples whose open chromatin profiles were available by ATAC-seq [28].Among 84 CLL samples, 34 of them are uCLL type and 50 of them are mCLL type. A total of 48,443 CTCF-associated open chromatin interactions and 23,633 RNA Pol II-associated open chromatin interactions were predicted based on the pooled open chromatin regions of all samples (Fig. 7a).RNA Pol II-associated chromatin interactions were better conserved across the CLL samples than CTCF-associated chromatin interactions (Fig. 7b), which could be attributed to that open chromatin regions in the CLL samples that overlapped with GM12878 RNA Pol II peaks were better conserved than those overlapping with GM12878 CTCF peaks (Fig. 7c).Using this set of ATAC-seq data in CLL samples, it was reported that regions with higher open chromatin signals in uCLL samples showed strong enrichment of binding sites of CTCF, RAD21 and SMC3 [28], which could also contribute to the high variability of CTCF chromatin interactions.Moreover, we again observed extensive patient heterogeneity of CTCF and RNA Pol II-associated predicted open chromatin interactions in these clinical samples. (See figure on previous page.)Fig. 6 Performances of the sequence-based models in new CLL samples.a Venn diagram of chromatin interactions identified by Juicer in unmutated and mutated CLL samples.b Uniqueness analysis of real Hi-C and predicted Hi-C chromatin interactions in new CLL samples.Hi-C, real Hi-C interactions; predicted, predicted chromatin interactions using CLL 401 model.c Precision-recall curves of the sequence-based models on distance-matched Hi-C datasets using only sequence features.d Across-sample performances as measured by area-under precision-recall curve (auPRC) of the models on distance-matched Hi-C datasets using only sequence features.e The importance scores of sequence features extracted from both directions (F, forward; RC, reverse complement) of the two anchors (left and right) by models trained on CLL 401 sample.The orange horizontal lines indicate average importance scores of the features from the strand of the anchor.Pearson correlations between feature importance scores of the two anchors are given in the table.f Validations of predicted chromatin interactions by 4C-seq at GREB1 gene region in MCF-7 cells.In the predicted Hi-C interaction panel, only those interactions connected to GREB1 promoter were shown.g Validations of predicted chromatin interactions by 4C-seq at SIAH2 gene region in MCF-7 cells.In the predicted Hi-C interaction panel, only those interactions connected to SIAH2 promoter were shown When applying the GM12878 Hi-C model to the CLL samples, a total of 758,407 Hi-C-associated open chromatin interactions were predicted (Fig. 8a).The phenomenon observed from the CTCF model also can be observed from the Hi-C model, for example, the chromatin interactions across the CLL samples and the overlapping peaks between CLL samples and GM12878 Hi-C peaks were not well conserved as that of RNA Pol II (Fig. 8b,c).The predicted open chromatin interactions by Hi-C model were able to separate mCLL and uCLL samples (Additional file 1: Fig. S10a).Most differential chromatin interactions were associated with changes in the occurrence of one anchor (Fig. 8d).Genes that were upregulated in uCLL were associated with uCLL-specific chromatin interactions (Fig. 8e).In the set of differential chromatin interactions whose anchors did not have the same level of changes as the chromatin interactions themselves between the two subtypes, the rate of co-occurrences of the two anchors within the same All, all open chromatin regions.d Distribution of differential CTCF and RNA Pol II chromatin interactions based on whether both anchors (both), one anchor (one-side), or neither anchors (neither) showed the same level of differences between uCLL and mCLL samples as the associated chromatin interaction.e Association of differences in chromatin interactions between uCLL and mCLL samples with differentially expressed genes identified from a set of microarray samples.IFC, the fold change of the average number of chromatin interactions observed at the gene promoter in uCLL samples over that in mCLL samples.p-values were calculated using the Kruskal-Wallis test.f, g Examples of genes, ZBTB20 and LPL, whose different connectivity are associated with differences in distal regions.The red bars and curves indicate significantly different open chromatin regions and chromatin interactions based on Fisher's exact test sample and the levels in chromatin interactions could change (Additional file 1: Fig. S10b).Examples of predicted open chromatin interactions are shown in Fig. 8f andg and Additional file 1: Fig. S10e-h.Thus, we observed extensive patient heterogeneity of Hi-C predicted open chromatin interactions in these clinical samples. Using the predicted open chromatin interactions, it was possible to separate mCLL and uCLL samples (Additional file 1: Fig. S11a).Variations in occurrences of open chromatin interactions between the two subtypes of CLL were associated with variations in occurrences of anchor regions.Most differential ChIA-PET chromatin interactions were associated with changes in the occurrence of one anchor (Fig. 7d).There was a small portion of differential chromatin interactions whose anchors did not have the same level of changes as the chromatin interactions themselves between the two subtypes.In this set of differential chromatin interactions, the rate of co-occurrences of the two anchors within the same sample could change, contributing to the levels of changes in predicted open chromatin interactions (Additional file 1: Fig. S11b).With the GM12878 Hi-C model, we were also able to see differences in connectivity at All pairs, all possible pairs used for prediction; y-axis, the proportion of total chromatin interactions that can be found in a particular number of samples.c Uniqueness analysis of open chromatin regions that overlap with Hi-C peaks from GM12878 cells in the CLL samples.All, all open chromatin regions; y-axis, the proportion of total chromatin interactions that can be found in a particular number of samples.d Distribution of differential Hi-C chromatin interactions based on whether both anchors (both), one anchor (one-side), or neither anchors (neither) showed the same level of differences between uCLL and mCLL samples as the associated chromatin interaction.e Association of differences in chromatin interactions between uCLL and mCLL samples with differentially expressed genes identified from a set of microarray samples.IFC, the fold change of the average number of chromatin interactions observed at the gene promoter in uCLL samples over that in mCLL samples.p-values were calculated using the Kruskal-Wallis test.f, g Examples of genes, ZBTB20 and LPL, whose different connectivity are associated with differences in distal regions.The red bars and curves indicate significantly different open chromatin regions and chromatin interactions based on Fisher's exact test transcription start sites associated with differences in the occurrences of the open chromatin regions at the transcription start sites (Additional file 1: Fig. S10d). Genes with higher expression in uCLL showed higher connectivity at the transcription start sites (Fig. 7e, Additional file 1: Fig. S11c, Fig. 6e, Additional file 1: Fig. S10c).The differences in connectivity at transcription start sites were associated with differences in the occurrences of the open chromatin regions at the transcription start sites between CLL subtypes (Additional file 1: Fig. S10d and Additional file 1: Fig. S11d), and also, differences in connectivity were sometimes associated with differences in distal interacting regions (Additional file 1: Fig. S11e, Fig. 7f).Examples of predicted open chromatin interactions are shown at important CLL prognostic markers, such as LPL (Fig. 7g), ZAP70 (Additional file 1: Fig. S11f), ZNF667 (Additional file 1: Fig. S11g), and CD38 (Additional file 1: Fig. S11h) [45][46][47][48].Taken together, our results indicate that different subtypes show different profiles of predicted open chromatin interactions.Different subtypes may be a source of patient heterogeneity in clinical samples. ",
"section_name": "Exploring open chromatin interactions in a cohort of patient samples",
"section_num": null
},
{
"section_content": "In this manuscript, we described two methods of predicting chromatin interactions, first, a functional genomics approach which uses local epigenomics data to accurately predict chromatin interactions, and second, a convolutional neural network, ChINN, which can extract sequence features and be coupled to classifiers to predict chromatin interactions between open chromatin regions using DNA sequences and distance. We showed that at resolutions limited by the experimental techniques, chromatin interactions between open chromatin regions could be predicted from 1-dimensional functional genomics data through the fact that the cross-sample model can capture the chromatin interactions.ChINN only requires the use of open chromatin data and showed good generalizability on the same type of chromatin interactions across different cell types.Thus, it has the potential to be applied to large sets of clinical samples with limited biological materials.In addition, ChINN can discover sequence features that are important for predicting chromatin interactions, including shared features such as the CTCF motif and cell-type specific features such as GATA3 binding motif in MCF-7, which is frequently mutated in breast cancer [49].Also, we could validate ChINN-identified chromatin interactions by 4C. In distance-controlled experiments, our prediction method using functional genomics data performed better on RNA Pol II chromatin interactions but worse on CTCF chromatin interactions compared to sequence-based ChINN.Such differences could be attributed to the lower functional genomic complexity at CTCF binding sites and functional genomic data might fail to capture the convergent CTCF binding motifs often observed at CTCF-mediated chromatin interactions. We also noticed that the models trained using sequence features of CTCF ChIA-PET data perform better than the models trained using functional genomics data from CTCF ChIA-PET in the cross-sample prediction.We reason that the difference of the performance may be explained by the different resolution of the data.ChIP-seq data can yield ChIP-seq peaks of over several hundred bp long (and they are further generalized into count data when preparing the input features), while the CTCF motif is only less than 20 bp (CTCF motif MA0139.1 from JASPAR database).In addition, the ChIPseq peaks cannot tell the orientation of the CTCF binding, while sequence can tell the direction of CTCF motif.As CTCF orientation is found to be important in chromatin interactions [41], the sequence feature can give more information of the binding site as well as the binding orientation.Therefore, the information of CTCF in these two crosssample prediction results is different. On the other hand, RNA Pol II binding sites do not have such distinctive DNA motifs, making it harder to predict RNA Pol II binding sites [13,14] and consequently harder to predict RNA Pol II-associated chromatin interactions from DNA sequences.However, RNA Pol II binding sites are usually occupied by many other transcription factors, making it easier to predict RNA Pol II-associated chromatin interactions using functional genomic data. The application of ChINN models with gradient boosted tree classifiers to a set of CLL ATAC-seq samples showed that several of the predicted open chromatin interactions could be validated by Hi-C.However, we note that the auPRC scores of ChINN reported, particularly the GM12878 and K562 models applied to explore chromatin interactions in patient samples, was around 0.26-0.6,which is consistent with crosssample testing of other epigenomics machine learning prediction methods such as DeepHistone [50].However, these auPRC scores are not very high, which could be due to several reasons. First, auPRC is a performance metric that is usually not very high, especially when the number of negative samples hugely overwhelm the positive samples.Because the number of chromatin interactions in the entire genome, relative to the number of genomic regions with no reported chromatin interactions by Hi-C, is not very high; therefore, the number of negative samples in our data hugely overwhelm the positive samples.As such, it is expected that the auPRC score will not be very high when applying GM12878 and K562 Hi-C models to other cell lines or patient samples. Second, the ChINN method only takes as input the sequences of DNA at open chromatin regions of the genome for prediction.If more types of data are input into the model, the performance of the model is likely to improve, but at the cost of requiring more datatypes which are expensive and labor-intensive to acquire. Third, while there were also chromatin interactions that were predicted but not validated by Hi-C, our results showing that 4C could validate predicted chromatin interactions in MCF-7 cells that were not identified by Hi-C suggest that these so-called \"false positives\" might potentially be real chromatin interactions that were simply not captured by Hi-C due to limited sequencing depth of Hi-C libraries. In future work, further development of Hi-C and other chromatin interaction sequencing methods to comprehensively capture chromatin interactions will allow for a better comparison with chromatin interaction predictions.Additionally, further development and refinement of ChINN to improve the accuracy of chromatin interaction prediction is warranted. Application of ChINN models in CLL revealed that although there were open chromatin interactions that were ubiquitous in all samples, there were a large number of patient-specific open chromatin interactions and also chromatin interactions that were found in fewer than half the samples.We note that chromatin interactions predicted using cross-sample models are likely to show less cell-type specificity, and the fact that sample heterogeneity can be seen in these predicted open chromatin interactions in spite of the lower likelihood of cell-type specificity due to the nature of the chromatin interaction prediction, suggests that chromatin interaction heterogeneity is widespread throughout the genome.Moreover, the observation of predicted open chromatin interaction heterogeneity agrees with our observations that there exist both ubiquitous chromatin interactions and patient-specific chromatin interactions in the 6 Hi-C libraries from the 6 CLL patient samples that we examined.While we previously observed patient-specific chromatin interactions at particular loci [51], here, we show that this phenomenon is widespread.To the best of our knowledge, this observation of widespread nature of patient-specific chromatin interactions is novel and has not been previously reported in the 3D genome organization field. One potential reason for these different chromatin interactions could be due to different patient subtypes.Importantly, we found systematic differences in chromatin interactions involving important CLL prognostic genes, such as LPL and CD38, between the IGHV-mutated and IGHV-unmutated subtypes.These results suggest that differences in chromatin interaction landscapes between CLL subtypes could have important functional implications in CLL biology.Moreover, differences in chromatin interaction presence or absence may lead to different expression of oncogenes in cancers. Our observation of widespread patient heterogeneity in patient cancer samples highlights the need for precision medicine and the need to understand chromatin interactions in individual patient samples.Machine learning offers one way for us to predict chromatin interactions in a cost-effective manner.The ChINN method may be useful in the future in understanding chromatin interactions in large cohorts of clinical samples and identifying chromatin interaction-based biomarkers that can be used to distinguish between different subtypes of cancer which may help in the development of precise therapies for the different subtypes of cancer. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "A functional genomics approach is able to predict chromatin interactions.The ChINN framework is able to predict chromatin interactions from open chromatin regions in the human genome, using DNA sequences and distances as features.This framework can be applied in other cell lines or clinical samples given the knowledge of open chromatin regions, making it a useful tool to interrogate chromatin interactions when large-scale functional genomics acquisition is not applicable due to limited biological materials. ",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "We performed machine learning, Hi-C interaction analysis, ATAC-seq, RNA-seq, and gene expression analyses as described in the following sections.The quality information of generated Hi-C, ATAC-seq, and RNA-seq libraries can be found in Additional file 1: Table S7, Additional file 1: Table S8, and Additional file 1: Fig. S12. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The development of the sequence models was divided into three stages.In the first stage, the distance-matched datasets were used to train the models consist of convolutional neural network (feature extractor) with fully connected layers as the classier, as shown in Fig. 2a.The first stage deep learning method works as a feature extractor to convert the raw sequence feature to numerical representation that can be used as input of the machine learning models.Stages 2 and 3 aim to train the different machine learning models to make the prediction.In the second and third stage, the feature extractors trained in the first stage were frozen and gradient tree boosting classifiers were used as classifiers.In the second stage, the gradient tree boosting classifiers were trained using the extended datasets.In the third stage, the gradient tree boosting classifiers were trained using all potential pairs of anchors generated from open chromatin data and annotated by existing ChIA-PET data.Thus, the final result was a program that took in a list of open chromatin regions and produced predictions of chromatin interactions between the open chromatin regions. The feature extractors took DNA sequences of both anchors of a potential interacting pair as input.The classier then took the features generated by the feature extractor and optionally the distance between anchors as input and produced a probability score of interaction.This final model was defined as the \"from open chromatin\" model.More details can be found from Additional file 1: Supplementary Methods [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69]. ",
"section_name": "Machine learning of ChIA-PET data",
"section_num": null
},
{
"section_content": "We collected the Hi-C interactions from 8 cell lines, including GM12878, HeLaS3, HMEC, HUVEC, IMR90, K562, KBM7, and NHEK.The construction of machine learning model using Hi-C data from cell lines follows the same procedures as described in that of ChIA-PET data, where the positive data is annotated according to the Hi-C interactions. ",
"section_name": "Machine learning of Hi-C data from cell lines",
"section_num": null
},
{
"section_content": "We collected the Hi-C interactions from 6 CLL clinical samples, including CLL 102, CLL 312, CLL 324, CLL 344, CLL 401, and CLL 484.The construction of machine learning model using Hi-C data from cell lines follows the same procedures as described in that of ChIA-PET data, where the positive data is annotated according to the Hi-C interactions.The CLL 401 model was used in the across-sample prediction. ",
"section_name": "Machine learning of Hi-C data from clinical samples",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia patient samples (either peripheral blood or bone marrow isolates) were obtained from the Leukemia Cell Bank at the National University Health System (NUHS) with patient consent, under Institute Review Board number H-20-022E.The CLL samples were either bone marrow aspirates (312,324,344,484 and 102) or peripheral blood (401).The samples were immediately frozen after collection and stored in liquid nitrogen until further use. The samples were taken out of the liquid nitrogen and thawed by dipping in a beaker containing water at 37 °C.Once the sample was thawed completely, the cells were immediately transferred to the 15 ml falcon and resuspended in 10 ml PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA.The cells were pelleted at 300×g for 5 min at room temperature and resuspended in 5 ml PBS containing 2% FBS and 2 mM EDTA.The cells were counted and checked for viability using Trypan Blue. RNA and genomic DNA were isolated from the CLL patient samples using AllPrep DNA/RNA/miRNA universal kit (Qiagen) according to the manufacturer's instructions.Briefly, cells lysate were homogenized by a 21-G needle and syringe together with lysis buffer and 1 M DTT.After that, the homogenized lysate were transferred into AllPrep DNA mini spin column for genomic DNA extraction.The genomic DNA were then eluted by water and proceeded for the IGHV mutation test.The flow through after the AllPrep DNA mini spin column was then proceeded into RNease Mini spin column with on-column digestion for RNA extraction.The RNA were eluted in water and further sent for RNA-seq. IGHV mutation test was performed following the method in Agathangelidis et al. [70].Briefly, IGHV-IGHD-IGHJ gene rearrangements were amplified by 5′ IGHV leader primers and 3′ IGHJ primers (primer sequences are provided in Additional file 1: Table S9) using genomic DNA (gDNA) from CLL patient samples.The PCR amplification was performed by PCR core kit (Qiagen).Final PCR products were imaged by agarose gel electrophoresis and purified by PCR purification kit (QIAGEN).Purified PCR products were confirmed through Sanger sequencing by 3′ IGHJ primers.The Sanger sequencing results were analyzed by IMGT/V-QUEST tools [71] to get the IGHV identity scores.If the identity score was larger than 98%, the CLL sample was considered an unmutated sample while if the score was lower than 98%, the CLL sample was considered a mutated sample. ",
"section_name": "Preparation of clinical samples",
"section_num": null
},
{
"section_content": "Hi-C libraries were prepared using the Arima Genomics kit (Arima Genomics, San Diego, CA) in conjunction with the Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Cat # 21024) and Swift Biosciences Indexing Kit (Cat # 26148) following the manufacturer's recommendations.In brief, 1X 10 6 cells were fixed with formaldehyde in the nucleus.Fixed cells were permeabilized using a lysis buffer and then digested with a restriction enzyme cocktail supplied in the Arima Hi-C kit.The resulting overhangs were filled in with biotinylated nucleotides followed by ligation.After ligation, crosslinks were reversed, and the DNA was purified from protein.Purified DNA was treated to remove biotin that was not internal to ligated fragments.Hi-C material was then sonicated using a Covaris Focused-Ultrasonicator M220 instrument to achieve 300-500 bp fragment sizes.The sonicated DNA was double-size selected using Ampure XP beads, and the sequencing libraries were generated using low input Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Cat # 21024) and Swift Biosciences Indexing Kit (Cat # 26148).The Hi-C libraries were loaded on an Illumina flow cell for paired-end 150-nucleotide read length sequencing on the Illumina HiSeq 4000 following the manufacturer's protocols. Cell culture MCF-7, a breast cancer cell line, was cultured in DMEM/F12 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin and maintained at 37 °C, 5% CO 2 humidified incubator.Before 4C-seq assays, MCF-7 cells were grown in hormone-free media: they were washed with PBS twice to remove any residual FBS or growth factors and incubated in phenol red-free medium (Invitrogen/Gibco) supplemented with 10% charcoal-dextran stripped FBS (Hyclone) and 1% pencillin-streptomycin for a minimum of 72 h.Hormone-depleted MCF-7 cells were then treated with estrogen (Sigma) to a final concentration of 100 nM for 45 min before 4C-seq assay.The control cells were treated with an equal volume and concentration of vehicle, ethanol (Sigma), for 45 min. Circular chromosome conformation capture (4C) 4C-seq assays were performed according to Splinter et al [72] with slight modifications.Briefly, 4 × 10 7 cells were crosslinked with 1% formaldehyde.The nuclei pellets were isolated by cell lysis with cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, 0.5% NP 40) supplemented with protease inhibitors (Roche).First step digestion was performed overnight at 37 °C with HindIII enzyme (NEB).Digestion efficiency was measured by RT-qPCR with HindIII site-specific primers.After confirmation of good digestion efficiency, DNA was ligated overnight at 16 °C by T4 DNA ligase (Thermo Scientific) and de-crosslinked.Following decrosslinking, DNA was extracted by phenol-chloroform and this is the 3C library.The DNA was then processed for second digestion with DpnII enzyme (NEB) overnight at 37 °C.After final ligation, 4C template DNA was obtained, and the concentration was determined using Qubit assays (Thermo Scientific).The 4C template DNA was then amplified using specific primers with Illumina Nextera adapters and sent for sequencing on the MiSeq system.All the 4C genome coordinates are listed in Additional file 1: Table S9. ",
"section_name": "In situ Hi-C",
"section_num": null
},
{
"section_content": "Total RNA was extracted from the CLL samples using the All Prep DNA/RNA kit (Qiagen).The RNA was quantified using the Qubit BR RNA Assay kit.RNA-seq libraries (strand specific and ribo zero) were constructed using Illumina Total RNA Prep kit (Illumina, San Diego, CA, USA) and sequenced 150 bases paired-end on the Illumina HiSeq 4000 following the manufacturer's instruction. ",
"section_name": "RNA-seq",
"section_num": null
},
{
"section_content": "ATAC-seq library was prepared as described previously [73].Briefly, 50,000 cells were lysed for nuclei isolation using ATAC-Resuspention Buffer containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin.Transposition reaction was performed for 30 min at 37 °C using Nextera DNA library preparation kit (NEB).Transposed fragments were amplified by eight PCR cycles for library preparation.Primer dimers and long DNA fragments were removed by AMPure XP beads purification step.DNA concentration was measured by Qubit fluorometric assay and library quality was determined by Bioanalyzer.The library was sequenced in Nextseq 500 76 bp paired-end configuration using Illumina platform. ",
"section_name": "ATAC-seq",
"section_num": null
}
] |
[
{
"section_content": "We would like to thank all members of the Fullwood Lab for helpful comments.We would like to thank the NUHS Leukemia Cell Bank for providing Chronic Lymphocytic Leukemia samples. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This research is supported by the National Research Foundation (NRF) Singapore through an NRF Fellowship awarded to M.J.F (NRF-NRFF2012-054) and NTU start-up funds awarded to M.J.F.This research is supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education Academic Research Fund Tier 3 awarded to Daniel Tenen as lead PI with M.J.F as co-investigator (MOE2014-T3-1-006).This research is supported by a National Research Foundation Competitive Research Programme grant awarded to V.T. as lead PI and M.J.F. as co-PI (NRF-CRP17-2017-02).This research is supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative.This research is supported by a Ministry of Education Tier II grant awarded to M.J.F (T2EP30120-0020). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All relevant data supporting the key findings of this study are available within the article and its Additional files.The datasets of RNA-Seq, ATAC-Seq and Hi-C, which are generated during the current study are available in GEO under accession number GSE163896 [74].The 4C data has been deposited with GEO accession number GSE135052 [75] and is publicly available.The source codes of ChINN are freely available under Apache License 2.0 at https://github.com/mjflab/chinn[76].The code used for this paper has also been deposited at Zenodo with DOI 10.5281/zenodo.5139249[77]. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1186/s13059-021-02453-5. Additional file 1. Supplementary text, methods, figures, and tables. The review history is available as Additional file 2. Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. All samples were collected by the NUHS Leukemia Bank with patient consent.Ethics approval for the study was given by National University of Singapore Institutional Review Board (IRB approval number: LH-20-022E).The authors confirm that experimental methods comply with the Helsinki Declaration. 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/s13059-021-02453-5. Additional file 1. Supplementary text, methods, figures, and tables. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional file 2. Review history.",
"section_num": null
},
{
"section_content": "The review history is available as Additional file 2. ",
"section_name": "Review history",
"section_num": null
},
{
"section_content": "Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. ",
"section_name": "Peer review information",
"section_num": null
},
{
"section_content": "All samples were collected by the NUHS Leukemia Bank with patient consent.Ethics approval for the study was given by National University of Singapore Institutional Review Board (IRB approval number: LH-20-022E).The authors confirm that experimental methods comply with the Helsinki Declaration. ",
"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
}
] |
10.20452/pamw.15898
|
Richter transformation of chronic lymphocytic leukaemia presenting as otitis externa
|
FIGurE 1 A -erythema, extensive oedema, and skin thickening causing obstruction of the left external auditory canal. Skin necrosis developed in the course of hospitalization.
|
[
{
"section_content": "lymphocytic leukemia (CLL) 3 years earlier (Rai stage I, Binet stage A).Following a sharp increase in lymphocyte number, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab was initiated 1 month previously.Despite the administration of combined systemic antimicrobial and topical treatment, the patient deteriorated (FIGurE 1A) and was referred for hospitalization.Meanwhile, she developed marked regional lymphadenopathy. Diagnostic workup on admission to hospital did not identify the specific pathogen and the patient received empiric antimicrobial treatment with meropenem and clindamycin.Biopsy of the external auditory canal revealed diffuse infiltration of the cutaneous lamina propria by neoplastic lymphoid cells with histologic and immunohistochemical characteristics compatible with high -grade non -Hodgkin lymphoma (NHL) (FIGurE 1B).The patient developed sepsis and died within 3 weeks of hospitalization. Numerous conditions may be misdiagnosed as otitis externa, and special considerations apply to immunocompromised patients. 1Chronic lymphocytic leukemia is the most frequently encountered type of leukemia in Western countries.It affects predominantly the elderly and is characterized by a highly heterogeneous prognosis.Transformation of CLL to an aggressive lymphoma (termed Richter transformation) is rare, with a cumulative 5 -year incidence of 2.8% and a dire prognosis. 2,3Reports of skin or ear infiltration are scant; prompt recognition may be key to prolong survival. 4ute otitis externa is a common condition which generates a high volume of urgent ear, nose, and throat (ENT) consultations, also during the COVID -19 pandemic.In this setting, a 75 -year -old woman was referred for an outpatient ENT consultation due to ear pain and hearing loss and diagnosed with acute otitis externa.The patient had been diagnosed with chronic Localization of NHL in the external auditory canal is generally a rare occurrence and misdiagnosis as otitis externa is a common pitfall. 5Although the clinical presentation further depends on the extent of disease and infiltration of adjacent anatomical structures, the most common clinical symptoms include severe otalgia, otorrhea with exudative discharge, erythema and oedema of the ear canal, sensorineural hearing loss, difficulty with mastication, and regional lymphadenopathy. 5The main therapeutic approach is immunopolychemotherapy; however, the management differs by NHL subtype, among other factors. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "PAtIENt CoNsENt The patient provided informed consent to publish the photographs. ",
"section_name": "ArtICLE INForMAtIoN",
"section_num": null
},
{
"section_content": "oPEN ACCEss This is an Open Access article distributed under the terms of the Creative Commons Attribution -NonCommercial -ShareAlike 4.0 International License (CC BY -NC -SA 4.0), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited, distributed under the same license, and used for noncommercial purposes only.For commercial use, please contact the journal office at [email protected]. ",
"section_name": "CoNFLICt oF INtErEst None declared.",
"section_num": null
},
{
"section_content": "",
"section_name": "How to CItE",
"section_num": null
}
] |
[] |
10.3390/biomedicines10112892
|
Non-Myelofibrosis Chronic Myeloproliferative Neoplasm Patients Show Better Seroconversion Rates after SARS-CoV-2 Vaccination Compared to Other Hematologic Diseases: A Multicentric Prospective Study of KroHem
|
<jats:p>Disease- and treatment-mediated immunodeficiency might render SARS-CoV-2 vaccines less effective in patients with hematologic diseases. We performed a prospective non-interventional study to evaluate humoral response after one and two doses of mRNA-1273, BNT162b2, or ChAdOx1 nCoV-19 vaccine in 118 patients with different malignant or non-malignant hematologic diseases from three Croatian treatment centers. An electrochemiluminescent assay was used to measure total anti-SARS-CoV-2 S-RBD antibody titers. After one vaccine dose, 20/66 (33%) achieved seropositivity with a median antibody titer of 6.1 U/mL. The response rate (58/90, 64.4%) and median antibody titer (>250 U/mL) were higher after two doses. Seropositivity varied with diagnosis (overall p < 0.001), with the lowest rates in lymphoma (34.6%) and chronic lymphocytic leukemia (52.5%). The overall response rate in chronic myeloproliferative neoplasms (CMPN) was 81.3% but reached 100% in chronic myeloid leukemia and other non-myelofibrosis CMPN. At univariable analysis, age > 67 years, non-Hodgkin’s lymphoma, active treatment, and anti-CD20 monoclonal antibody therapy increased the likelihood of no vaccine response, while hematopoietic stem cell recipients were more likely to respond. Age and anti-CD20 monoclonal antibody therapy remained associated with no response in a multivariable model. Patients with the hematologic disease have attenuated responses to SARS-CoV-2 vaccines, and significant variations in different disease subgroups warrant an individualized approach.</jats:p>
|
[
{
"section_content": "In a time span of months from its first isolation as the cause of a local respiratory disease outbreak, novel coronavirus (SARS-CoV-2) became the leading cause of death from infectious disease worldwide.Initial reports demonstrated several-fold higher overall mortality in patients with hematologic malignancy compared to the general population in the same pandemic period [1][2][3][4].While mortality varied between different hematologic conditions and was highest in patients with acute leukemia and bone marrow failure, it still exceeded 30% in every disease group [1]. With a prompt response from the scientific community, landmark phase III randomized controlled trials (RCTs) administering two doses of SARS-CoV-2 vaccines demonstrated over 90% efficacy in preventing symptomatic coronavirus disease (COVID-19) [5][6][7].Following authorization from the European Medicines Agency, the BNT162b2 mRNA (BioNTech, Mainz, Germany/Pfizer, New York, NY, USA) became available in Croatia in late December 2020, followed by ChAdOx1 nCoV-19 (AstraZeneca, Cambridge, UK) and mRNA-1273 (Moderna, Cambridge, MA, USA) in January 2021.Patients with hematologic conditions were among those prioritized for early vaccination.However, as initial RCTs excluded immunocompromised populations, it was unknown how their vaccine response would compare to immunocompetent trial subjects.Hematologic malignancy is already associated with an attenuated response to other vaccines [8][9][10], and those who had received B-cell-depleting therapy had no antibody response with prolonged viral shedding following SARS-CoV-2 infection [11].Several single-center studies soon raised concern about suboptimal seroconversion rates following a standard two-dose regimen of SARS-CoV-2 vaccines [12][13][14][15]. Immune response in patients with hematologic disorders depends on disease-specific immunologic environments and targeted therapeutic modalities.Identifying which subgroups of these high-risk patients might not respond to classic vaccination schemes would influence clinical decision-making as they could be candidates for alternative interventions.We aimed to examine quantitative and qualitative humoral vaccine response in a prospectively enrolled cohort with different malignant and non-malignant hematologic diseases in a multicentric setting.An additional explorative analysis was performed to identify potential predictors of no humoral response after two vaccine doses.The study was conducted by the Croatian Cooperative Group for Hematologic Diseases (KroHem). ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Between January and July 2021, we prospectively enrolled patients from University Hospital Centre Zagreb, University Hospital Centre Osijek, and General Hospital \"Dr.Josip Benčević\" Slavonski Brod.All patients were adults (≥18 years) with a history of the malignant or non-malignant hematologic disease who had received at least one dose of either mRNA-1273 (Moderna, Cambridge, MA, USA), BNT162b2 mRNA (BioNTech, Mainz, Germany/Pfizer, New York, NY, USA), or ChAdOx1 nCoV-19 (AstraZeneca, Cambridge, UK) COVID-19 vaccine.The exclusion criterion was previous COVID-19 infection.Patients followed the recommended dosing intervals: 21 days for BNT162b2, 28 days for mRNA-1273, and 12 weeks for ChAdOx1 nCoV-19.The response was evaluated at least 7 days following the last vaccine dose.The study was approved by hospital ethics committees, and all participants provided written informed consent. Seroconversion after vaccination was determined using a serological immunoassay registered for quantitative measurement of antibodies against the SARS-CoV-2 spike protein receptor-binding domain (RBD).This assay, Elecsys Anti-SARS-CoV-2 S (Roche Diagnostics, Mannheim, Germany), measures total antibodies against SARS-CoV-2 S glycoprotein by using SARS-CoV-2 S RBD recombinant antigens that predominantly capture anti-SARS-CoV-2 S immunoglobulin G (IgG), but also IgA and IgM [16]. In order to identify the previous infection, anti-SARS-CoV-2 nucleocapsid antigen antibodies (including IgG) were qualitatively assessed.The used reagent, Elecsys Anti-SARS-CoV-2 (Roche Diagnostics, Mannheim, Germany), consists of a recombinant protein representing the N antigen in a double-antigen sandwich assay format. Both assays were performed by Cobas e801 analyzer (Roche Diagnostics, Mannheim, Germany) according to manufacturer instructions and by following principles.Biotinylated and ruthenylated antigens, in the presence of corresponding antibodies, create doubleantigen sandwich immune complexes.The complexes bind to the solid phase by an interac-tion between biotin and streptavidin after the addition of streptavidin-coated microparticles.Microparticles are magnetically captured to the electrode surface in the measuring cell.Electrochemiluminescence is then induced by applying a voltage and measured with a photomultiplier.The signal yield increases with the antibody titer.A positive response was defined as >0.8 U/mL with lower and upper limits of quantification of 0.4 U/mL and 250 U/mL, respectively [16].These numerical results in U/mL are equivalent to the 1st WHO International Standard for anti-SARS-CoV 2 immunoglobulin BAU/mL [17]. We reviewed in-hospital electronic records for demographic and clinical characteristics, including underlying hematologic disease, date of diagnosis, current and previously received treatment, the number of received treatment lines, application of anti-CD20 monoclonal antibodies (mAbs) or corticosteroid therapy in six months before vaccination, hematopoietic stem cell transplantation (HSCT) and total serum IgG levels prior to vaccination.Participants were followed until December 2021 for outcomes that included symptomatic SARS-CoV-2 infection, severe forms of COVID-19 requiring oxygen supplementation or ICU admission, and death. Categorical variables were summarized with counts and frequencies, and continuous variables with medians and interquartile ranges.Response after the first versus second dose was compared with Wilcoxon sign-rank test for antibody titers and McNemar's χ 2 test for seropositivity rates.Subgroup comparisons for those who received two doses were performed with Mann-Whitney U, χ 2 , or Fisher's exact test as appropriate.ROC curve analysis was used to find optimized cut-off values of numerical variables regarding response to the second dose.Zou's modified Poisson regression was performed to compute risk ratios (RRs) for no humoral response after two vaccine doses [18].Variables of interest for regression analysis were: age > 67 years, sex, time from the second dose to antibody assessment, vaccine type, diagnosis of Non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), multiple myeloma/amyloidosis (MM), acute leukemia (AL) or chronic myeloproliferative neoplasms (CMPN) including chronic myeloid leukemia (CML), polycythemia vera (PV), chronic eosinophilic leukemia or myelofibrosis (MF), time from diagnosis to the second dose, low total serum IgG, active treatment, any prior therapy, line of therapy, HSCT, HSCT > 1 year prior to vaccination, antiCD20 mAb therapy, corticosteroid therapy six months prior to vaccination and prednisone equivalent dose > 120 mg.Statistically significant variables at univariable analysis were included in a multivariable model.The final model was constructed with backward elimination. Statistical significance was determined at an α level of 0.05 throughout the analysis.All p values are based on two-sided tests.MedCalc statistical software (version 20.008, MedCalc Software Ltd., Ostend, Belgium) was used for inter-group comparisons; regression analysis and data visualization were performed with RStudio for OS X (version 1.2.1335,RStudio, PBC, Boston, MA, USA). ",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "We initially enrolled 141 participants.After excluding 23 patients for prior SARS-CoV-2 infection, as evidenced by the presence of anti-nucleocapsid antigen antibodies, 118 remained who had received at least one dose.Of those, 55.9% were male, with a median age of 65.2 years (IQR 52.3-71.9).The majority had received the Pfizer-BioNTech vaccine (66.9%), followed by Oxford-AstraZeneca (23.7%) and Moderna (9.3%).Lymphoid conditions were most prevalent with NHL in 32 participants (27.1%),CLL in 28 (23.7%), and Hodgkin lymphoma (HL) in three (2.5%), followed by MM in 17 (14.4%),AL in 15 (12.7%),CML in seven (5.9%), other CMPNs in nine (7.6%), myelodysplastic syndrome (MDS) in four (3.4%), aplastic anemia (AA) in two (1.7%) and immune thrombocytopenia (ITP) in one (0.8%).More than half were in active therapy at the time of vaccination (66.9%), while only 5.9% had not received any previous therapy. Data on the humoral response after the first dose were available in 66 patients.Median time from vaccination to evaluation after the first dose was 20 days (IQR [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].Twenty participants (30.3%) achieved a positive response with a median specific antibody titer of 6.1 U/mL.After receiving the first dose, two patients died from their primary disease, two received HSCT and were not eligible to receive a second dose, and two contracted COVID-19.Data after the second dose were not available for 22 patients.Of the 90 patients who had evaluable responses following a second dose, 58 (64.4%) achieved seropositivity with median specific antibody levels at the upper limit of quantification (250 U/mL).All firstdose responders maintained seropositivity after the second dose.In a subgroup analysis of those who provided samples after both doses (n = 38), the second dose significantly improved both the response rate (p = 0.041) and specific antibody titer (p < 0.001). Seropositivity rates after two doses differed significantly with diagnosis (Figure 1, overall p < 0.001).Lymphoma patients had the lowest response rate (34.6%), followed by CLL (52.5%).Patients with CMPNs achieved an overall seropositivity rate of 81.3%.Response in the CML subgroup was 100% after two vaccine doses.Patients with PV and chronic eosinophilic leukemia all achieved response as well.Seronegative patients with CMPNs (n = 3) all had secondary MF and were on ruxolitinib therapy at the time of vaccination.Seropositive patients (n = 13) received either tyrosine-kinase inhibitors (TKIs), and hydroxyurea or had no specific chemoimmunotherapy.Antibody titers significantly varied with diagnosis as well (overall p < 0.001): lowest median titer levels were observed in lymphoma patients (0.4 U/mL, IQR 0.4-5.69)and those with CLL (2.6 U/mL, IQR 0.4-250), while other subgroups had median titers at the upper limit (Figure 1). (MDS) in four (3.4%), aplastic anemia (AA) in two (1.7%) and immune thrombocytopenia (ITP) in one (0.8%).More than half were in active therapy at the time of vaccination (66.9%), while only 5.9% had not received any previous therapy. Data on the humoral response after the first dose were available in 66 patients.Median time from vaccination to evaluation after the first dose was 20 days (IQR [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].Twenty participants (30.3%) achieved a positive response with a median specific antibody titer of 6.1 U/mL.After receiving the first dose, two patients died from their primary disease, two received HSCT and were not eligible to receive a second dose, and two contracted COVID-19.Data after the second dose were not available for 22 patients.Of the 90 patients who had evaluable responses following a second dose, 58 (64.4%) achieved seropositivity with median specific antibody levels at the upper limit of quantification (250 U/mL).All first-dose responders maintained seropositivity after the second dose.In a subgroup analysis of those who provided samples after both doses (n = 38), the second dose significantly improved both the response rate (p = 0.041) and specific antibody titer (p < 0.001). Seropositivity rates after two doses differed significantly with diagnosis (Figure 1, overall p < 0.001).Lymphoma patients had the lowest response rate (34.6%), followed by CLL (52.5%).Patients with CMPNs achieved an overall seropositivity rate of 81.3%.Response in the CML subgroup was 100% after two vaccine doses.Patients with PV and chronic eosinophilic leukemia all achieved response as well.Seronegative patients with CMPNs (n = 3) all had secondary MF and were on ruxolitinib therapy at the time of vaccination.Seropositive patients (n = 13) received either tyrosine-kinase inhibitors (TKIs), and hydroxyurea or had no specific chemoimmunotherapy.Antibody titers significantly varied with diagnosis as well (overall p < 0.001): lowest median titer levels were observed in lymphoma patients (0.4 U/mL, IQR 0.4-5.69)and those with CLL (2.6 U/mL, IQR 0.4-250), while other subgroups had median titers at the upper limit (Figure 1).Characteristics of responders and non-responders after two vaccine doses are summarized in Table 1.At univariable analysis, age > 67 years, a diagnosis of NHL, active treatment, and anti-CD20 mAb therapy 6 months prior to vaccination increased the Characteristics of responders and non-responders after two vaccine doses are summarized in Table 1.At univariable analysis, age > 67 years, a diagnosis of NHL, active treatment, and anti-CD20 mAb therapy 6 months prior to vaccination increased the likelihood of no humoral response.HSCT overall and HSCT > 1 year prior to vaccination were associated with a significantly decreased risk of no response (Table 2).While the diagnosis of CMPN (RR 0.48, 95% CI 0.17-1.38;p = 0.172) did not reach statistical significance, as seroconversion of CML patients was 100%, risk could not be computed for this subgroup alone.CLL patients were at increased risk of no response (RR 1.46, 95% CI 0.82-2.61;p = 0.2).Incidentally, participants with AL (RR 0.21, 95% CI 0.03-1.40;p = 0.106) and MM (RR 0.46, 95% CI 0.13-1.66;p = 0.237) were more likely to respond, although none reached statistical significance.Any corticosteroid therapy or >120 mg equivalent prednisone dose did not significantly influence response, nor did vaccine type.In a multivariable model, only age > 67 years and anti-CD20 mAb therapy remained significantly associated with a lack of response (Table 2).After a median follow-up of 8.2 months (IQR 7.5-8.8),five (4.2%) patients tested positive for SARS-CoV-2 after vaccination, of which two had received only one dose, and three had no humoral response despite receiving two doses.Two patients required in-hospital treatment and oxygen supplementation, and two died of COVID-19. ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Although a second vaccine dose was associated with a significantly better humoral response, the overall seroconversion rate of 64.4% in our cohort of hematologic patients was much lower than those reported in phase I/II RCTs, where virtually all participants seroconverted [19][20][21].We observed the lowest seroconversion rates and antibody titers in patients with lymphoid malignancies, with only half of CLL and a third of lymphoma patients reaching the threshold for a positive response.On the other hand, overall seropositivity in CMPNs was 81.3%, and rates observed in CML and non-myelofibrosis CMPN patients were comparable to healthy trial subjects. All chronic myeloproliferative disorders are associated with innate immune inhibition.However, CML patients in major and deep molecular responses seem to regain immunocompetency irrespective of TKI therapy or treatment-free remission [22].Adequate disease control typically prevails in current-day CML cohorts, resulting in COVID-19 mortality similar to that of the general population in the same era of the pandemic [23], consistent reports of high seroconversion rates [12,14], and production of both humoral and cellular response to SARS-CoV-2 vaccines [24].In contrast, BCR-ABL-1 negative CMPNs are associated with an increased risk of severe forms of COVID-19, most prominently MF, where case-fatality risk is 48% [25,26].In our CMPN subgroup, those who failed to respond were all secondary MF patients on active treatment with ruxolitinib.Ruxolitinib was consistently associated with no humoral vaccine response [12,[27][28][29], raising the question of treatment cessation in select high-risk patients during a standard SARS-CoV-2 vaccination schedule.In theory, immunomodulation could even be beneficial during the immune system hyperactivation phase of COVID-19, and discontinuation of ruxolitinib in active COVID-19 infection was associated with excess mortality [25].However, Caocci et al. reported similar seroconversion rates in MF patients treated with ruxolitinib compared to other regimens [30], and discerning JAK1/2 inhibitor action from the effect of immune phenotypes in BCR-ABL1 negative CMPNs is difficult as both are known to cause deep immune system dysregulation [31,32].Sudden ruxolitinib suspension is associated with severe inflammatory hyper reactions [33], and a phase III RCT of ruxolitinib plus standard of care versus placebo plus standard of care did not meet the composite endpoint of death, respiratory failure, or ICU admission in patients hospitalized for COVID-19 [34].As the sum of current findings shows no clear benefit of withholding ruxolitinib in light of either SARS-CoV.2vaccination or infection, the present agreement is to continue all active treatment in CMPNs [35]. To the extent of our knowledge, we report the lowest response rate (34.6%) in lymphoma patients to date.They were predominantly NHL patients, shown to have worse responses compared to HL [36], with a high proportion of those actively or recently treated with B-cell depleting agents.Comparably, Ghione et al. recorded a positive response in 36.3% of B-cell lymphoma patients, of whom almost a third received B-cell-directed treatment nine months before vaccination [37].We observed a biologically plausible total failure of B-cell activation with median antibody titers below the lower limit of quantification in almost all participants who had received anti-CD20 mAbs.Surprisingly, the diagnosis of MM was associated with a tendency towards seropositivity.Previously reported seropositivity rates in MM patients widely varied (66-84%) [12,13,38,39], and so did their antibody titer range.In many seropositive patients with MM, antibody titers do not reach the upper limit of quantification even after two vaccine doses [39], revealing a more pronounced difference in immune response than observed by qualitative measurement alone. In early reports of humoral response after two doses of the SARS-CoV-2 vaccine in HSCT recipients, 78% had quantifiable IgG (S-RBD) [40], and subsequent larger cohorts from Spanish and French registries reported similarly favorable seroconversion rates overall and up to 85% after autologous HSCT [41,42].Of note, approximately 60% of HSCT recipients had antibody levels in a range likely to neutralize the virus [40,41].In our study, prior HSCT was associated with a positive vaccine response.By withholding vaccination for the first three months, which is according to national guidelines, the majority produced detectable antibodies.Seroconversion rates in previous studies increased with time after transplantation.However, factors beyond the scope of our studies, such as lymphopenia, active GVHD, or immunosuppression, were independently predictive of response [40][41][42].We propose that successful immune reconstitution after HSCT, disease remission, and no active chemoimmunotherapy, contributed to the result we observed, a similar pattern to that of the Lithuanian national cohort [27].A confounding effect may also explain the high response rates observed in the AL subgroup since AL was the most common indication for HSCT. The study period coincided with the third pandemic wave in Croatia and the rise of the SARS-CoV-2 B.1.1.7 (Alpha) variant for which attenuated vaccine efficacy was observed, a continuing trend for variants of concern that followed [43][44][45].Compared to healthy controls, hematologic patients showed an even faster decline in neutralizing antibody titers for B.1.351(Beta) and B.167.2 (Delta) variants [46].While routine serological response monitoring was not implemented even in high-risk populations, EMA and ECDC recommend administering a booster dose in all adults and, as of recently, even a second booster in the elderly and those with predisposing conditions.Administering a three-dose vaccine regimen was associated with lower hospitalization rates in the immunocompromised [47].In hematologic patients, the additional dose produces a more robust humoral response in responders, although a fraction of non-responders seems to remain non-responsive [48][49][50].Monin et al. observed a higher proportion of cellular versus humoral response in hematologic patients [51].Subgroups with low seroconversion rates, such as rituximab-treated patients and those with lymphoid neoplasms, might still have some degree of SARS-CoV-2 vaccine protection from an independently induced cellular vaccine response [52][53][54]. Although most anti-RBD antibodies share neutralizing potential [55], without neutralization assays, we cannot definitively confirm the viral neutralization capacity of those who had seroconverted.A relatively small cohort, no control group, and real-world convenience sampling limit the interpretation of our results, and multiple treatment modalities in disease subgroups with few participants inevitably yielded residual confounding on the effect of disease and treatment. In conclusion, the original two-dose regimen of SARS-CoV-2 vaccines produces lower seropositivity rates overall in patients with hematologic disease.Response rates range significantly across different disease subgroups, from severely attenuated in lymphoid neoplasms to almost universal in BCR-ABL1-positive and non-myelofibrosis BCR-ABL1 negative CMPNs.Seroconversion rates after SARS-CoV-2 vaccination and the incidence of breakthrough infection in patients with hematologic disease should be investigated further in an interventional study design for which we stress the need for single-disease groups. ",
"section_name": "Discussion",
"section_num": "4."
}
] |
[
{
"section_content": "The data presented in this study is available upon request from the corresponding author.The data is not publicly available due to privacy restrictions. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Funding: This study was financially supported by fund allocation from the Croatian Cooperative Group for Hematologic Diseases (KroHem). The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of University Hospital Centre Zagreb (02/21-JG, 12 April 2021). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The authors declare no conflict of interest.The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Funding: This study was financially supported by fund allocation from the Croatian Cooperative Group for Hematologic Diseases (KroHem). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of University Hospital Centre Zagreb (02/21-JG, 12 April 2021). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.3390/cancers15184508
|
ZFP64 Promotes Gallbladder Cancer Progression through Recruiting HDAC1 to Activate NOTCH1 Signaling Pathway
|
<jats:p>The lack of meaningful and effective early-stage markers remains the major challenge in the diagnosis of gallbladder cancer (GBC) and a huge barrier to timely treatment. Zinc finger protein 64 (ZFP64), a member of the zinc finger protein family, is considered to be a promising predictor in multiple tumors, but its potential effect in GBC still remains unclear. Here, we identified that ZFP64 was a vital regulatory protein in GBC. We found that ZFP64 expressed higher in GBC gallbladder carcinoma tissues than in normal tissues and was positively correlated with poor prognosis. Furthermore, ZFP64 was responsible for the migration, invasion, proliferation, anti-apoptosis, and epithelial mesenchymal transition (EMT) of GBC cells in vitro and in vivo. Mechanistically, through Co-IP assay, we confirmed that ZFP64 recruits HDAC1 localized to the promoter region of NUMB for deacetylation and therefore inhibits NUMB expression. The downregulation of NUMB enhanced the activation of the Notch1 signaling pathway, which is indispensable for the GBC-promotion effect of ZFP64 on GBC. In conclusion, ZFP64 regulated GBC progression and metastasis through upregulating the Notch1 signaling pathway, and thus ZFP64 is expected to become a new focus for a GBC prognostic marker and targeted therapy.</jats:p>
|
[
{
"section_content": "Gallbladder cancer (GBC) is one of the six major gastrointestinal malignancies and is a common solid tumor in malignant biliary tract cancer (BTC) [1].In recent years, the incidence of GBC has continuously increased in several countries, particularly in Southeast Asia [2].Due to the lack of characteristic early symptoms and tumor markers, GBC patients are often diagnosed at an advanced stage, with a poor prognosis and high mortality rate [3].The pathogenesis of GBC is extremely complex, and most GBC patients also have concomitant gallbladder inflammation and gallstones, leading to the uncontrolled and rapid growth of GBC under this multi-disease setting [2].Furthermore, common treatments such as surgical resection and chemotherapy are ineffective in the management of advanced GBC [4].As a result, it is of great therapeutic importance to discover novel targeted therapies for GBC and to prolong the survival time of GBC patients. Transcriptional dysregulation is a crucial feature of tumor cells and transcription factors have a direct impact on this process [5].Zinc finger proteins, as the largest family of transcription factors, have been shown to regulate the development of multiple cancers, including gallbladder cancer [6,7].Zinc finger protein 64 (ZFP64) is a member of the zinc-finger type C2H2 transcription factors and, like other members of this family, the predominant function of ZFP64 is thought to regulate transcription [8].Early studies identified ZFP64 as a coactivator of Notch1.ZFP64 interacts with the Notch intracellular domain (NICD) and participates in the regulation of Notch1 signaling [9].In most instances, ZFP64 functions as a tumorigenesis promoter.ZFP64 was significantly upregulated in various types of human cancers, including gastric cancer (GC), hepatocellular carcinoma (HCC), and oesophageal cancer [10,11].As a transcription factor, ZFP64 also activates the promoter of the mixed spectrum leukaemia (MLL) gene, thereby maintaining this oncogene at a high expression level [12].In addition, by binding to and increasing the expression of promoters such as Gal-1, PD-1, and CTLA-4, ZFP64 enables cancer cells to acquire stemness and resist immunosuppression; ZFP64 possesses the capability to affect tumor microenvironment and renders tumor cells resistant to immunosuppression [10,13].These findings are an indication that the targeting of ZFP64 may be a potential cancer therapy. Notch is a family of receptors that is widely distributed and highly conserved in multicellular organisms.In mammals, the Notch family consists of four receptors, Notch1-4, which can influence organ development and repair organismal damage by regulating cell proliferation and differentiation [14].Notch1 has complex regulatory functions in cancer.On the one hand, Notch1 has been reported to promote cancer cell migration and invasion and inhibit apoptosis in many tumors, including tongue cancer [15], pancreatic cancer [16], and breast cancer [17], while mechanistic studies in bladder cancer have revealed that Notch1 also serves as an oncogene [18].The Notch1 signaling pathway can be suppressed by its negative regulator NUMB through several mechanisms, such as Notch1 ubiquitination, Notch1 endocytosis, and degradation of the NICD [19,20].Considering the possible roles of Notch1 in cancer, NUMB is promised to exhibit the inhibitory potential for tumor progression and has been clarified in lung cancer and pancreatic cancer.Another study found that high Notch1 expression was closely related to poor prognosis in GBC patients [21], but the exact mechanism of Notch1's regulatory role in GBC has not been fully illustrated. Here, through in vivo and in vitro assays, we confirmed that ZFP64 was an unfavorable prognostic factor in GBC and that ZFP64 promoted GBC development by supporting malignant biological behaviors such as GBC cell proliferation, anti-apoptosis, migration, invasion, and epithelial mesenchymal transition (EMT).Furthermore, we reported the Notch1 signaling pathway as a downstream mechanism of ZFP64 and further confirmed that ZFP64 activated the Notch1 signaling pathway by recruiting HDAC1 to the NUMB promoter region and modifying H4K77 and H3K18 through deacetylation and delactylation, respectively.Our findings provided insights into the concrete mechanisms of GBC and suggested that targeting ZFP64 promised to be an efficient therapeutic option for GBC. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Human GBC tissues and adjacent normal tissues were acquired from the Department of Biliary Surgery of the Western China Hospital of Sichuan university with informed consent of the patients and approval of the institutional research ethics committee.Fresh tissues were stored at -80 • C for subsequent research or embedded with paraffin.Corresponding clinicopathological features including age, sex, liver function index, tumor marker, and lymph node metastasis were collected by clinical follow-up.None of the patients received radiotherapy, chemotherapy, and any other adjuvant treatment before surgery.GBC-SD and NOZ cell line were purchased from Shanghai Key Laboratory of Biliary Disease Research and cultured in Dulbecco's Modified Eagle's medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) and 100 unit/mL penicillin and 100 µg/mL streptomycin. ",
"section_name": "Human Specimens and Cell Culture",
"section_num": "2.1."
},
{
"section_content": "Lentivirus for overexpression and knockdown of ZFP64 was purchased from Shanghai Genechem Co., Ltd.(Shanghai, China) GBC-SD or NOZ cells were implanted into the six-well plate, and when the cells grew to 70% full, lentivirus was added and infected for 24 h.Tumor cells with ZFP64 overexpression or knockdown were subsequently screened using complete culture medium supplemented with 10 µg/mL puromycin. ",
"section_name": "Construction of Stable Cell Lines",
"section_num": "2.2."
},
{
"section_content": "Total RNAs of cells and tissues were extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and then reversely transcribed with SuperScript TMII reverse transcriptase (Vazyme, Nanjing, China).Quantitative real-time PCR was performed with SYBR Green qPCR Master Mix (Vazyme) on Bio-Rad CFX96TM (Hercules, CA, USA) using the 2 -∆∆Ct method. Primer pairs: ",
"section_name": "Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR)",
"section_num": "2.3."
},
{
"section_content": "Tissues or cells were splatted on ice for 40 min with RIPA supplemented with 1% protease inhibitor and 1% phosphatase inhibitor, then the protein was quantified using a BCA kit (Epizyme, Shanghai, China).After denaturation, the proteins with different weights were separated via 10% SDS-PAGE and then transferred onto a PVDF membrane (Merck, Darmstadt, Germany).The membrane was blocked with 5% skim milk dissolved in TBST at RT for 1 h, incubated in specific primary antibody solution at 4 • C overnight, and then incubated in corresponding second antibody solution at RT for 1 h.The results were detected using the enhanced chemiluminescence (Epizyme, Shanghai, China).The details of all primary antibodies are listed in Table S3. ",
"section_name": "Western Blot",
"section_num": "2.4."
},
{
"section_content": "The cell slipper was placed into a 24-well plate.GBC-SD and NOZ cells stably overexpressing or knocking down ZFP64 were seeded into a 24-well plate and incubated overnight.Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% TritonX-100.Next, cells were incubated in NICD primary antibody at 4 • C overnight and then incubated in second antibody conjugated with FITC.The nucleus was stained with DAPI solution and the fluorescence intensity of each group of cells was digitized using Image J (v2021.8.0, National Institutes of Health, Bethesda, MD, USA). ",
"section_name": "Immunofluorescence Staining",
"section_num": "2.5."
},
{
"section_content": "3 × 10 4 or 5 × 10 4 cells were resuspended in 200 µL serum-free DMEM and then seeded into the transwell upper chamber with or without Matrigel (Corning, New York, NY, USA).The transwell lower chamber was filled with 600 µL medium with 10% FBS and incubated for 24 h.Cells on the inside of chamber were gently removed with cotton swabs, and cells on the outside of chamber were fixed with 4% paraformaldehyde and then stained using 1% crystal violet.Five random fields were photographed under the microscope and the average number of cells in the five fields was calculated using Image J. ",
"section_name": "Cell Migration and Invasion Assay",
"section_num": "2.6."
},
{
"section_content": "The cell proliferation ability was examined using a EdU kit (KeyGEN BioTECH, Nanjing, China) according to the instructions.GBC-SD and NOZ cells were seeded into a 96-well plate and cultured to a suitable density.An amount of 10 µM EdU solution was added into cells and then the cells were incubated for 2 h.Next, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% TritonX-100.Cells labeled with EdU developed color by the addition of a Click-iT reaction mixture.The nuclei were stained with 1 × Hoechst33342 solution for 10 min.Five fields were randomly photographed under an inverted fluorescence microscope and the proportion of EdU-positive cells to total cells was calculated using Image J. ",
"section_name": "Cell Proliferation Assay (EdU)",
"section_num": "2.7."
},
{
"section_content": "ChIP assay was performed used a ChIP kit (Bersin BioTM, Guangzhou, China).Following the instructions, the cells were crosslinked with 1% formaldehyde and then DNA were broken into 100-500 bp lengths using non-contact ultrasound, followed by the addition of anti-ZFP64, anti-H3K18lac, or anti-H4K77ac antibodies and incubated at 4 • C for 12 to 16 h.An amount of 20 µL protein A/G-beads were added and incubated at room temperature for 30 min, and then the DNA fragments were collected using elution buffers.Three primers were designed according to the binding sites (BS) of ZFP64 in the NUMB promoter region predicted by JASPAR (http://jaspar.genereg.net/,accessed on 22 February 2023).Quantitative PCR was used to detect the target DNA fragment (Table 1).PierceTM Co-IP kit (Thermo SCIENTIFIC, Carlsbad, CA, USA) was used to confirm the interaction between ZFP64 with HDAC1.Briefly, ZFP64 or HDAC1 antibody were crosslinked with magnetic beads.Tumor cells were lysed with cell lysate, then centrifuged at 12,000× g for 5 min to remove the precipitation and finally total protein of the cells was obtained.The total protein was incubated with magnetic bead-conjugated antibodies at RT for 1 h.Antigens coupled to magnetic beads were eluted and analyzed using mass spectrometry and Western blot. ",
"section_name": "Chromatin Immunoprecipitation (ChIP)-qPCR Assay",
"section_num": "2.8."
},
{
"section_content": "Subcutaneous xenograft assay had required approval of the Animal Ethics Committees of Western China Hospital of Sichuan university.Five-week-old BALB/C nude male mice were purchased from Beijing HFK Bioscience Co., Ltd.(Beijing, China) and kept in a sterile, pathogen-free environment.Then, 2 × 10 6 GBC-SD or NOZ cells in the logarithmic growth phase were injected into the right underarm of the mice.Each group consisted of 6 mice.The mice were sacrificed, and the tumors were removed and then weighed and measured after 20 or 28 days. ",
"section_name": "Mouse Subcutaneous Xenograft Model",
"section_num": "2.10."
},
{
"section_content": "All statistics in this study were analyzed using unpaired Student's t test, one-way ANOVA, two-way ANOVA, or Kaplan-Meier survival analysis.p values less than 0.05 were considered statistically significant.All the statistics in this paper were conducted by GraphPad Prism 8.0. ",
"section_name": "Statistical Analysis",
"section_num": "2.11."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "As mentioned before, ZFP64 has been implicated in promoting multiple cancers.To clarify the possible role of ZFP64 in GBC, we evaluated and compared the expression difference of ZFP64 between GBC tissues and adjacent normal tissues from 50 GBC patients.Indeed, the RT-qPCR results showed that ZFP64 mRNA levels were evidently much higher in gallbladder carcinoma tissues than in adjacent normal tissues from GBC patients (Figure 1A,C).In 50 gallbladder carcinoma tissues, the mRNA levels of ZFP64 in metastatic gallbladder carcinoma tissues were higher than that in non-metastatic gallbladder carcinoma tissues (Figure 1B).Similarly, Western blot analysis showed the analogous results in protein levels: the expression of ZFP64 was significantly higher in gallbladder carcinoma tissues than in adjacent normal tissues in all 10 GBC patient samples (Figure 1D).Overall, ZFP64 is highly expressed in GBC tissues, especially in metastatic GBC tissues.To further investigate the relationship between the clinicopathological features of GBC patients and ZFP64 expression, we divided 50 GBC patients with detailed clinicopathological information into a high ZFP64 expression group (n = 25) and a low ZFP64 expression group (n = 25), and then analyzed their pathological features.The analysis revealed that patients with high expression of ZFP64 had higher CA19-9 levels and higher rates of lymph node metastasis, liver invasion, nerve invasion, and lower tumor differentiation (Table 2).These aggressive characteristics may directly contribute to the poor prognosis in these GBC patients.Subsequently, we went on to look at whether ZFP64 had an impact on patient survival.We found that patients with high ZFP64 expression had shorter overall survival (OS) and recurrence-free survival (RFS) compared with the low ZFP64 expression group (Tables S1 andS2; Figure 1E,G).In both univariate and multivariate Cox proportional hazards regression models, the expression level of ZFP64 was an independent prognostic factor for OS and RFS (Tables S1 andS2, Figure 1F,H).Taken together, these results suggest that ZFP64 is a potential predictive biomarker for GBC and is strongly associated with GBC prognosis and metastasis. ",
"section_name": "ZFP64 Is Overexpressed in GBC Patients and Associated with Poor Prognosis",
"section_num": "3.1."
},
{
"section_content": "To further elucidate the specific biological functions of ZFP64 in GBC progression, we generated GBC-SD and NOZ cell lines with stable overexpression or the knockdown of ZFP64 via lentivirus-mediated infection.The RT-qPCR and Western blot results demonstrated that ZFP64 was successfully overexpressed or knocked down at both the mRNA and protein level in both cell lines (Figure S1).The 5-ethynyl-20-deoxyuridine (EdU) assays demonstrated that the overexpression of ZFP64 significantly enhanced GBC cells proliferation and ZFP64 knockdown leading to opposing results (Figure 2A,B).Simultaneously, a transwell assay showed that the migration and invasion ability of tumor cells was significantly enhanced in the overexpressed ZFP64 group, while it was significantly decreased after ZFP64 was knocked down (Figure 2C,D).Western blot also showed a dramatic increase in BCL-2, cyclinD1, N-cadherin, and vimentin expression with ZFP64 overexpression, and a downregulation in BAX and E-cadherin, while ZFP64 knockdown exhibited opposing results (Figure 2E).All the results above meant that ZFP64 had a promotive effect on GBC cell anti-apoptosis, proliferation, migration, invasion, and EMT, thereby contributing to the procession of GBC tumors.As our results above have revealed that ZFP64 promotes GBC cell progression in vitro, it is necessary to further verify whether ZFP64 has the same promotive effect on GBC cell in vivo through subcutaneous tumorigenesis assays.GBC-SD and NOZ cells stably overexpressing or knocking down ZFP64 were injected into BALB/C nude male mice.In terms of tumor growth rate and final tumor weight, the ZFP64 overexpression group showed the opposite results to the ZFP64 knockdown group: in the ZFP64 overexpression group, the subcutaneous tumor growth rate was faster than that in the control group and the tumors were heavier (Figure 2F), whereas the ZFP64 knockdown group exhibited an As our results above have revealed that ZFP64 promotes GBC cell progression in vitro, it is necessary to further verify whether ZFP64 has the same promotive effect on GBC cell in vivo through subcutaneous tumorigenesis assays.GBC-SD and NOZ cells stably overexpressing or knocking down ZFP64 were injected into BALB/C nude male mice. In terms of tumor growth rate and final tumor weight, the ZFP64 overexpression group showed the opposite results to the ZFP64 knockdown group: in the ZFP64 overexpression group, the subcutaneous tumor growth rate was faster than that in the control group and the tumors were heavier (Figure 2F), whereas the ZFP64 knockdown group exhibited an obviously slower tumor growth rate and smaller tumors (Figure 2G).Therefore, ZFP64 could promote gallbladder cancer growth in vivo. ",
"section_name": "ZFP64 Promotes Gallbladder Cancer Progression In Vitro and In Vivo",
"section_num": "3.2."
},
{
"section_content": "HEY1 and HES1 are endogenous Notch1 target genes and previous studies have shown that ZFP64 is a coactivator of NICD that can locate on the promoter regions of HEY1 and HES1 and then facilitate transcription [9].In addition, in GBC patients, the Notch1 signaling pathway has been implicated in poor prognosis [21].Therefore, ZFP64 may be involved in the regulation of GBC development through the Notch1 signaling pathway.Western blot assays showed that the increased expression of Notch1, NICD, HES1, and HEY1 is due to the overexpression of ZFP64 (Figure 3A), whereas the expression of these essential molecules diminished when ZFP64 was knocked down (Figure 3B).NICD is a product of Notch hydrolysis by γ-secretase.The entry of NICD into the nucleus and the regulation of downstream gene transcription are two key events in the activation of Notch1 signaling pathways [22].Therefore, we performed immunofluorescence to detect the protein level of NICD in the nucleus.The results showed that the level of NICD in the nucleus was significantly increased in the ZFP64 overexpression group (Figure 3C), while it decreased in the ZFP64 knockdown group (Figure 3D).Thus, ZFP64 is capable of activating the Notch1 signaling pathway.obviously slower tumor growth rate and smaller tumors (Figure 2G).Therefore, ZFP64 could promote gallbladder cancer growth in vivo. ",
"section_name": "ZFP64 Activates Notch1 Signaling Pathways",
"section_num": "3.3."
},
{
"section_content": "HEY1 and HES1 are endogenous Notch1 target genes and previous studies have shown that ZFP64 is a coactivator of NICD that can locate on the promoter regions of HEY1 and HES1 and then facilitate transcription [9].In addition, in GBC patients, the Notch1 signaling pathway has been implicated in poor prognosis [21].Therefore, ZFP64 may be involved in the regulation of GBC development through the Notch1 signaling pathway.Western blot assays showed that the increased expression of Notch1, NICD, HES1, and HEY1 is due to the overexpression of ZFP64 (Figure 3A), whereas the expression of these essential molecules diminished when ZFP64 was knocked down (Figure 3B).NICD is a product of Notch hydrolysis by γ-secretase.The entry of NICD into the nucleus and the regulation of downstream gene transcription are two key events in the activation of Notch1 signaling pathways [22].Therefore, we performed immunofluorescence to detect the protein level of NICD in the nucleus.The results showed that the level of NICD in the nucleus was significantly increased in the ZFP64 overexpression group (Figure 3C), while it decreased in the ZFP64 knockdown group (Figure 3D).Thus, ZFP64 is capable of activating the Notch1 signaling pathway. ",
"section_name": "ZFP64 Activates Notch1 Signaling Pathways",
"section_num": "3.3."
},
{
"section_content": "To further validate that ZFP64-promoted GBC procession through activating the Notch1 signaling pathway, we treated normal GBC cells and ZFP64-overexpressing GBC cells with or without the Notch1 signaling pathway inhibitor DAPT.We then used Western blot and immunofluorescence to detect the expression of key proteins in the Notch1 pathway.The results showed that, compared with the normal GBC cells, the protein levels of Notch1, NICD, HES1, and HEY1 in the ZFP64-overexpressing GBC cells were markedly increased, while the corresponding protein levels of the DAPT-treated GBC cells were substantially decreased.The protein levels of the combined treatment group of ZFP64 overexpression and DAPT were between those in the separate ZFP64 overexpression or DAPT groups (Figure 4A,B).Meanwhile, Western blot analysis also showed that with ZFP64 overexpressing, the expression levels of BCL-2, cyclinD1, N-cadherin, and vimentin were significantly elevated, while BAX and E-cadherin were downregulated.Within the treatment of DAPT, the expression levels of the above proteins were opposite to that of the ZFP64-overexpressing GBC cells.In addition, combined treatment with ZFP64 overexpression and DAPT led to the middle expression level of these critical proteins between the ZFP64-overexpressing GBC cells and the DAPT-treated GBC cells (Figure 4C).We also conducted EdU assays to examine the proportion of proliferating tumor cells in each group.The proportion of EdU-positive cells in the ZFP64-overexpressing GBC cells appeared to be higher than that in normal GBC cells, while the DAPT group had fewer proliferating cells.Consistent with the protein expression results, the EdU result of the DAPT and ZFP64 combined treatment group was between the two (Figure 4D).Furthermore, transwell assays demonstrated that the overexpression of ZFP64 significantly promoted the proliferation and invasion of GBC cells, while the use of DAPT obviously blocked GBC cell proliferation and invasion.The combined treatment of ZFP64 overexpression and DAPT demonstrated that DAPT obviously weakened the promotive effect of ZFP64 on GBC cells (Figure 4E).In summary, ZFP64 promotes the progression of gallbladder cancer through activating the Notch1 signaling pathway. ",
"section_name": "ZFP64 Promotes Gallbladder Cancer Proliferation, Migration, and Invasion In Vitro via Activating Notch1 Signaling Pathway",
"section_num": "3.4."
},
{
"section_content": "To further investigate how ZFP64 activated the Notch1 signaling pathway, we speculated about the possible promoter region of the gene to which ZFP64 might bind and affect via JASPAR (http://jaspar.genereg.net/,accessed on 22 February 2023).As expected, we found that the promoter region of NUMB, a known inhibitor of the Notch1 signaling pathway, had the modification to which ZFP64 could bond (Figure 5A).Then, we designed three pairs of primers of the NUMB promoter region for further qPCR analysis according to the sites to which ZFP64 might bind.ChIP-qPCR assay further confirmed that ZFP64 directly interacted with the NUMB promoter region (Figure 5B,C).Thus, ZFP64 might influence the transcription of NUMB to subsequently affect the Notch1 signaling pathway.RT-qPCR and Western blot assays showed that the overexpression of ZFP64 down-regulated the mRNA and protein level of NUMB, whereas the knockdown of ZFP64 elevated NUMB expression (Figure 5D-F).ZFP64 acted as an inhibitory transcription factor to control NUMB expression. Moreover, the mechanism of how ZFP64 inhibited NUMB expression was still unclear, so we combined co-immunoprecipitation (Co-IP) with mass spectrometry analysis to define the proteins that interacted with ZFP64 and might play the crucial role in the ZFP64 inhibition of NUMB expression.The 20 proteins with the highest abundance among the pull-down proteins were listed in Table S4.The results showed that, among these high abundance proteins, HDAC1 was identified as one of the potential ZFP64-interacting proteins because it has been widely recognized that HDAC1 is a key component of the histone deacetylase complex and is able to regulate gene expression using deacetylating histone [23] and delactylation [24].The direct interaction of ZFP64 with HDAC1 probably meant that ZFP64 recruited HDAC1 to the promoter region of NUMB, where HDAC1 catalyzed deacetylation and delactylation to prevent NUMB from proper transcription and expression.Co-IP and Western blot assay further confirmed the interaction between ZFP64 and HDAC1(Figure 5G).Furthermore, we conducted ChIP assays and evaluated the influence of ZFP64 expression on the level of histone acetylation and the lactylation of the NUMB promoter region.Consistent with our speculation, the overexpression of ZFP64 apparently impaired the histone lactylation level at the H3K18 site and the histone acetylation level at the H4K77 site, while the results performed on ZFP64 knockdown GBC cells exhibited the reverse results (Figure 5H-K).These data indicated that the repressive effect of ZFP64 on NUMB was facilitated by the deacetylation and delactylation of the NUMB promoter region, which was due to the recruitment of ZFP64 to HDAC1. In order to further elucidate the effect of the ZFP64-HDAC1 axis on the NUMB-Notch1 signaling pathway, HDAC1 was overexpressed in gallbladder cancer cell lines with ZFP64 knockdown.Western blot results showed that the overexpression of HDAC1 suppressed NUMB expression only in the control group.The inhibitory effect of HDAC1 on NUMB expression disappeared when ZFP64 was knocked down (Figure 6A), indicating that this inhibitory effect was dependent on the expression level of ZFP64.On the other hand, Western blot assays also demonstrated that, in GBC cells simultaneously overexpressing ZFP64 and knocking down HDAC1, the prohibitive effect of ZFP64 on NUMB and the promotive effect on the Notch1 signaling pathway were significantly reversed by HDAC1 knockdown (Figure 6B,C).The immunofluorescence of NICD further drew the same conclusion (Figure 6D). ",
"section_name": "ZFP64 Activates the Notch1 Signaling Pathway by Recruiting HDAC1 to Inhibit NUMB Expression",
"section_num": "3.5."
},
{
"section_content": "The lack of early markers and the poor treatment outcomes in the intermediate and advanced stage settings remain the most considerable obstacles to cure in GBC.Our study ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The lack of early markers and the poor treatment outcomes in the intermediate and advanced stage settings remain the most considerable obstacles to cure in GBC.Our study focused on the vital role of ZFP64 in GBC for the very first time.Through the RT-qPCR and the western blot assays, we demonstrated that ZFP64 expressed evidently higher in gallbladder carcinoma tissues from GBC patients than in normal tissues, and that GBC patients with high ZFP64 expression appeared to have an extremely worse prognosis.The expression level of ZFP64 was directly correlated with the prognosis of GBC patients and might positively serve as a reference indicator to aid in the prognosis of GBC and a biomarker for the early diagnosis of clinical GBC.In our exploration of more detailed mechanisms, we found that the overexpression of ZFP64 remarkably enhanced the proliferation, anti-apoptosis, invasion, migration, and EMT in both GBC-SD and NOZ cells in vitro and in vivo, which in turn promoted the metastasis and malignant development of GBC tumors.In addition, GBC can invade adjacent tissues through liver metastases, lymph node metastases, and vascular metastases, which has become another severe difficulty in the treatment of GBC [25,26].Patients with high expression level of ZFP64 showed higher rates of lymph node metastasis, liver invasion, nerve invasion, and lower tumor differentiation, which might also be related to the facilitative effect of ZFP64 on the migration and invasion of GBC cells. Histone acetylation, one of the most common histone epigenetic modifications, is in connection with multiple physiological and pathological diseases, in particular cancer.Two prime proteins engage in mediating this histone modification: histone acetyltransferases (HATs) add acetyl groups to histone lysine residues for acetylation modification, while histone deacetylases (HDACs) remove acetyl groups from histone lysines [27].Belonging to the class I Rpd3-like family of HDACs, HDAC1 widely distributes in cells and is Zn 2+dependent for its catalytic function.HDAC1 is considered to be primary to mediate the regulation of epigenetic inheritance [28].As the research progresses, HDAC1 has also been demonstrated to be apparently upregulated in a variety of cancers and the loss of HDAC1 interdicts tumor progression and induces apoptosis [29,30].In addition, HDAC1 strengthens the repressive effect of some transcription factors on oncoprotein promoters by deacetylating these transcription factors.For example, in breast cancer, HDAC1 deacetylated SREBP1 and inhibited E-calmodulin transcription, thereby suppressing the EMT [31].In hepatoblastoma (HBL), HDAC1 delivered to the promoter of the p21 gene by another transcription factor, sp5, and activated p21 expression, thereby promoting the proliferation and metastasis of HBL cells [32].In addition, inhibitors of HDAC1 have been developed and placed in more well-established pre-clinical and clinical systems to treat cancers, including colorectal cancer, hepatocellular carcinoma, and neuroblastoma [33,34].In GBC, HDAC1 was first shown to interact with the transcription factor TCF-12, driving GBC tumor invasion and leading to poor prognosis [35].Combined treatment with the HDAC inhibitor SAHA and the BRD4 inhibitor JQ1 could resist GBC cell proliferation and viability and induced apoptosis, thereby reducing the tumorigenic capacity of GBC cells in vivo [36]. Histone lactylation is another epigenetic modification present in histones, and like histone acetylation modifications, it imposes gene transcription with analogous molecular mechanisms [37].As the Warburg effect describes, tumor cells produce large amounts of lactic acid through aerobic glycolysis [38].The degree of lactylation is dependent on the production of endogenous lactic acid, and the accumulation of endogenous lactic acid provides the indispensable substrate for lactylation [37,39].Recent studies have expounded the role of histone lactylation in some cancers.In ocular melanoma, a lactylation increase in H3K18 sites at the YTHDF2 promoter region improved YTHDF2 expression and sustained ocular melanoma malignization [40].In addition, two lactylation sites, H3K9 lac and H3K56 lac, were also known to contribute to HCC development.Meanwhile, the demethylzeylasteral (DML) has been shown to be equipped to control glycolysis and gluconeogenesireduce, therefore reducing lactate levels in hepatocellular carcinoma stem cells (LCSC) and preventing LCSC tumorigenesis [41].In addition to deacetylating histones, HDAC1 also acts as a lysine delactylase to delactylate histone lysines [24].However, the function of HDAC1 as a delactylase in tumors still remains unknown.Here, we proved that HDAC1 interacted with ZFP64 directly.In GBC cell lines overexpressing ZFP64, mass spectrometry identification confirmed lessened levels of H3K18 lactylation and H4K77 acetylation in the NUMB promoter region.Further Co-IP confirmed that HDAC1 principally mediated this process: ZFP64 recruited HDAC1 to locate on the promoter region of NUMB and modified it through deacetylation and delactylation.Subsequently, this modification suppressed NUMB expression, thereby activating the Notch1 pathway and upregulating the expressions of Notch1 pathway-related proteins HEY1, HES1, and NICD.The Notch1 signaling pathway inhibitor DAPT abrogated the promotive effects of ZFP64 on the proliferation and metastasis of GBC cells, which further demonstrated that ZFP64 regulated GBC development through the Notch1 signaling pathway. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Motivated by the current poor therapeutic efficacy of GBC, our study aims at developing new methods for GBC treatment.In conclusion, we provide evidence to support that ZFP64 is a provital regulatory protein in GBC progression.Furthermore, we also demonstrated that ZFP64-HDAC1-NUMB-Notch1 is an essential oncogenic axis in GBC that promotes multiple malignant biological behaviors of GBC cells.We highlight that targeting ZFP64 may be an effective avenue for future GBC therapy. Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of West China Hospital, Sichuan University (2023-1269).The animal study protocol was approved by the Animal Ethics Committee of West China Hospital, Sichuan University (20230807007). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "We sincerely appreciate all the staffs from the Core Facilities, West China Hospital, Sichuan University, including Jingyao Zhang, Cong Li, Huifang Li, Jian yang, Yan Wang, and Liwen Qin for their technical guidance to this study. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYJC21046, funder: Fuyu Li); 1.3.5 project for disciplines of excellence-Clinical Research Incubation Project, West China Hospital, Sichuan University (2021HXFH001, funder: Fuyu Li); Natural Science Foundation of Sichuan Province (2022NSFSC0806, funder: Fuyu Li); National Natural Science Foundation of China for Young Scientists Fund (82203650, funder: Wenjie Ma; 82203782: funder Haijie Hu), Sichuan Science and Technology Program (2021YJ0132, funder: Wenjie Ma; 2021YFS0100, funder: Haijie Hu); the fellowship of China Postdoctoral Science Foundation (2021M692277, funder: Haijie Hu); Sichuan University-Zigong School-local Cooperation project (2021CDZG-23, funder: Fuyu Li); Sichuan University-Sui Lin School-local Cooperation project (2022CDSN-18, funder: Fuyu Li); Science and Technology project of the Health planning committee of Sichuan (21PJ046, funder: Fuyu Li); Post-Doctor Research Project, West China Hospital, Sichuan University (2020HXBH127, funder: Haijie Hu). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Data Availability Statement: All data are contained within the article or Supplementary Materials. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers15184508/s1, Figure S1.GBC-SD and NOZ cells were generated to stably overexpress or knockdown ZFP64 via lentivirus transfection for 24 h, and then 10 µg/mL of puromycin was added to screen successfully infected cells; Figure S2.Uncropped Western Blot images for Figure 1D; Figure S3.Uncropped Western Blot images for Figure 2E; S1.Prognostic factors for overall survival by the univariate and multivariate cox proportional hazards regression model; Table S2.Prognostic factors for disease-free survival by the univariate and multivariate cox proportional hazards regression model; Table S3.Summary of primary antibody information used in this article; Table S4 ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
10.4172/2155-9864.1000220
|
BAALC Gene Expression in Adult B-precursor Acute Lymphoblastic Leukemia: Impact on Prognosis
|
en el presente estudio es analizar la expresión de BAALC y evaluar su impacto pronóstico en el resultado clínico de la LLA de precursores B en adultos. Métodos Pacientes Doscientos recién diagnosticados como LLA de precursores B adultos se inscribieron entre 2005 y 2014 en el Centro de Oncología Mansoura, Mansoura, Egipto. Las muestras se seleccionaron de forma consecutiva
|
[
{
"section_content": "Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease with distinct manifestations and prognostic and therapeutic implications [1].ALL in adults is a rare disease.The results of therapy remain unsatisfactory, and progress has been relatively slow [2].B-cell ALL (B-ALL) is a clonal malignant disease originated in a single cell and characterized by the accumulation of blast cells that are phenotypically reminiscent of normal stages of B-cell differentiation [3]. Outcome of adult B-precursor ALL has considerably improved because of identification of clinical and genetic risk factors stratifying patients to different treatment groups [4].Commonly accepted risk factors in B-precursor ALL include age, performance status, white blood cell (WBC) count, lactate dehydrogenase concentration, the immunophenotype, response to induction therapy, level of minimal residual disease, cytogenetics and genetic aberrations [5,6].Patients lacking clinical and molecular risk factors are considered standard risk (SR).Outcome for SR patients is still unsatisfactory [5,7] indicating the clinical and biologic heterogeneity of these patients.Therefore, the identification of novel predictive molecular markers in adult B-precursor ALL may improve treatment stratification of this subgroup. The Brain And Acute Leukemia, Cytoplasmic (BAALC) gene is highly expressed in normal uncommitted progenitor cells and downregulated with the onset of differentiation [8].An elevated expression of the BAALC gene was originally discovered in a gene expression profiling study of acute myeloid leukemia (AML) with trisomy 8, but was later also found in other AML and in ALL [9,10].Over expression of BAALC predicts an inferior outcome in AML and ALL patients [9][10][11]. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Molecular analyses",
"section_num": null
},
{
"section_content": "To assess molecular responses, total RNA was extracted from PB or BM blood cells.BCR-ABL and internal control transcript levels were quantified using real-time PCR analysis (TaqMan) on an ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA).Specific PCR products were amplified and detected using dual-fluorescent non-extendable probes labeled with 6-carboxyfluorescein (FAM), reporter and 6-carboxytetramethylrhodamine (TAMRA), quencher at 5`-end and 3`-end, respectively.The relative mRNA expression of BCR-ABL transcript was calculated using the comparative cycle threshold (Ct) method [12]. ",
"section_name": "Real-time quantitative polymerase chain reaction (RTQ-PCR) for BCR-ABL",
"section_num": null
},
{
"section_content": "From each patient and healthy subject 3 ml of PB or BM samples were collected in sterile EDTA vacutainers.BAALC mRNA expression was normalized to the simultaneously analyzed glucose phosphate isomerase (GPI) gene.The relative BAALC expression was determined using the comparative cycle threshold (CT) method.Glucose phosphate isomerase (GPI) and BAALC were coamplified in the same tube using 1 μL cDNA, 1× master mix (IQ Mix; BioRad, Munich, Germany), GPI probe (5'-HEX-TTCAGCTTGACCCTCAACACCAAC-TAM-RA-3') with GPI forward (5'-TCTTCGATGCCAACAAGGAC-3') and reverse (5'-GCATCACGTCCTCCGTCAC-3') primers, and BAALC probe (5'-FAMCTCTTTTAGCCTCTGTGGTCTGAAGGCCAT-TAMRA-3') with BAALC forward (5'-GCCCTCTGACCCAGAAA-CAG-3') and reverse (5'-CTTTTGCAGGCATTCTCTTAGCA-3') primers.Reactions were performed using real-time PCR 7000 sequence detection system (Applied Biosystems, Foster City, USA).Positive and negative controls were included in all assays. ",
"section_name": "Real-time quantitative PCR analysis for BAALC",
"section_num": null
},
{
"section_content": "The statistical analysis of data was done by using excel program and SPSS (statistical package for social science) program (SPSS, Inc, Chicago, IL) version 16.Qualitative data were presented as frequency and percentage.Chi square test was used to compare groups.Quantitative data were presented as mean and standard deviation.For comparison between two groups; student t-test and Mann-whitney test (for non-parametric data) were used.Kaplan-Meier test was used for survival analysis and the statistical significance of differences among curves was determined by Log-Rank test.Prediction of survival was done using multivariate analysis.For gene expression quantification, we used the comparative Ct method.First, gene expression levels for each sample were normalized to the expression level of the housekeeping gene encoding GPI within a given sample (∆Ct).Results were evaluated by using 2-∆∆CT method as relative gene expression values.N.B: p is significant if ≤ 0.05 at confidence interval 95%. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "ALL Patients were divided into 2 BAALC expression groups; high BAALC expression (above median) and low BAALC expression (below median).The clinicohematological features differed in patients with high BAALC and those with low BAALC mRNA expression levels.Patients expressing high BAALC gene were significantly older (P =0.037), had higher WBC (P =0.019), LDH concentration (p=0.007)patients who had sufficient material available.They were 106 males (53%) and 94 females (47%) with mean age 42.32 ± 10.675 years.Patients were diagnosed according to standard diagnostic methods including clinical, cytomorphological, cytochemical, immunophenotypic methods.In addition, 20 healthy subjects, of matched age (39 ± 11.202 years) and sex (12 males (60%) and 8 females (40%)); with normal laboratory findings; were selected as a control group.Informed consent was obtained from all patients.Risk groups were assigned as follows: SR (no risk factor), high risk (≥ 1 risk factor), and very high risk (presence of t (9;22)/BCR-ABL). ",
"section_name": "BAALC expression and relationship with clinical, laboratory and molecular features",
"section_num": null
},
{
"section_content": "ALL cases were treated according to our risk adapted chemotherapy protocol: The patients were stratified according to their prognostic factors into standard, high, and very high risk groups.The treatment plan included: Prephase for patients with high WBC and/or organomegaly.Induction phase I: Four drugs: Vincristine, Doxorubicin, L-Asparaginase and prednisone with intrathecal MTX.Patients that attained complete remission (CR) were subjected to cranial irradiation with 24 Gy and intrathecal MTX for four injections.Phase II induction with Cyclophosphamide and Cytarabine.Consolidation phase I: Vincristine, Doxorubicine and prednisone with Triple intrathecal.Phase II consolidation: Cyclophosphamide, Cytarabine and Etoposide with triple intrathecal.Maintenance therapy: two years with 6 mercaptopurine and methotrexate.For patients with high and very high risks who were not planned to stem cell transplantation, one cycle of high dose cytarabine and mitoxantrone (HAM regimen) was added between induction and consolidation.Bone marrow aspirate was done to evaluate response to chemotherapy (status post induction).Cases who died before treatment or who didn't receive treatment due to poor performance status or elderly cases kept on supportive treatment were excluded, as well as patients who received SCT in first CR. ",
"section_name": "Treatment protocol",
"section_num": null
},
{
"section_content": "Complete remission (CR) was assessed after completion of induction chemotherapy.CR was defined as follows: granulocyte count of at least 1.5 × 10 9 /L, platelet count of at least 100 × 10 9 /L, no peripheral blood (PB) blasts, bone marrow (BM) cellularity of at least 20% with maturation of all cell lines and less than 5% blasts, and no extramedullary leukemia.Primary therapy failure (refractory disease) was defined as persistence of PB blasts or at least 25% blasts in BM after induction therapy.Relapse was defined as reappearance of PB blasts, more than 5% blasts in BM, or appearance of extramedullary manifestations after CR was achieved.OS was defined as the time from diagnosis to date of death.For patients achieving CR, DFS was the time from the date of first CR to an event (death in first CR or relapse). ",
"section_name": "Criteria of response and survival definitions",
"section_num": null
},
{
"section_content": "Immunophenotypic analyses were performed by flow cytometry on fresh pretreatment BM and PB samples.A wide panel of monoclonal antibodies (Mo Abs) was used.Lymphoid markers included CD19, CD22 for B Lineage and CD1, CD2, CD3, CD4, CD5 and CD7, for exclusion of T-lineage and other markers used included CD45, HLA-DR, CD10, and CD34.All the monoclonal antibodies were obtained from Coulter Hialeah, FL.A cell-surface antigen was considered positive when at least 20% of cells showed fluorescence intensity greater than the negative control.and CD34 positivity (p=0.011)compared with patients with low BAALC expression group.In addition, higher BAALC expression was associated with the presence of BCR-ABL (P=0.011).There was no significant association between BAALC expression level and sex, hemoglobin concentration, platelet count, peripheral or marrow blasts, or immunophenotypic subgroups of B-precursor ALL.No other association was seen between BAALC expression and clinical features (Table 1). ",
"section_name": "Immunophenotypic analysis",
"section_num": null
},
{
"section_content": "Complete remission rate did not differ significantly between high and low BAALC expression groups (p=0.120),although patients with high BAALC expression showed a marginally significant lower CCR rate than low expression group (30% versus 43% respectively; p=0.056) and a higher incidence of primary resistant disease (34% versus 14% respectively; p=0.001).No influence on the relapse rate (P =0.674) was observed with respect to BAALC expression.There were no significant differences regarding deaths in induction therapy (P =0.203) between the two BAALC expression groups.Total mortality rates during the entire period of the study were higher in high BAALC expression group when compared to low expression group, but did not reach significant level (P =0.063) (Table 2). When applying BAALC expression, age, marrow blasts, immunophenotypes and BCRABL as covariates for prediction of resistant disease, BAALC remained a predictive factor for primary therapy resistance (P = 0.003; OR= 3.133; 95% confidence interval [CI], 1.482-6.623),as well as immunophenotype (common versus pre B-ALL) (P = 0.015; OR= 2.326; 95%CI, 1.431-7.956)(Table 3). Overall survival (OS) was significantly shorter in patients with higher BAALC expression compared with those with lower BAALC expression (34.39 versus 50.11 months; 4-year cumulative OS %, 39.6%, 53.6% respectively; p=0.010).In addition, high BAALC expression group showed significantly shorter DFS than those with low expression (27.93 versus 58.82 months; 51.7% versus 64.9% respectively; p<0.001) (Figure 1).BAALC expression was independently predictive for OS and DFS in multivariate analysis (p=0.039,HR=1.652, 95%CI=1.025-2.663;p=0.002,HR=1.214, 95%CI=1.013-4.368respectively).In addition, BCR-ABL is an independent unfavourable prognostic factor for OS (p<0.001,HR=2.749, 95%CI=1.124-4.940),as well as common B-ALL immunophenotype for DFS (p<0.001,HR=0.254, 95%CI= 0.187-2.872)(Table 4). ",
"section_name": "Outcome in B-precursor ALL patients with respect to BAALC expression",
"section_num": null
},
{
"section_content": "All studied patients were stratified according to BCR-ABL into BCR-ABL-and BCR-ABL+ groups (n =133, 67 respectively).We analyzed the prognostic impact of BAALC expression in these low-and high-risk subgroups of BCR-ABL B-precursor ALL.In the BCR-ABLgroup, no significant differences were obtained regarding CR, CCR, relapse, induction death and total mortality rates.Refractory disease was significantly higher in patients with high BAALC expression versus those with low BAALC expression (p=0.039).In addition, in the BCR-ABL+ group, no significant differences were obtained regarding CR, CCR, relapse and total mortality rates.Resistance to chemotherapy was higher in patients expression high BAALC expression versus those with low BAALC expression group (p=0.003).Death during induction therapy was significantly higher in low versus high BAALC expression groups (p=0.025)(Table 2).Multivariate analysis showed that BAALC gene expression was independent risk factor for resistance to chemotherapy in BCR-ABL-and BCR-ABL+ groups (OR=2.359,95% CI=1.004-5.538,p=0.049;OR= 2.672, 95% CI=1.824-3.326,p=0.014 respectively) (Table 5). OS in BCR-ABL -patients was shorter in high versus low BAALC expression group, although did not reach significant level (4-year OS: 58.5%, 66.2%; 47.63, 60.48 months; p=0.102).Higher BAALC expression was significantly associated with an inferior DFS when compared to low BAALC expression group for BCR-ABL -patients (4year DFS: 69.1%, 51.2%; 60.36, 28.21 months P <0.001).BCR-ABL+ patients showed no significant differences between BAALC expression groups for OS and DFS (p=0.991,0.671 respectively) (Figure 2).In the Cox regression analysis, BAALC expression was an independent adverse factor regarding DFS in BCR-ABL-group (p=0.001,HR=3.774, 95% CI= 1.831-5.448)(Table 6). ",
"section_name": "BAALC expression and outcome according to BCR-ABL in B-precursor ALL patients",
"section_num": null
},
{
"section_content": "Acute lymphoblastic leukemia (ALL) remains one of the most challenging adult malignancies, especially with respect to therapy.Immunophenotyping, cytogenetic-molecular studies [12].However, most of the studies focused on children and therefore a deep molecular characterization of adults is still challenging, especially for those cases lacking high-risk markers (eg, BCR-ABL).In this study, we have evaluated the prognostic significance of BAALC expression in B-precursor ALL. In B-precursor ALL, higher BAALC expression was associated with older age, higher WBC, LDH concentration and BCR-ABL, which are known risk factors in accordance with others [13].However, no significant differences were found between low and high BAALC expression groups regarding clinical presentations, sex, hemoglobin concentration, platelet count, peripheral or marrow blasts, or immunophenotypic subgroups of B-precursor ALL. Patients with low BAALC gene expression continued CR at a rate marginally significantly higher than those with high BAALC gene expression.High BAALC gene expression group confer higher resistance to chemotherapy than those with low BAALC gene expression in agreement with Kohnl [13].Total mortality rate was marginally significantly higher in high versus low BAALC gene expression groups.This may be due to lower CCR rate, higher resistance to chemotherapy, more aggressive disease having more immature cells.In multivariate analysis, high BAALC expression retains its high risk for primary therapy failure, in accordance with other reports [13]. When stratifying all patients according to BCR-ABL, 133 cases were BCR-ABL negative and 67 patients were BCR-ABL+.High BAALC gene expression group had significantly higher resistance to chemotherapy in BCR-ABL and BCR-ABL + groups.In multivariate analysis, high BAALC gene expression is still independent prognostic factor for resistance to chemotherapy in BCR-ABL and BCR-ABL + groups. In addition, higher BAALC expression was independently predictive for DFS and OS all patients and for DFS in BCR-ABL -patients. In previous studies, high BAALC expression was associated with an unfavorable outcome and inferior long-term survival in adult CN-AML and T-ALL [9,14,15].Thus, BAALC characterizes a more aggressive, immature, highly proliferative, and chemoresistant leukemic phenotype. Our data suggest that BAALC may identify patients with an immature, chemoresistant leukemic phenotype associated with an unfavorable outcome and shorter survival of adult B-precursor ALL. BAALC expression could better discriminate patients into various prognostic groups and identify patients who might benefit from doseintensified induction chemotherapy. Stratification of adult B-precursor ALL into 2 distinctive groups of patients with outcome and survival characteristics, might in the future facilitate treatment stratification for adult B-precursor ALL. ",
"section_name": "Discussion",
"section_num": null
}
] |
[] |
10.3857/roj.2021.00815
|
Impact of somatic mutations on clinical and pathologic outcomes in borderline resectable and locally advanced pancreatic cancer treated with neoadjuvant chemotherapy and stereotactic body radiotherapy followed by surgical resection
|
<jats:p>Purpose: The purpose of this study was to determine if somatic mutations are associated with clinical and pathologic outcomes in patients with borderline resectable pancreatic cancer (BRPC) or locally advanced pancreatic cancer (LAPC) who were treated with neoadjuvant chemotherapy and stereotactic body radiotherapy (SBRT). Materials and Methods: Patients treated with neoadjuvant chemotherapy and SBRT followed by surgical resection from August 2016 to January 2019 and who underwent next generation sequencing of their primary tumor were included in the study. Next-generation sequencing was performed either in-house with a Solid Tumor Panel or with FoundationOne CDx. Univariate (UVA) and multivariable analyses (MVA) were performed to determine associations between somatic mutations and pathologic and clinical outcomes. Results: Thirty-five patients were included in the study. Chemotherapy consisted of modified FOLFIRINOX, gemcitabine and nab-paclitaxel, or gemcitabine and capecitabine. Patients were treated with SBRT in 33 Gy in 5 fractions. On UVA and MVA, tumors with KRAS G12V mutation demonstrated better pathologic tumor regression grade (TRG) to neoadjuvant therapy when compared to tumors with other KRAS mutations (odds ratio = 0.087; 95% confidence interval [CI], 0.009–0.860; p = 0.036). On UVA and MVA, mutations in NOTCH1/2 were associated with worse overall survival (hazard ratio [HR] = 4.15; 95% CI, 1.57–10.95; p = 0.004) and progression-free survival (HR = 3.61; 95% CI, 1.41–9.28; p = 0.008). On UVA, only mutations in NOTCH1/2 were associated with inferior distant metastasis-free survival (HR = 3.38; 95% CI, 1.25–9.16; p = 0.017). Conclusion: In BRPC and LAPC, the KRAS G12V mutation was associated with better TRG following chemotherapy and SBRT. Additionally, NOTCH1/2 mutations were associated with worse overall survival, distant metastasis-free survival, and progression-free survival. </jats:p>
|
[
{
"section_content": "",
"section_name": "",
"section_num": ""
}
] |
[
{
"section_content": "",
"section_name": "Supplementary Materials",
"section_num": null
}
] |
10.3390/biomedicines10061229
|
Dysregulation of Small Nucleolar RNAs in B-Cell Malignancies
|
<jats:p>Small nucleolar RNAs (snoRNAs) are responsible for post-transcriptional modification of ribosomal RNAs, transfer RNAs and small nuclear RNAs, and thereby have important regulatory functions in mRNA splicing and protein translation. Several studies have shown that snoRNAs are dysregulated in human cancer and may play a role in cancer initiation and progression. In this review, we focus on the role of snoRNAs in normal and malignant B-cell development. SnoRNA activity appears to be essential for normal B-cell differentiation and dysregulated expression of sno-RNAs is determined in B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell non-Hodgkin’s lymphoma, and plasma cell neoplasms. SnoRNA expression is associated with cytogenetic/molecular subgroups and clinical outcome in patients with B-cell malignancies. Translocations involving snoRNAs have been described as well. Here, we discuss the different aspects of snoRNAs in B-cell malignancies and report on their role in oncogenic transformation, which may be useful for the development of novel diagnostic biomarkers or therapeutic targets.</jats:p>
|
[
{
"section_content": "B-cells are essential for the humoral immune response [1].B-cells mature via strictlyregulated mechanisms in the bone marrow (BM) from stem cells to naive B-cells (antigenindependent) and mainly in lymph nodes from naive B-cells to memory B-cells and plasma cells (antigen-dependent) [1].Each B-cell maturation stage can undergo an oncogenic transformation, resulting in a variety of B-cell malignancies, ranging from acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-non-Hodgkin's lymphoma (B-NHL) to multiple myeloma (MM).B-cell malignancies present with a wide range of molecular abnormalities and recurrent cytogenetic aberrations [2][3][4][5], often related to the B-cell specific processes, such as VDJ gene arrangements, somatic hypermutation and class switching. Small noncoding RNA (sncRNA) are highly abundant RNAs that have key regulatory functions in various cellular processes [6].MicroRNAs (miRNAs), the most studied endogenous sncRNAs, are small single-stranded RNAs of approximately 22 nucleotides (nts) in length and target messenger RNAs (mRNAs) for post-transcriptional silencing [7].Another class of sncRNAs are the small nucleolar RNAs (snoRNAs), which are 50-300 nts in length and are mainly localized in the nucleoli.SnoRNAs are responsible for post-transcriptional modification of ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs) [8]. Several studies have shown that snoRNAs are involved in oncogenesis [9][10][11][12][13].For instance, a large study including over 10,000 samples across 31 cancer types identified 46 snoRNAs that showed clinical relevance in at least 12 cancer types.SnoRNAs were associated with DNA damage response and mitotic nuclear division, suggesting a role in cancer initiation and progression.Furthermore, genes that are associated with the biogenesis and functions of snoRNAs were dysregulated in multiple types of cancer [11]. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "The C/D box snoRNAs are the largest group of snoRNAs, with more than 400 entities present in the human genome [14].C/D box snoRNAs are 50-100 nts in length and they contain the highly-conserved motifs box C (RUGAUGA) and box D (CUGA) [21,22].C/D box motifs are critical for the characteristic secondary helix-bulge-helix structure, the so-called K-turn [21,23].In addition to the C/D box, these snoRNAs contain less conserved copies of the C and D motifs, the C' and D' motifs [24].In between the C, C' motifs and D, D' motifs are the so-called guide sequences that direct the snoRNA to reverse complementary motifs within the target rRNAs [17,23] (Figure 1). C/D box snoRNA are mainly located in introns of host genes coding for proteins of the ribosomal biogenesis, proteins involved in translation, or proteins with a nuclear or nucleolar function [25].The biogenesis of C/D box snoRNAs largely depends on the splicing of pre-mRNA [26,27].Within the intron, C/D box snoRNAs are preferentially positioned approximately 70 nts of the 3 splice site [28].The transcription of C/D box sno-RNAs is performed by RNA polymerase II and includes the addition of a 5 monomethyl guanosine CAP structure [29].After transcription, the snoRNA sequence in the pre-mRNA is bound to a complex of various proteins [30][31][32][33], and the small nucleolar ribonucleoprotein (snoRNP) complex then recruits nucleolar protein 56 (NOP56) and fibrillarin (FBL).Subsequently, the pre-mRNA is spliced and the immature snoRNP complex is then transported to the Cajal bodies, where the 5 cap is hypermethylated by the trimethylguanosine synthase (TGS)-coilin complex [34].Next, the mature snoRNPs are transported to the nucleoli by the 140 kDa nucleolar phosphoprotein (NOPP140) [35,36].The snoRNA guides the snoRNP complex to the target rRNA by the interaction with reverse complementary motifs [24,[37][38][39], whereas the methylation reaction is performed by the methyltransferase FBL [24] (Figure 1).[31,40].The RUVBL1 and RUVBL2 subunits of the R2TP complex bind, together with nuclear fragile X mental-retardation-interacting protein 1 (NUFIP), zinc finger HIT domain-containing protein 3 (ZNHIT3), and ZNHIT6, the core protein nucleolar protein 58 (NOP58) and small nuclear ribonucleoprotein 13 (SNU13) to form a new protein complex [35].This newly assembled complex then binds and recruits fibrilarin (FBL), which replaces ZNHIT3 in the complex.The complex binds the C/D box snoRNA sequence during the splicing of the pre-mRNA.RUVBL1, RUVBL2, NUFIP, and ZNHIT6 are released from the complex and the immature snoRNP is translocated to the Cajal body for further maturation before it is translocated to the nucleolus.The mature C/D box snoRNAs are associated with four proteins, SNU13, NOP56, NOP58, and FBL [41,42].C/D box snoRNA guide the 2′O-methylation of targets rRNAs. ",
"section_name": "Structure and Biogenesis of C/D Box snoRNAs",
"section_num": "3."
},
{
"section_content": "H/ACA box snoRNAs are the second main group of snoRNAs.Up to now, more than 200 different human H/ACA box snoRNAs have been discovered [14], all consisting of a specific H-(ANANNA) and an ACA-box motif [18,23,37].The ACA-box motif is always located three nucleotides from the 3' end of the snoRNA strand [18].The H/ACA box snoRNAs possess a characteristic secondary structure, consisting of two 60-75 nucleotidelong hairpins [18,23,36,37], in which the H-box motif is located in-between [18,23,37].Both hairpins contain a pseudouridylation pocket which is essential for target interaction, as it contains the reverse complementary sequence to the target rRNA [18,23,36] (Figure 2).[31,40].The RUVBL1 and RUVBL2 subunits of the R2TP complex bind, together with nuclear fragile X mental-retardation-interacting protein 1 (NUFIP), zinc finger HIT domain-containing protein 3 (ZNHIT3), and ZNHIT6, the core protein nucleolar protein 58 (NOP58) and small nuclear ribonucleoprotein 13 (SNU13) to form a new protein complex [35].This newly assembled complex then binds and recruits fibrilarin (FBL), which replaces ZNHIT3 in the complex.The complex binds the C/D box snoRNA sequence during the splicing of the pre-mRNA.RUVBL1, RUVBL2, NUFIP, and ZNHIT6 are released from the complex and the immature snoRNP is translocated to the Cajal body for further maturation before it is translocated to the nucleolus.The mature C/D box snoRNAs are associated with four proteins, SNU13, NOP56, NOP58, and FBL [41,42].C/D box snoRNA guide the 2 O-methylation of targets rRNAs. ",
"section_name": "Structure and Biogenesis of H/ACA Box snoRNAs",
"section_num": "4."
},
{
"section_content": "H/ACA box snoRNAs are the second main group of snoRNAs.Up to now, more than 200 different human H/ACA box snoRNAs have been discovered [14], all consisting of a specific H-(ANANNA) and an ACA-box motif [18,23,37].The ACA-box motif is always located three nucleotides from the 3' end of the snoRNA strand [18].The H/ACA box snoRNAs possess a characteristic secondary structure, consisting of two 60-75 nucleotidelong hairpins [18,23,36,37], in which the H-box motif is located in-between [18,23,37].Both hairpins contain a pseudouridylation pocket which is essential for target interaction, as it contains the reverse complementary sequence to the target rRNA [18,23,36] (Figure 2). H/ACA box snoRNA can be positioned both near the 5′ and the 3′ splice site of the intronic region [28].After transcription of the host gene and splicing of the pre-mRNA, the immature H/ACA box snoRNA is translocated to the Cajal bodies, where it binds to a protein complex, resulting in H/ACA box snoRNP [35].The H/ACA box snoRNP is translocated to the nucleolus, where it pseudouridylates target RNA.The H/ACA box sno-RNAs guide the RNP complex to the target rRNAs, whereas the pseudouridylation reaction is carried out by the pseudouridine synthase dyskerin (DKC1) [23,24,37,39].Biogenesis of H/ACA box snoRNP.The generation of a H/ACA box snoRNP complex starts with the binding of the SHQ1 protein to dyskerin (DKC1).Subsequently, three additional proteins (nuclear assembly factor 1 (NAF1), H/ACA RNP complex subunit 3/nucleolar protein 10 (NOP10) and H/ACA ribonucleoprotein complex subunit 2 (NHP2)) bind the DKC1-SHQ1 complex, mediated by the RUVBL1-RUVBL2-TAH1-PIH1 (R2TP) complex [20,35].After the release of SHQ1, this new complex is then transported to the nucleus where it binds the snoRNA sequence on the pre-mRNA.After splicing, the immature H/ACA box snoRNP is translocated to the Cajal bodies where NAF1 is exchanged by H/ACA ribonucleoprotein complex subunit 1 (GAR1).The H/ACA box snoRNP is translocated to the nucleolus by NOPP140, where it can perform the pseudouridylation of target rRNA. ",
"section_name": "Structure and Biogenesis of H/ACA Box snoRNAs",
"section_num": "4."
},
{
"section_content": "ScaRNAs are the third type of snoRNAs and are, other than C/D box and H/ACA box snoRNAs, specifically localized in the Cajal bodies.ScaRNAs include C/D box or H/ACA box domains [21,23,43], but there are also compound scaRNAs that contain both C/D boxes and H/ACA boxes [38,39].Currently, 24 human scaRNAs are known, four C/D box scaRNAs, four C/D-H/ACA mixed scaRNAs, and 16 H/ACA box scaRNAs [19].The Cajal bodies localization of scaRNAs is due to two specific Cajal body localization signals: the Biogenesis of H/ACA box snoRNP.The generation of a H/ACA box snoRNP complex starts with the binding of the SHQ1 protein to dyskerin (DKC1).Subsequently, three additional proteins (nuclear assembly factor 1 (NAF1), H/ACA RNP complex subunit 3/nucleolar protein 10 (NOP10) and H/ACA ribonucleoprotein complex subunit 2 (NHP2)) bind the DKC1-SHQ1 complex, mediated by the RUVBL1-RUVBL2-TAH1-PIH1 (R2TP) complex [20,35].After the release of SHQ1, this new complex is then transported to the nucleus where it binds the snoRNA sequence on the pre-mRNA.After splicing, the immature H/ACA box snoRNP is translocated to the Cajal bodies where NAF1 is exchanged by H/ACA ribonucleoprotein complex subunit 1 (GAR1).The H/ACA box snoRNP is translocated to the nucleolus by NOPP140, where it can perform the pseudouridylation of target rRNA.H/ACA box snoRNA can be positioned both near the 5 and the 3 splice site of the intronic region [28].After transcription of the host gene and splicing of the pre-mRNA, the immature H/ACA box snoRNA is translocated to the Cajal bodies, where it binds to a protein complex, resulting in H/ACA box snoRNP [35].The H/ACA box snoRNP is translocated to the nucleolus, where it pseudouridylates target RNA.The H/ACA box sno-RNAs guide the RNP complex to the target rRNAs, whereas the pseudouridylation reaction is carried out by the pseudouridine synthase dyskerin (DKC1) [23,24,37,39]. ",
"section_name": "Structure and Biogenesis of scaRNAs",
"section_num": "5."
},
{
"section_content": "ScaRNAs are the third type of snoRNAs and are, other than C/D box and H/ACA box snoRNAs, specifically localized in the Cajal bodies.ScaRNAs include C/D box or H/ACA box domains [21,23,43], but there are also compound scaRNAs that contain both C/D boxes and H/ACA boxes [38,39].Currently, 24 human scaRNAs are known, four C/D box scaRNAs, four C/D-H/ACA mixed scaRNAs, and 16 H/ACA box scaRNAs [19].The Cajal bodies localization of scaRNAs is due to two specific Cajal body localization signals: the CAB Box domains [21,23,43,44].Each CAB box domain contains a CAB consensus (UGAG) sequence in the hairpin structures of the H/ACA or C/D domain [43].ScaRNAs are associated with COILIN, a Cajal body's specific protein, and H/ACA or C/D box core proteins [45,46].C/D box and H/ACA box scaRNAs are assembled as their C/D box and H/ACA box snoRNA counterparts [19], whereas the assembly of composite box scaRNAs is less well understood.ScaRNPs are involved in the 2 O-methylation (C/D box scaRNA) and/or pseudouridylation (H/ACA box scaRNA) of snRNAs, which are involved in splicing of pre-mRNAs [17]. ",
"section_name": "Structure and Biogenesis of scaRNAs",
"section_num": "5."
},
{
"section_content": "The 2 -O methylation and pseudouridylation of rRNA and snRNAs are an important part of the maturation of spliceosomal RNPs [47], and these modifications increase their stability [48,49].Modifications on target RNAs are not randomly distributed, but are found on highly conserved nucleotides [50].Besides the protection against degradation, modifications on rRNA regulate the binding of ribosomal proteins to the rRNA to form the ribosomes [51][52][53].Ribosomes were thought to be homogenous structures in the cell [52], but it was recently shown that ribosomes occur in different compositions.These differences occur by the high heterogeneity of post-transcriptional modifications of rRNA [48,50,52,[54][55][56][57][58] and subsequent binding of different ribosomal protein analogs to the ribosomes, a concept known as ribosomal heterogeneity [52].These different ribosomes have a unique regulation of translation.For example, different ribosomes have specific preferences for the translation of specific mRNAs [52,53].Furthermore, it has been shown that ribosomes can be partially modified and that there are different modification signatures in cellular stress conditions compared to normal [56] and between health and disease state [55,57,59].These findings suggest an important snoRNA-driven mechanism of translational control and protein expression [48]. ",
"section_name": "Canonical Functions of snoRNAs",
"section_num": "6."
},
{
"section_content": "Besides the canonical housekeeping and regulatory functions, snoRNAs may have other functions as well.First, it was discovered that some snoRNAs can be processed by DICER and that the sno-derived small RNAs (sdRNA) have miRNA-like functions [60][61][62].sdRNAs processed from C/D snoRNAs are mainly at 17-19 nts or larger than 27 nts in size and predominantly originate from the 5 end of the snoRNA [62,63].In contrast, sdRNAs processed from H/ACA snoRNAs are predominantly 20-24 nts in length and originate mainly from the 3 end of the snoRNA [62]. Second, it has been shown that there is a direct interaction between argonaute (AGO) 1 and AGO2 with NOP56 and FBL in HEK293 cells, indicating a possible link between RNA-induced silencing complex (RISC) and snoRNA function [64].In 2008, Ender et al. identified that the human snoRNA scaRNA15/ACA45 is processed by DICER into a small regulatory RNA, which is bound to AGO1/AGO2 and silences the expression of cyclindependent kinase 19 (CDK19, CDC2L6) [60].By regulating the translation of CDK19, scaRNA15/ACA45 plays a potential role in cell proliferation.Thus, some sdRNAs are loaded in RISC and have important regulatory functions.Consequently, dysregulation of snoRNA may not only affect splicing and ribosomal function but may also directly have an effect on cellular proliferation. ",
"section_name": "Noncanonical Functions of snoRNAs",
"section_num": "7."
},
{
"section_content": "In recent years, new targets of snoRNAs have been discovered [65].First, like rRNAs, tRNAs undergo post-transcriptional modifications [65,66].It was found that the human snoRNAs SNORD97 and scaRNA97 have a sequence complementarity with the anticodon loop of tRNA Met at cytidine 34 (C34) [65].This may indicate that C34 is a target for 2 Omethylation by SNORD97 and scaRNA97.Indeed, it was shown in vertebrates that C34 is methylated, and that the modification prevents the cleavage of tRNA Met by angiogenin.A single and double SNORD97/scaRNA97 knockout human HAP-1 cell model showed that the methylation of C34 is guided by SNORD97 in cooperation with scaRNA97.However, this double knockout cell model did not inhibit proliferation of HAP-1 cells, suggesting that C34 methylation is not essential for canonical tRNA Met function. Second, Huang et al. found that SNORD50A negatively regulated the mammalian mRNA 3 processing complex by binding to the polyadenylation site and competing with Pre-mRNA 3'-end-processing factor FIP1 (FIP1) [67].The mRNA 3 processing complex is involved in the maturation of mRNA and the regulation of translation [68,69].Indeed, downregulation of SNORD50A optimized the processing of mRNA 3 -ends, which increased mRNA levels [65,69].These data suggest that these snoRNAs may have a regulatory function on the mRNA level as well [67]. ",
"section_name": "Noncanonical Targets of snoRNAs",
"section_num": "8."
},
{
"section_content": "Mounting evidence shows that snoRNAs are essential for normal hematopoiesis [10,70].Genes associated with snoRNA functions and biogenesis were found mutated in hematopoietic disease.For instance, DKC1 is mutated in inherited syndromes including X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson syndrome, a clinically severe variant of dyskeratosis congenita (DC) and characterized by severe bone marrow failure [71,72].Strikingly, only the expression of SNORNA15 and SNORNA67 was downregulated in DKC1-mutant CD34+ cells compared with normal CD34+ cells [70].Variable expression of different H/ACA snoRNAs was found in X-DC patient cells with different mutations and the dysregulation of H/ACA box snoRNAs in X-DC patients was cell type dependent.These results indicate the complexity of snoRNA dysregulation in the X-DC patients [70].In addition, it has been shown that CD34+ cells with a catalytically inactive mutant of DKC1 (DKC1 D125A ) were greatly impaired in their capacity to differentiate in myeloid and erythroid colony assays as compared to CD34+ control cells [70].An acquired mutation at arginine 525 of DKC1 (DKC1 R525H ) and loss of function mutations in the DDX41 gene, a factor that is required for proper snoRNA processing in hematopoetic stem and precursor cells (HSPCs), contribute to hematopoietic defects leading to myelodysplasia [73].DDX41 mutated cells showed decreased expression levels of snoRNAs, resulting in reduced pseudouridylation of rRNA target uridines and impaired formation of ribosomes [73].R525H is hypomorphic for DDX41 function and may allow for HSC survival, whereas the rapid proliferative hematopoietic progenitors are more sensitive to loss of DDX41 activity due to their dependency on high protein synthesis [73].Loss of snoRNA function in HSPCs causes cell cycle arrest and apoptosis caused by dysregulation of pathways including p53 stress response, phosphatidylinositol 3-kinase (PI3K/AKT and Wnt/β-catenin [73].Together, these results indicate that snoRNA activity is essential for normal hematopoiesis. Only a few studies describe the expression levels of snoRNA in normal hematopoiesis.In the study of Warner et al., the expression of snoRNA was analyzed in different hematopoietic cell fractions and hierarchical clustering analysis of snoRNA expression data showed grouping of the samples dependent on cellular lineage and developmental state [10].For instance, it was found that two imprinted snoRNA-containing loci, namely the DLK-DIO3 locus (containing a large number of sncRNAs including 41 snoRNAs) and the SNURF/SNRPN locus (containing 82 snoRNAs), were expressed at high levels in CD34+ progenitor cells and strongly downregulated during granulocytic differentiation.Interestingly, the DLK-DIO3 locus snoRNAs were also expressed at reduced levels in B and T-cells, whereas the expression of SNURF/SNRPN snoRNAs remained high [10], suggesting for cell type-specific functions of these snoRNAs.The regulation of snoRNA expression was not correlated with splicing of host genes.In addition, no evident correlation between snoRNA expression and the expression of the host gene has been found in hematopoietic cells [10].These results indicate that other, yet unknown factors are involved in the regulation snoRNA expression and stability.In another study, snoRNA expression was compared between different types of normal mature B-cells [74].Germinal center (GC), naïve (N), marginal zone (MZ) and memory B-cells (SM), all isolated from tonsils, expressed similar levels of snoRNA, whereas peripheral blood B-cells (pBC) showed a distinct snoRNA expression pattern with reduced expression of SNORD116-1, SNORD116-23, SNORD116-29, SNORD94, and SNORA36A [74].Liuksiala et al. showed that SNORA25 (ACA25) and SNORA61 (ACA61) are highly expressed in naïve B-cells compared to HSCs, naive T cells and pediatric leukemia (T-ALL, BCP-ALL and AML), suggesting B-cell specific functions [75].SNORA25 is a well-conserved snoRNA and guides the pseudouridylation of residue U801 of the human 18S rRNA subunit (18S-801) [76].Computational sequence analysis suggested 18S:U814 as another potential target of SNORA25 [39].However, Deryusheva et al. found that the antisense interaction of SNORA25 to 18S:U814 did not form a functional pseudouridylation pocket within SNORA25 and is therefore unable to modify 18S:U814 [76].SNORA61 guides the pseudouridylation of the 28S rRNA subunit at residue U2496 [39].The functional consequences of these rRNA modifications remains to be elucidated. ",
"section_name": "SnoRNAs in Normal Hematopoiesis",
"section_num": "9."
},
{
"section_content": "In BCP-ALL, expression of snoRNAs was investigated with Sequencing by Oligonucleotide Ligation and Detection (SOLiD)-based next generation sequencing (NGS) by Teittinen et al. [77].In total, 46 snoRNAs were differentially expressed between BCP-ALL and T-ALL.Of these snoRNAs, 29 were upregulated and 17 were downregulated.A subset of snoRNAs were validated by qPCR and only scaRNA9 (up) and SNORD24 (down) were significantly differentially expressed in BCP-ALL compared to T-ALL.ScaRNA9, a C/D box scaRNA, guides the 2 -O-methylation of the U2:G19 and U2:A30 on the U2 snRNA [38].Besides the canonical function, scaRNA9 can be cleaved by DROSHA into an sdRNA with unknown functions.This processing can be enhanced upon stress signaling, such as cisplatin treatment [78].Unfortunately, differential snoRNA expression between BCP-ALL and normal precursor B-cells was not investigated in this study. In BCP-ALL, intragenic deletions of the erythroblast transformation-specific related gene (ERG) occur in 3-5% of patients [79].These intragenic deletions occur almost exclusively in BCP-ALL without a recurrent genetic aberration, known as BCP-ALL not otherwise specified (NOS) [80].This ERG-related subtype is characterized by aberrant expression of DUX4 and ERG transcription factors, which are involved in the differentiation of B-cells [81].SnoRNA expression analysis showed that SNORD109A, SNORD64, SNORD107 and 12 snoRNAs in the SNORD116 cluster (SNORD116-11, 14-18, 20-24, 27) were upregulated in ERG-related BCP-ALL patients compared to non-ERG-related BCP-ALL NOS patients [80].Most upregulated SNORD116 family members belong to the snoRNA group (SNOG)-2 of SNORD116 snoRNAs.All these snoRNAs are genomically located in the 15q11-q13 region [82], which deletion is associated with various myelocytic malignancies, such as AML and Chronic Myelomonocytic Leukemia (CMML) [83].These findings may suggest a role for the 15q11-13 region in lineage specific differentiation.Furthermore, the gene coding the multifunctional protein necdin (NDN) is located in the 15q11-13 region and NDN plays an important role during hematopoietic regeneration by inhibiting excessive HSC proliferation [84].This was confirmed in a NDN-deficient mice model, which exhibited an enhanced number of proliferating HSCs [84]. In addition to the profiling of snoRNAs, the expression of critical compounds of the rRNA methylation complex, including FBL, NOP56, NOP58, NHP2L1, nucleolin (NCL), and cMYC, which is a regulator of C/D box snoRNA expression, was studied in pediatric BCP-ALL [85].In this study, no significant correlations between the expression of the compounds of the rRNA methylation complex and leukemic blast counts in peripheral blood were found.Interestingly, expression levels of FBL, NOP56, cMYC, SNORD35b, and SNORD46 were significantly higher in patients who experienced relapse [85].In addition, high expression of FBL, cMYC, SNORD35B, and SNORD46 was found to be associated with reduced leukemia free survival, indicating that these factors may be used as prognostic markers.SNORD46 is upregulated in multiple types of cancer [86], whereas inhibition of SNORD46 in A549 cells led to a decrease in cell viability, migration and tissue invasion, suggesting an oncogenic role for SNORD46 in pediatric BCP-ALL. ",
"section_name": "Dysregulation of snoRNAs in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)",
"section_num": "10."
},
{
"section_content": "CLL is the most common adult leukemia in the Western world [87].It is characterized by the clonal expansion of CD5+ B-cells and mainly elderly people are affected [88].CLL is a heterogeneous disease and most patients survive decades, while others have a rapid disease progression with poor outcome [89].In a study of Ronchetti et al., snoRNA expression was profiled in CLL patients and different snoRNAs were identified as potential biomarkers [74].SNORD116-1, SNORD116-23, SNORD116-29, SNORD94, and SNORA36A were expressed at low levels in CLL and N, MZ and SM B-cells compared to GC B-cells, suggesting that CLL cells are not from GC origin.SNORA6, SNORA31, SNORA62, SNORA71C, SNORD37, and SNORD50B were significantly downregulated in CLL compared to normal total tonsillar B-cells.Downregulation of SNORD50B has also been observed in a variety of other human cancers, including breast cancer [90], lung cancer [91], melanoma [91] and T-cell lymphoma [91].The expression of SNORA31 in CLL patients correlated with the expression of its host gene, tumor protein translationally controlled 1 (TPT1), which is an important target of TP53 [92].Thus, reduced TP53 activity in CLL may explain the low SNORA3 expression.SNORD50A and SNORD50B both directly bind and inhibit K-RAS.Deletion of these snoRNAs leads to an increased activity of oncogenic K-RAS signaling, suggesting a functional role for loss of these snoRNAs in CLL development [90,91].In addition, the snoRNA host gene 5 (SNHG5), which harbors SNORD50A/B, is confirmed to regulate chemotherapy resistance by modulating the SNHG5/miR-32/DNAJB9 axis in AML patients [93].Also in CML, SNHG5 is associated with chemotherapy resistance by modulating miR-205-5p function [92].Whether SNHG5 also plays a role in CLL remains to be analyzed. Ronchetti et al. also compared the expression of snoRNAs in different genetic subgroups of CLL.They showed that CLL patients with a deletion of chromosome 11q23 (del11) have reduced expression of scaRNA9 compared to other CLL subgroups, which can be explained by its location within the deleted region [74].In addition, SNORA70F was downregulated by unknown mechanisms in CLL patients with trisomy 12 (12+), del11, and CLL patients which are ZAP-70 positive or CD38 positive [74].In immunoglobulin heavy chain gene (IGHV) unmutated CLL (UM-CLL), the expression of SNORA70F and SNORA70C was decreased, whereas SNORA71C levels were increased compared to IGHV mutated CLL.In addition, CLL with 12+ showed downregulation of scaRNA17 and upregulation of SNORA2b, SNORD59a and SNORD59b compared to non-12+ CLL patients, which can be explained by the extra chromosome 12.Finally, CLL patients with deletion of chromosome 13p13 (del13) were characterized by 19 downregulated snoRNAs.Strikingly, none of these snoRNAs was located at chromosome 13 [74].In CLL patients, high expression of SNORA74A and SNORD116-18 and low expression of SNORD56 were significantly associated with shorter progression free survival (PFS) [74], suggesting for a role of these snoRNAs in disease progression.In gastric cancer, knockdown of SNORA74A inhibits cell proliferation [94].Furthermore, SNORA74B, which shares the same target sites as SNORA74A, contributes to the overactivation of AKT/mTOR signaling, a pathway that is involved in cell survival, proliferation and cell migration [95] and that is frequently aberrant in human cancer [96,97].These results suggest that overexpression of SNORA74A may increase proliferation and has a potential oncogenic role in CLL. In a study by Berquet et al. [98], differential expression of SNORD35b, SNORD71, SNORD116-11 and SNORD116-25 was found in CLL compared to normal CD19-positive B-cells.In this study, the majority of the included patients had an adverse prognosis. The difference in patient characteristics and the different cell populations used for the comparative analyses may explain the discrepancies between the differentially expressed snoRNAs determined in this study and the ones found in study of Ronchetti, et al. [74].In the study of Berquet et al., differentially expressed snoRNAs were also assessed in CLL patients with different chromosomal alterations or mutations in the IGHV.In IGHVmutated CLL patients two groups with significant different treatment free survival (TFS) (32 versus 144 months) could be identified based on differential expression of 20 snoR-NAs.Of these 20 snoRNAs, eight snoRNAs (SNORA12, SNORA22, SNORA27, SNORA56, SNORA70, SNORD8, SNORD105B, and scaRNA8) were upregulated and two snoRNAs (SNORA80 and SNORD1A) were downregulated in both proliferating normal B-cells and proliferating CLL cells, suggesting that these snoRNAs could be functionally relevant for proliferation [98]. Together, these data suggest that snoRNAs are aberrantly expressed in CLL compared to healthy mature B-cells.Furthermore, different cytogenetic aberrations are associated with distinct snoRNA expression profiles.Finally, functional studies indicate a role for snoRNAs in proliferation of CLL cells. ",
"section_name": "Dysregulation of snoRNAs in CLL",
"section_num": "11."
},
{
"section_content": "B-cell lymphomas are classified as a variety of hematological diseases originating from mature B-cells [99].B-cell lymphomas are characterized by chromosomal translocations of regions with important oncogenes and tumor suppressor genes, including C-MYC, Cyclin D1 (CCND1), B-cell lymphoma 2 (BCL2) and B-cell lymphoma 6 (BCL6) [99,100].In some cases, translocations involve genes that harbor snoRNAs.For instance, a diffuse large B-cell lymphoma patient was identified with t(1;3)(q25;q27), fusing growth arrest-specific transcript-5 (GAS-5) to BCL6, generating the GAS5-BCL6 fusion gene [101].GAS5 is a noncoding gene that harbors multiple snoRNAs, including SNORD81, SNORD47, SNORD80, SNORD79, SNORD78, SNORD44, SNORD77, SNORD76, SNORD75 and SNORD74 [102].Despite oncogenic functions of dysregulated GAS5 sno-RNAs in different types of cancer [103][104][105], it remains unknown how t(1;3)(q25;q27) affects the expression levels of GAS5 snoRNAs.The t(3;6)(q27;q15) is another example of a translocation involving snoRNAs in human B-cell lymphoma and involves BCL6 and U50HG (also known as SNHG5) [106,107].Again, the effect of the t(3;6)(q27;q15) translocation on SNORD50A/B expression has not been studied yet, which is essential to understand a possible role for these snoRNAs in oncogenesis of B-cell lymphoma. ",
"section_name": "Dysregulation of snoRNAs in B-Cell Lymphomas",
"section_num": "12."
},
{
"section_content": "Multiple myeloma (MM) is an incurable malignancy of plasma cells [108,109] with recurrent chromosomal aberrations in the majority of patients [110].For instance, the t(4;14)(p16.3;q32.3) is detected in 20% of MM patients and it is associated with reduced overall survival [111][112][113].This translocation results into the fusion of immunoglobulin heavy chain region enhancer elements to the Wolf-Hirschhorn syndrome candidate 1 gene (WHSC1).WHSC1 encodes 3 protein isoforms, of which two are methyltransferases with H4K20, H3K36, and H3K27 as targets [111].The t(4;14)(p16.3;q32.3)causes upregulation of WHSC1, but this event is not sufficient to drive oncogenic transformation of plasma cells [111].ScaRNA22 (ACA11), which is located within the WHSC1 gene, is also upregulated in t(4;14) positive MM.The overexpression of scaRNA22 in t(4;14) positive MM cells was associated with the downregulation of 60S ribosomal protein L13a (RPL13A) and snoRNA U30 [111].Furthermore, lentiviral overexpression of scaRNA22 resulted in lower levels of H 2 O 2 in t(4;14)-negative dexamethasone sensitive MM cells (MM1.S) and enhanced MM cell proliferation compared to nontransduced MM1.S cells [111].The response to oxidative stress (H 2 O 2 ) is mediated by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor [114].In normal conditions, NRF2 is located in the cytosol, while during cellular stress conditions with increased levels of reactive oxygen species (ROS), NRF2 translocates to the nucleus where antioxidant genes are transcribed. Mahajan et al. found that mRNA and protein levels of NRF2 were not upregulated after lentiviral scaRNA22 overexpression.However, it was observed that the translocation rate of NRF2 to the nucleus was increased [114].This could explain the lower ROS levels in scaRNA22 overexpressing t(4;14) MM cells.In addition, scaRNA22 controls ribosome biogenesis in a ROS dependent manner [115] and patients with scaRNA22 overexpression respond better to Bortezomib treatment, suggesting that elevated expression of scaRNA22 is a potential prognostic marker for MM. In addition to the expression based on cytogenetic subgroups, snoRNA expression was studied at different stages of disease progression, such as monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and MM [116,117].In MGUS, SMM and MM patients, a total of 17 snoRNAs (of which 15 reside in the SNORD115 cluster: SNORD115-3, -4, -5, -6, -7, -9, -10, -11, -12, -13, -23, -24, -25, -32, -44, SNORA64, and SNORA46) were downregulated compared to normal plasma cells [116].In addition, the expression of eight snoRNAs (SNORA73A, SNORD32A, SNORA68, SNORA5A, SNORD15B, SNORD47, SNORA3, and SNORA71A) were more strongly expressed in patients with more severe disease (MGUS < SMM < MM) [116].Some of these snoRNAs were functionally investigated.For instance, SNORD32A controlled the response to oxidative stress (H 2 O 2 ) and endoplasmic reticulum (ER) stress [118].An additional study showed that overexpression several snoRNAs was correlated with shorter time to progression in MM patients (SNORD25, SNORD27, SNORD30: median PFS 24 months versus not reached; SNORD31: median PFS 17 months versus 36 months) [117].These studies indicate that different snoRNAs are dysregulated during disease progression and prognosis of MM and that the prognosis can be predicted based on SNORD25, SNORD27, SNORD30, and SNORD31 expression levels. ",
"section_name": "Dysregulation of snoRNAs in Multiple Myeloma",
"section_num": "13."
},
{
"section_content": "As reported above and summarized in Figure 3 and Table 1, several studies have shown that snoRNAs are differentially expressed in different types of B-cell malignancies compared to healthy B-cells or other hematological malignancies.However, most studies consisted of expression profiling analyses without functional characterization of the dysregulated snoRNAs.In addition, the effects of snoRNA dysregulation on the target modifications and additional downstream mechanisms is generally not studied.Furthermore, the functional role of sdRNAs in B-cell malignancies is not elucidated yet. In different B-cell malignancies, specific snoRNAs were dysregulated.However, the regulation of snoRNA processing and stability is not understood yet.For example, the expression of different snoRNAs of the SNORD116 cluster was dysregulated in ALL and CLL, suggesting the presence of additional, yet unknown mechanisms of snoRNA expression regulation. Further studies are warranted to enhance our understanding on snoRNAs in B-cell malignancies and to characterize their functions in normal and malignant B-cell differentiation.To achieve this, cellular models with modulated snoRNA expression need to be generated.Overexpression models of snoRNAs can be generated using lentiviral transduction [111].In addition, novel technologies as CRISPR-Cas9 genome editing allow to generate snoRNA knockout cell and animal models [119].Furthermore, assays to evaluate modifications of putative snoRNA targets have been developed.2 O-methylation can be assessed using reverse transcription at low deoxy-ribonucleoside triphosphate (dNTP) concentrations followed by polymerase chain reaction (PCR) (RTL-P) [120].Pseudouridylation of target RNAs can be studied by treatment with N-Cyclohexyl-N'(2-morpholinoethyl)carbodiimide (CMC) and subsequent reverse transcriptase PCR amplification [121].In our opinion, these technologies help to reveal the role of snoRNA dysregulation in B-cell malignancies. ",
"section_name": "Conclusions and Future Prospective",
"section_num": "14."
}
] |
[
{
"section_content": "Funding: This research received no external funding. Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Funding: This research received no external funding. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"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": "Author Contributions: Writing-original draft preparation, M.W.C.V., S.J.E. and V.H.J.v.d.V.; writingreview and editing, M.W.C.V., S.J.E. and V.H.J.v.d.V.; visualization, M.W.C.V.; supervision, S.J.E. and V.H.J.v.d.V.; funding acquisition, V.H.J.v.d.V.All authors have read and agreed to the published version of the manuscript. ",
"section_name": "",
"section_num": ""
}
] |
10.17344/acsi.2016.2970
|
Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome
|
Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.
|
[
{
"section_content": "Despite of rapid development of medicine and other sciences, timely and reliable diagnosis of diseases is sometimes still a pretentious task.In addition when it comes to ascertaining the health status of an individual some diagnostic procedures are very invasive.A solution to such difficulties could lie in new methods for human plasma analysis.Human plasma serves as an important source of information about human health.Various diseases are often associated with biomarkers, which have been defined by Hulka et al. as biochemical, molecular or cellular changes that are measureable in human fluids, tissues and cells. 1 The human plasma proteome consists of more than 3000 proteins and peptides, therefore the search for potential disease biomarkers, and their identification in the plasma proteome is a very complicated procedure. 2Only ten proteins make up 90% of the mass of plasma proteins and albumin (HSA) and immunoglobulins (Ig) represent 75% out of these ten proteins.Majority of biomarkers in blood plasma are often scarce and small in size.Consequently, their detection can be limited by the presence of more abundant and larger proteins such as HSA or IgG 2 .Because of the complexity of plasma no single technique can fully exploit the information plasma has to offer.Until now mass spectrometry and 2D-electrophoresis have been routinely used for detection and characterization of specific biomarkers. 3Chaires et al. have discovered the poten-tial use of differential scanning calorimetry (DSC) for plasma analysis. 2Using a DSC to analyze plasma yields a thermogram that is sensitive to differences in thermodynamic properties of the most abundant plasma proteins.Chaires et al. have shown that thermograms of plasma from diseased individuals differ significantly from thermograms of plasma from healthy individuals. 2,4Another tool to facilitate the search for biomarkers is liquid chromatography, in particular affinity chromatography and ion-exchange chromatography.Several researchers have demonstrated that depletion of most abundant plasma proteins amplifies the contribution of least abundant plasma proteins, thus creating the opportunity to uncover and study specific proteins/peptides. 5,6Polymethacrylate chromatographic monoliths afford flow rate independent binding capacity and resolution for large biomolecules due to the convective nature of the flow, which allows relatively short analysis times compared to traditional chromatographic supports. 7Having these advantages in mind we decided to partially deplete human serum albumin from plasma sample using chromatographic monoliths bearing immunoaffinity ligand.The purpose of our research was to develop and optimize procedures for combined use of DSC and immunoaffinity chromatography (IAC) for the investigation of different plasma samples from diseased individuals. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Experimental",
"section_num": "2."
},
{
"section_content": "We investigated plasma samples from three individuals with three different haematological diseases, namely chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and acute myeloid leukemia (AML).Individuals diagnosed with MM and AML were females and the one with CLL was a male.Due to ethical issues further information about these individuals cannot be revealed.Chaires et al. 8 have shown that the main shape of plasma thermograms is unbiased by age, gender and ethnicity, however small deviations in HSA unfolding signal amplitudes can be observed due to higher HSA concentrations in male plasma samples.For the control sample blood plasma from a healthy child was used.At the University Medical Centre Ljubljana (UMCL) the peripheral blood samples were withdrawn in tubes with EDTA and centrifuged at 2500 RPM for 10 minutes to obtain plasma.The use of plasma was approved by National Medical Ethics Committee (Approval number: MZ 0120-299/2016-2, KME 83/05/16) Because only 3 plasma samples were investigated we have to emphasize that this is only a preliminary study in which our main aim was to identify the effectiveness of our approach and not to generalize differences for the three investigated diseases.It should be clear that for validation of our method more plasma samples should be analyzed.Even though only one set of measurements was performed for CLL and AML samples, the robustness and reproducibility of our method was tested with other plasma samples (data not shown) and two separate fractionations of MM sample (Figure S1). ",
"section_name": "1. Plasma Samples",
"section_num": "2."
},
{
"section_content": "The buffer solutions used in our experiments consisted of 20 mM disodium phosphate and 300 mM NaCl.HCl was added to disodium phosphate to reach pH of 7.4.All buffers were filtered before use.Samples were first diluted 5-to 7-fold and then extensively dialyzed against the buffer at 4 °C (three changes of buffer solution in 24 h) using a dialysis tube Float-A-Lyzer with a 3500 Da cut-off.Total protein concentration of plasma samples was determined spectrophotometrically according to the bicinchoninic acid method procedure (Sigma-Aldrich). 9 ",
"section_name": "Sample Preparation",
"section_num": "2."
},
{
"section_content": "Separations were performed using a Knauer highperformance liquid chromatography (HPLC) workstation, consisting of two pumps, an autosampler injection system with a sample loop volume of 100 μL and a UVdetector at a wavelength of 280 nm.Samples were loaded by using a mobile phase consisting of 20 mM disodium phosphate and 300 mM NaCl, pH = 7.4 at a flow rate of 1 mL/min.For experimental data acquisition, Eurochrom 2000 software was used.The hydrazide-modified monolithic chromatographic column (CIM ® HIDA) with 1 mL bed volume and 6 μm pore size was provided by BIA Separations d.o.o.The immobilization of polyclonal anti-HSA was performed according to the procedure described by Tarasova et al. 10 and the determined dynamic binding capacity at 50% breakthrough of prepared CIM αHSA column for pure HSA in phosphate buffered saline was 0.35 mg/mL.Before injection, diluted and dialyzed plasma samples were filtered through 0.45 μm membrane filters (Sartorius) and diluted with dialysate to a total protein concentration of approximately 4 to 5 mg/mL.100 μl loop was used to load the sample and flow-through and bound fractions of plasma were collected.Succeeding collection of flow-through fraction the bound fraction was eluted using 0.1 M HCOOH, pH = 2.5, followed by immediate neutralization by 200 mM disodium phosphate, 300 mM NaCl, pH = 8.5.Both fractions were transferred to Vivaspin sample concentrators with MWCO of 3500 Da and centrifuged to final volume of 1 to 1.5 mL at 5000 RPM.This was repeated 18 times. ",
"section_name": "3. IAC Protocol",
"section_num": "2."
},
{
"section_content": "DSC thermograms were obtained using nano DSC II (CSC).Dialyzed plasma samples were diluted 3-to 4-fold with dialysate and degassed for 15 min.DSC scans were Brudar et al.: Use of Differential Scanning Calorimetry ... recorded from 20 °C to 100 °C at 2 °C/min.Corresponding buffer scans were also recorded.Raw data was analyzed with NanoAnalyze software and Microsoft Excel.The corresponding baseline (buffer-buffer) scans were subtracted from the plasma scans and normalized to total protein concentration to obtain partial heat capacity as a function of temperature. ",
"section_name": "4. DSC Protocol",
"section_num": "2."
},
{
"section_content": "Fractions were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Plasma fractions from IAC were diluted to a final concentration between 0.5 and 1.0 mg/mL.Separation of proteins was performed on 12% resolving gel and 4% stacking gel.Electrophoresis was carried out under reducing conditions according to the basic Laemmli SDS-PAGE procedure. 11Gels were run at 200 V and 15 °C for 75 min.Visualization of protein bands was done by silver staining. 12Gel images were altered to achieve more efficient interpretation of result (color was changed to black&white and positions of lanes on the same gel were shuffled).Unaltered images can be seen in Figure S2. ",
"section_name": "5. SDS-PAGE",
"section_num": "2."
},
{
"section_content": "",
"section_name": "Results and Discussion",
"section_num": "3."
},
{
"section_content": "Figure 1 shows that plasma thermograms belonging to diseased individuals differ significantly from one another and from \"healthy\" plasma thermograms.The thermo-grams of plasma display multiple peaks that can be associated to the melting temperatures (T m ) characteristic of main plasma proteins. Chaires et al. have performed thermal denaturation of \"healthy\" blood plasma at heating rate of 1 °C/min and in 10 mM phosphate buffer with 150 mM NaCl to mimic physiological conditions.Several peaks were identified corresponding to transition temperatures of fibrinogen (T m ∼51 °C), HSA (T m ∼63 °C), IgG (T m ∼70 °C) and transferrin (T m ∼85 °C). 2 Thermograms in our research were recorded at heating rate of 2 °C/min to increase the number of processed samples and 20 mM phosphate buffer with 300 mM NaCl was used to avoid non-specific plasma-column interactions.Higher heating rate and salt concentration shifted the melting temperatures of majority of plasma proteins to higher values (Figure 1).Thus T m of fibrinogen shifts to ∼53 °C, T m of HSA to ∼67 °C, T m of IgG to ∼72 °C) and T m of transferrin to ∼86 °C.Melting temperatures are not the only obtainable data from thermograms.The integration of area under the thermograms of plasma yields the enthalpy of plasma proteins denaturation (Table 1).Figure 1 shows that thermograms obtained from thermal denaturation of blood plasma from diseased individuals are shifted to higher temperatures.The shift in unfolding temperatures is in agreement with previously reported results for other diseases. 2,14,15,16Proteins in plasma from diseased individuals seem to unfold at higher temperatures for a yet undefined reason.Chaires et al. had hypothesized that such stabilization of proteins occurs due to interactions between major plasma proteins and specific disease biomarkers. 2 Thermal denaturation of plasma from an individual with CLL yields a thermogram, where at least four transitions could be identified.These could correspond to denaturation of fibrinogen (T m ∼53 °C), HSA (T m ∼68 °C), IgG (T m ∼72 °C) and transferrin (T m ∼91 °C).The transitions are shifted from 0 to 5 °C towards higher temperatures when compared to thermogram of \"healthy\" plasma.For instance, T m of fibrinogen does not change, but T m of transferrin is shifted for approximately 5 °C.The denaturation enthalpy of non-fractionated CLL plasma is 3.80 ± 0.19 cal/g.This value is approximately the same as the denaturation enthalpy of plasma proteins from healthy individuals.Figure 1 shows that DSC thermograms of CLL and AML plasma are distinct even though both diseases are characterized by prominent changes in blood leukocytes not in plasma protein composition.In the thermogram of plasma from an individual with AML an additional endothermic peak can be observed at 62 °C.We hypothesize this peak is present due to thermal denaturation of haptoglobin, as it is supposed to unfold in the same temperature region as HSA. 2 T m values of fibrinogen and transferrin from AML plasma denaturation seem to be at approximately the same position as T m values from \"healthy\" plasma denaturation.The denaturation enthalpy of non-fractionated blood plasma from an individual with AML is 3.96 ± 0.20 cal/g, which is comparable to denaturation enthalpy of CLL and \"healthy\" plasma.Lastly Figure 1 shows a specific thermogram of plasma from an individual with MM.Three main transition peaks for most abundant plasma proteins can be characterized.A similar thermogram for this disease was obtained by Todinova et al. 17 Thermally induced peaks are shifted to higher temperatures to an even greater extent than those characteristic for proteins from CLL and AML plasma samples.Also in contrast to AML and CLL samples protein concentration in MM plasma was increased above reference range (94 g/L) due to monoclonal IgG fraction (M spike) typical for MM.Thermogram exhibits three distinct peaks (T m1 ∼62 °C, T m2 ∼71 °C and T m3 ∼82 °C) and is dramatically different from other thermograms.The determined enthalpy of denaturation for non-fractionated MM plasma was 4.57 ± 0.23 cal/g, which is approximately 20% higher from denaturation enthalpies of other samples.These results suggest that the amount of unfolding enthalpy is not a determining factor for disease conditions.We believe that only the shape of plasma thermograms can serve as a diagnostic tool for disease determination. ",
"section_name": "1. Comparison of Blood Plasma Thermograms",
"section_num": "3."
},
{
"section_content": "To gain further insight into thermal behavior of different blood plasma samples, fractionation of plasma samples and thermal denaturation of obtained fractions were carried out.The thermogram of the flow-through fraction of plasma from an individual with CLL in Figure 2 shows a distinct drop in signal intensity in the region characteristic of HSA, as a result of HSA depletion by a CIM αHSA column.As a result, transition peaks for other proteins are more pronounced.Along with enrichment of less abundant proteins we can observe an entirely new peak at approximately 65 °C, which seems to have been previously masked by HSA.We hypothesize that this peak is a result of haptoglobin denaturation. The obtained unfolding enthalpy of the flowthrough fraction of CLL plasma was 2.94 ± 0.15 cal/g (Table 1), which is 22% lower than that of non-fractionated plasma.The thermogram of the bound fraction in Figure 2 shows a major exothermic effect above 82 °C due to protein aggregation.We hypothesize that this phenomenon is due to elution of HSA with HCOOH, even though this fraction was intercepted by high capacity buffer to neutralize acid.Very acidic conditions are nonphysiological for HSA and can change its structure in a way that favors aggregation.Similar behavior of bound fractions was observed in all plasma samples and reported by other researchers 18,19 and will not be discussed any further.Together with the thermogram of the flow- through fraction, results of SDS-PAGE (Figure 2) show that only partial HSA depletion was achieved even though the applied concentration was bellow theoretical binding capacity.This was most likely due to complexity of plasma samples and overuse of our CIM αHSA column.Even though HSA is still present in the flowthrough fraction, the corresponding thermogram seems to be missing peak assigned to HSA denaturation.We have to assume that HSA unfolding is masked by the unfolding of other plasma proteins.CLL F2 lane (bound fraction) displays an intensive band at 66 kDa, characteristic of HSA, no slower migrating bands and some low intensity bands in lower MW region that could correspond to heavy (∼50 kDa) and light (∼25 kDa) IgG chains.Results of flow-through fraction PAGE can be seen in CLL F1 la-ne.An intensive band at ∼180 kDa, representing a subunit of α 2 -macroglobulin, is present in the high MW γ-region.Below in the β-region lies a distinct band at 80 kDa, characteristic of transferrin.HSA band is less intensive but still present because in order to reduce the number of fractionations, high concentration of plasma with HSA concentration on the limit of binding capacity was injected on CIM αHSA column.Beneath the band representing HSA there is a broader band consisting of Bβ-fibrinogen at ∼56 kDa, transthyretin at ∼55 kDa and heavy Ig-G chains at ∼50 kDa.The band at ∼15 kDa could not be identified. 20It is clear that intensities of all bands, which were weaker in the whole plasma sample are noticeably increased in the CLL F1 lane, suggesting that enrichment of less abundant proteins was successful.The thermogram of the flow-through fraction of AML plasma (Figure 3) shows that depletion of HSA from blood plasma resulted in well-defined peak at 62 °C which in our opinion is a result of haptoglobin denaturation.Signals for other plasma proteins, such as fibrinogen at 53 °C and transferrin at 86 °C are also slightly enriched.The remaining peak at 72 °C can be attributed to IgG denaturation.The obtained unfolding enthalpy for the flowthrough fraction was 2.89 ± 0.14 cal/g (Table 1), which is approximately 27% lower than for non-fractionated AML plasma.Thermogram obtained from thermal denaturation of the bound fraction of AML plasma (Figure 3) displays an intensive endothermic peak, followed by a steep exothermic effect characteristic of protein aggregation.Results of SDS-PAGE of AML plasma (Figure 3) show an intensive HSA band in AML F2 lane, suggesting that we have successfully depleted the majority of HSA from the AML plasma.Some faster migrating bands corresponding to heavy (∼50 kDa) and light (∼25 kDa) IgG chains can also be seen in AML F2 lane.AML F1 lane corresponds to the flow-through fraction and contains a band at 66 kDa showing that not all of HSA was removed.The intensities of other bands are increased, proving that we have succeeded in increasing the relative concentration of other proteins. When comparing thermograms of non-fractionated sample and flow-through fraction of the MM plasma (Figure 4), a distinct drop in signal intensity at temperature of 71 °C and increase in signal intensity at temperature of 82 °C are observed.The denaturation enthalpy of the flow-through fraction was 4.27 ± 0.21 cal/g, which is only 7% lower than for non-fractionated MM plasma.SDS-PAGE of MM flow-through fraction, MM F1 , shows decreased intensity of HSA band at 66 kDa and increased intensities of other bands (Figure 4).On the other hand SDS-PAGE of MM bound fraction, MM F2 , shows strong band, characteristic of HSA along with weaker bands representing heavy and light IgG chains.SDS-PAGE results suggest that almost all of the HSA is removed from flow through fraction which could be associated to drop in DSC signal intensity at temperature of 71 °C.Thermograms of MM plasma show that HSA and IgG unfolding have shifted to higher temperature, perhaps due to interactions with drugs 21 or interactions with monoclonal immunoglobulin. 22About 25 % drop in DSC signal intensity at temperature of 71 °C does not correspond to drop in band intensity observed in SDS-PAGE which means that HSA is unfolding in the same region as other proteins.Comparison of lanes with bound fractions in Figures 2, 3 and4 reveals that the intensities faster migrating bands are strongest in the case of MM plasma.This could be a result of HSA-IgG complex formation, non-specific interactions or column degradation and should be further investigated.The stained gel (Figure 4) gives information not only about the effectiveness of MM plasma fractionation, but also of multiple myeloma signs.This is especially evident from several protein bands in MM NF lane.A weak band, characteristic of HSA and abnormally intensive bands, characteristic of heavy and light IgG chains can be observed.Both phenomena are typical of multiple myeloma 20 and it should be emphasized that MM plasma was obtained from patient with IgG clonality and light chain kappa restriction. ",
"section_name": "2. Fractionation and Characterization of Proteins in Blood Plasma",
"section_num": "3."
},
{
"section_content": "DSC provides a unique signature of the three examined diseases (chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia), thus showing potential for plasma proteome investigation.Because only 3 plasma samples were investigated we have to emphasize that obtained results may not be used to generalize differences for the three investigated diseases.IAC with the use of a CIM αHSA column enables enrichment of less abundant plasma proteins, thus providing the tool to enhance the relative presence and/or influence of potential disease biomarkers.Enrichment of plasma proteins in flow-through fractions was verified with SDS-PAGE and analyzed with DSC.The obtained results suggest that combination of DSC and IAC could be introduced as a possible novel and non-invasive diagnostic tool, although further research in this field is required. ",
"section_name": "Conclusions",
"section_num": "4."
}
] |
[
{
"section_content": "We acknowledge the financial support of the Slovenian Research Agency through Program Groups P1-0201, P4-0121 and P4-0369.We are also grateful to Iva Hafner Bratkovi~ for contributing important knowledge on making and staining SDS-PAGE gels.This work was partially supported by the European Commission FP7 projects Prot-HiSPRA (grant number 282506) and HTP-GlycoMet (grant number 324400). ",
"section_name": "Acknowledgements",
"section_num": "5."
}
] |
10.3389/fonc.2024.1364958
|
Molecular characterization of the evolution of premalignant lesions in the upper aerodigestive tract
|
<jats:sec><jats:title>Introduction</jats:title><jats:p>Early relapse and development of metastatic disease are some of the primary reasons for the poor prognosis of patients with head and neck squamous cell carcinoma (HNSCC). HNSCC is a heterogeneous disease which may develop in large premalignant fields of genetically altered cells. Yet knowing which individuals will progress and develop clinically significant cancers during their lifetimes remains one of the most important challenges of reducing HNSCC morbidity and mortality. To further elucidate the molecular mechanisms, we performed a focused analysis of the genome and immune microenvironment from multiple, matched normal squamous tissue, premalignant lesions, as well as primary and recurrent tumors from seven patients with p16-negative HNSCC.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We performed targeted panel Next Generation Sequencing (161 genes) to analyze somatic variants from sequentially collected, matched formalin-fixed paraffin-embedded tissue (normal, premalignant, HNSCC) from two patients. These samples plus samples from five additional patients were analyzed with the Nanostring PanCancer Immune Panel. In addition, we performed shallow whole genome sequencing (0.5x coverage on average) on samples from three of these patients. Patients were, apart from one case, primarily treated with curative-intent surgery, and received subsequent adjuvant treatment, if indicated.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The most frequently mutated genes were <jats:italic>TP53</jats:italic> and <jats:italic>NOTCH1</jats:italic>. Other mutated genes included <jats:italic>NOTCH3</jats:italic> and <jats:italic>CDKN2A</jats:italic>, among others. A significant number of mutations were private to dysplasia and invasive carcinoma, respectively, however, almost 20% were shared between them. Increasing genomic instability was observed when comparing histologically normal squamous mucosa with higher levels of dysplasia. High-grade dysplasia showed similarly rearranged genomes as invasive carcinoma. Pathways related to interferon alpha and gamma response were upregulated even in moderate dysplastic lesions with increasing expression in higher grades of dysplasia and carcinoma. <jats:italic>SPINK5</jats:italic>, a known tumor suppressor gene in HNSCC, was already downregulated in low-grade dysplastic lesions, indicating an early deactivation in the evolution of the disease.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>Genomic alterations as well as aberrant immune gene expression can be observed early in the evolution of tumors of the upper aerodigestive tract, highlighting the potential for targeting early mechanisms of disease progression.</jats:p></jats:sec>
|
[
{
"section_content": "Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease that may develop in large carcinogeninduced premalignant fields of genetically altered cells.Field cancerization was first observed and described by Slaughter et al. in 1953 (1).At that time, the investigators concluded that the mucosa of the upper aerodigestive tract had undergone changes which were likely due to continued carcinogen exposure and could therefore become more susceptible to the development of multiple foci of malignant transformation (2).At its core, HNSCC evolution involves a multifaceted interplay of genetic mutations and interactions with the local microenvironment.It is well established that the process of tumor evolution progresses through stages of premalignancy wherein cells acquire abnormal geno-and phenotypes that increase their propensity for malignant transformation.Clonally unrelated premalignant cells in a cancerized field can often be found distant from each other and contribute to local disease recurrence or the development of a second primary tumor in the upper aerodigestive tract (3)(4)(5)(6)(7)(8)(9).However, the exact order and timing of these genetic changes remains to be determined for HNSCC pathogenesis.Frequent alterations in gene copy number, for example, are a hallmark of advanced cancers (10).Normal epithelium on the other hand displays very rarely copy number alterations (11) but may instead harbor numerous somatic mutations (12)(13)(14).Much less is known about the process of transformation from normal epithelium to premalignant lesions and subsequent evolution into malignant tumors.Evidence suggests that copy number alterations may play a key role in driving early malignancy (11,15,16).This concept adds another layer of complexity to the understanding of how malignancies develop in the upper aerodigestive tract and is particularly significant in the context of HNSCC, as it underscores the importance of not only treating the primary tumor but also monitoring and addressing potential premalignant changes in the surrounding tissue.Understanding the biology of premalignancy is therefore a critical enabler to developing the right approaches to intercepting the disease.Yet knowing which individuals with premalignancies will progress and develop clinically significant cancers during their lifetimes remains one of the most important challenges of reducing HNSCC morbidity and mortality.Simultaneously, it has become increasingly evident that the progression of tumors and the immune responses are profoundly interconnected, extending beyond genetic mutations and cellular transformations (17)(18)(19).Recent advancements in immune checkpoint therapy have demonstrated improved overall survival in a proportion of patients with recurrent/metastatic HNSCC (20)(21)(22)(23)(24).Given the impact of immunotherapy in advanced disease, modulation of the immune microenvironment might prove beneficial as a prevention strategy in patients with high-risk premalignant mucosal lesions.Available data indicates that immune surveillance may play an important role in preventing the transition of premalignancy to cancer (25)(26)(27).However, the extent to which an active immune system influences the genome evolution of a tumor (or vice versa) remains unknown, particularly with respect to the extensive genomic and transcriptomic heterogeneity within a tumor and its surrounding microenvironment (28,29). Here, we retrospectively analyzed archived FFPE tissue samples from primary and recurrent tumors as well as matched premalignant and normal squamous mucosa from patients with p16-negative HNSCC.We used targeted panel Next Generation Sequencing (NGS) and shallow whole genome sequencing (WGS) on a selected number of tissues to profile the mutational landscape of multi-regionally and sequentially collected samples.Additional patients' samples were analyzed with the Nanostring PanCancer Immune Panel to explore the mechanisms underlying immune evasion.The aims of this study were to provide a characterization of the preconditioned squamous mucosa in a cancerized field and their corresponding primary and, if available, recurrent tumors to shed light into their genomic, immune microenvironmental and evolutionary relationship.We confirm that genomic alterations, along with dysregulation in immune-related gene expression, are detectable at an early stage in the development of upper aerodigestive tract tumors.These observations underscore the potential for targeting the mechanisms involved in the early stages of disease progression, offering new avenues for therapeutic intervention. ",
"section_name": "Introduction",
"section_num": "1"
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": "2"
},
{
"section_content": "A retrospective cohort study was conducted to profile nucleic acids from resected tumor tissue, dysplastic lesions, and normal squamous mucosa.All samples were previously obtained and archived from a cohort of seven patients with HNSCC and/or dysplastic lesions in the upper aerodigestive tract.Patients with p16-negative HNSCC and/or dysplastic lesions of the oral cavity, pharynx, or larynx were included.Patients with distant metastasis (cM1) or other active malignancies as well as p16-positive cases were excluded.All patients were staged to exclude distant metastasis with computed tomography (CT) and/or magnetic resonance imaging (MRI).Patients with confirmed HNSCC received primary surgery with curative intent and adjuvant radio(chemo) therapy, or primary radiochemotherapy, according to the National Comprehensive Cancer Network guidelines (NCCN Guidelines) (30) and following recommendation by the local multidisciplinary tumor board.Immunohistochemical staining for p16 was done as part of the routine histopathological work-up. ",
"section_name": "Case collection",
"section_num": "2.1"
},
{
"section_content": "Formalin-fixed paraffin-embedded (FFPE) tumor blocks of the resected specimen, if available, as well as corresponding dysplastic lesions and normal squamous mucosa were obtained from the archives.Sections from FFPE tumor tissue samples were prepared followed by hematoxylin-eosin (H&E) staining of one slide.Tumor areas were microdissected from subsequent unstained sections and used for nucleic acid preparation.For dysplastic lesion and normal squamous mucosa analysis, twenty sections were cut including three slides for H&E staining (first, middle and last slides) and the residual ones for further analysis.The final histopathological status was confirmed by an experienced head and neck pathologist.DNA was extracted from the FFPE tissue samples using the GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany).RNA was extracted from the FFPE tissue samples using the RNeasy FFPE Kit (Qiagen, Hilden, Germany).Nucleic acid concentrations were measured using a Qubit 2.0 fluorometer (Invitrogen, CA, USA) and the Qubit dsDNA HS (High Sensitivity) Assay Kit (Thermofisher, MA, USA). Ten ml blood was collected into K2 EDTA tubes (BD Biosciences) and processed to buffy coat, i.e. leukocytes, for extraction of germline DNA, respectively, as described previously (31). ",
"section_name": "Nucleic acid extraction",
"section_num": "2.2"
},
{
"section_content": "Library preparation with the AmpliSeq for Illumina Oncomine Comprehensive Assay v3 (Illumina, CA, USA), targeting 161 cancer-associated genes (Supplementary Table 1), subsequent sequencing, and variant calling were performed as described in detail previously (31).Briefly, sequencing was performed on an Illumina NextSeq 500 system using NextSeq 500/550 High Output Kits v2.5 according to the manufacturer's protocols.Analysis of the results was performed with the Illumina Local Run Manager, subsequent annotation of VCF-files using wANNOVAR (32) and an in-house python script filtering for relevant mutations.Sequencing quality parameters are shown in Supplementary Table 2. Alterations were confirmed with the Integrative Genomics Viewer (IGV, Broad Institute, MA, USA).Variants were judged as relevant based on the interpretation criteria utilized in ClinVar (33).Pathogenicity prediction algorithms and other publicly available databases were used for variant interpretation (VarSome (34), dbSNP (Available from: https:// www.ncbi.nlm.nih.gov/snp/)).Only likely pathogenic and pathogenic mutations as well as VUS (variant of unknown significance or not evaluated in ClinVar with a prediction trend of being likely pathogenic) were reported (Supplementary Table 3).Single-nucleotide variants (SNV), multi-nucleotide variants (MNV), small insertions, deletions, indels, and copy number variation (CNV) were analyzed. ",
"section_name": "Sequencing library preparation, Next Generation Sequencing and variant interpretation",
"section_num": "2.3"
},
{
"section_content": "Sequencing libraries were prepared using 4-60ng DNA by the ThruPLEX Tag-Seq Kit (Takara Bio, France).Quantity and quality of the libraries were assessed by Qubit dsDNA High Sensitivity Assay Kit (Thermofisher, MA, USA).Sequencing libraries were pooled at equal amounts.WGS at an average coverage of 0.5x was performed on Illumina NexSeq 500 using 2 x 150 bp paired-end sequencing.A normal reference was created from shallow WGS of leukocytederived genomic DNA of the respective patients as well as a panel of healthy donors sequenced at 0.5x coverage.High-quality and nonduplicate reads were aligned to human reference genome GRCh38 using BWA (35).Aligned reads were pre-processed, removing unreliable or badly mapped reads, and excluding PCR artifacts.Reads were corrected for GC content.Copy number analysis was performed using GATK (http://software.broadinstitute.org/gatk/ ) and the ichorCNA software (36) for tumor fraction and ploidy estimation. ",
"section_name": "Shallow whole genome sequencing and copy number calling",
"section_num": "2.4"
},
{
"section_content": "We performed gene expression profiling on 36 sequentially collected RNA samples from seven patients using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, U.S.A.), targeting 770 transcripts including up to 40 reference genes (Supplementary Table 4).150 ng RNA was used for gene expression analysis according to the manufacturer's instructions.Data analysis was performed using the nSolver 4.0 software (https:// www.nanostring.com/products/analysis-software/nsolver).The geometric mean of the counts relative to each sample, the mean plus two standard deviations and the total sum of counts were used to correct the data for technical, background, and batch effects, respectively.The expression profiles of housekeeping genes and quantile normalization were used to account for inter-sample variations within the panel (Supplementary Table 5).Heatmaps of the most significantly, differentially expressed genes using log2 fold change of normalized gene expression values were generated using ClustVis (37).Differentially expressed gene analysis was performed using the Gene Set Enrichment Analysis (GSEA) v4.3.2 software (38).Significant p-values and false discovery rates (FDR) were obtained.The criteria for differentially expressed genes have been set as FDR<0.25.The thresholds were set to log2 fold change > 1 and adjusted p-value < 0.05 for further analysis.Lists of ranked genes in differentially expressed signaling pathways are presented in Supplementary Table 6.Supplementary Tables 789show the most significantly upregulated signaling pathways (c6 oncogenic pathways, c7 immune pathways, hallmark pathways) for each phenotype. ",
"section_name": "Gene expression profiling",
"section_num": "2.5"
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3"
},
{
"section_content": "Seven patients who were diagnosed with a p16-negative HNSCC and/or premalignant lesion(s) of the upper aerodigestive tract (low-/moderate/high-grade dysplasia) and primarily treated by surgery or radiochemotherapy were included into this study.Most patients (71%) were male, and all had a diagnosis of invasive squamous cell carcinoma at some point during their treatment.Apart from one patient, all were scheduled for curative-intent surgery and, if applicable, received adjuvant treatment according to the recommendations of the multidisciplinary tumor board.Patients were followed up and received multiple resections or biopsies, respectively, throughout their treatment.4/7 patients developed a histologically confirmed recurrence after completing their treatment.A total of 36 retrospectively collected primary tumor, premalignant and normal mucosal tissue samples from seven patients were selected for further analysis.A schematic overview of the patient cohort, including types of analyses used on each sample, is shown in Figure 1. ",
"section_name": "Patient characteristics",
"section_num": "3.1"
},
{
"section_content": "Each patient is described in brief below and in more detail in the Supplementary Material (Supplementary Table 10). Patient 1 was an 81 y/o female smoker who received surgical treatment for a pT1 pN0 squamous cell carcinoma (SCC) of the soft palate with a surrounding field of high-grade dysplasia which had been confirmed by multiple biopsies. Patient 2 was a 72 y/o male smoker who initially received primary radiochemotherapy for a cT3 cN2b hypopharyngeal carcinoma.This was followed by multiple resections of dysplastic lesions at the left glossotonsillar sulcus until eventually a large cT4a cN2b carcinoma of the base of the tongue extending into the left lateral tongue was diagnosed 9 years later.The patient received palliative radiotherapy. Patient 3 was an 87 y/o male smoker who presented with dysphonia and initial biopsy of the right vocal cord showed a moderate dysplastic lesion.Control panendoscopies with multiple biopsies taken confirmed different grades of dysplasia on both vocal cords until eventually 7 months later an invasive cT1a laryngeal SCC was confirmed histologically and subsequently resected. Patient 4 was a 54y/o male smoker who received surgical treatment for an invasive pT2 pN0 hypopharyngeal carcinoma (right sinus piriformis) on a background of a high-grade dysplasia at age 54.Following initial tumor resection and a bilateral neck dissection, a high-grade dysplastic lesion at the right glossotonsillar sulcus was subsequently histologically confirmed and resected 8 months later, and at the right aryepiglottic fold 2 years later.Another 7 months later a pT1 carcinoma of the base of the tongue was confirmed and treated by transoral laser microsurgery.In addition, high-grade dysplastic lesions at the posterior wall of the pharynx were resected.The patient presented with a rpT3 rpN1 hypopharyngeal carcinoma 12 months later.Following a pharyngolaryngectomy, bilateral node picking and reconstruction using a pectoralis major flap, the patient subsequently received adjuvant radiotherapy. Patient 5 was a 57y/o male smoker who was surgically treated for a high-grade dysplasia of the right vocal cord and 4, 7, and 11 years later for a high-grade dysplasia of the left vocal cord.Three years following the previous resection, the patient was diagnosed with an invasive cT1b laryngeal carcinoma on the background of a high-grade dysplasia for which he received primary radiotherapy.Sixteen months later a pT1 carcinoma of the epiglottis was treated by transoral laser resection.The patient received a laryngectomy 18 months later due to a local recurrence with extensive dysplasia throughout the larynx.Another local recurrence on the background of the cancerized field as well as a pulmonary metastasis were diagnosed 12 months later, and systemic treatment was initiated. Patient 6 was a 62y/o female non-smoker who had multiple biopsies of the left lateral tongue within 3-and 4-years following diagnosis of a lateral tongue lesion that showed hyperplastic mucosa without evidence of dysplasia or invasive carcinoma.A pT3 pN0 carcinoma of the left lateral tongue was confirmed 5 years later and the patient received a transoral tumor resection and bilateral neck dissection and subsequent adjuvant radiotherapy.A local recurrence yrpT3 was confirmed 5 months later and treated by surgical resection.Only 3 months later the patient presented with an extensive loco-regional as well as distant recurrence that was unresectable and the patient was subsequently started on systemic treatment. Patient 7 was a 68y/o male smoker who received surgical treatment for a pT3 pN3b oropharyngeal carcinoma (left base of tongue and glossotonsillar sulcus) and subsequent adjuvant radiochemotherapy.A high-grade dysplasia was confirmed at the larynx (arytenoid cartilages) 3 years later for which the patient received photodynamic therapy multiple times.Several biopsies confirmed extensive dysplasia throughout the larynx and pharynx. ",
"section_name": "Patient summaries",
"section_num": "3.2"
},
{
"section_content": "DNA was isolated from sequentially collected, matched FFPE tissue (normal squamous mucosa, premalignant mucosal lesions, HNSCC).Pathological review ensured the correct histological diagnosis before proceeding with the analysis.We performed targeted panel NGS (161 genes) to analyze somatic variants from 12 samples that were obtained from two patients (patient 4 and patient 5).DNA sequencing was successful in all samples with a mean DNA sequencing depth of 3670.4.The average identified number of somatic variants per tumor after applying our filtering criteria was 3.3 (range 1 -5).For dysplastic lesions we identified on average 8.375 somatic mutations (range .No somatic mutations were identified in normal squamous mucosa (sample 15) after applying our filtering criteria.The most frequently mutated genes were TP53 (9/12) with predominantly missense and truncating mutations (Table 1) and NOTCH1 (9/12) with missense and truncating mutations as well as splicing variants (Table 2) identified (Figure 2).Other mutated genes included CDKN2A (6/12), CREBBP (7/12) and NOTCH3 (4/12).CTNNB1, PDGFRA, PIK3R1, FGFR3, FGFR4, NF1, MSH6, ATRX, FANCD2, TSC2, PIK3CA, and AKT2, among others, were each mutated in one or two samples.Many variants at low variant allele fraction (VAF) were identified in samples 19 and 20 and are likely due to extensive field cancerization (Supplementary Table 3).In total 43/53 variants were private to dysplastic lesions and not detected in invasive carcinoma whereas only 2/53 variants were private to invasive carcinoma and not detected in dysplastic lesions.8/53 variants were found in both dysplasia as well as invasive carcinoma.The median VAF of all detected somatic variants was 3.4% (range 1.7% -95.2%).The median VAF of TP53 in all tumors was 28% (range 22.9 -83.0) and of all dysplastic lesions 9.8% (range 1.8 -47.5), respectively.For NOTCH1, the median VAF in all tumors was 3.7% (range 2.6 -25.8) and of all dysplastic lesions 2.7% (range 1.7 -35.3), respectively. In addition to targeted panel sequencing, we performed shallow WGS (0.5x coverage on average) on 20 samples from three patients (patients 4, 5 and 6).For case 6, increasing genomic instability could be observed when comparing histologically normal squamous mucosa (sample 31) with hyperplastic mucosa (sample 26) and primary and recurrent tumors of the lateral tongue (samples 28, 30 and 32).The latter displayed an increasing loss of chromosome arm 3p over time (Figure 3).High-grade dysplastic samples (samples 14 and 17) from case 4 had similarly rearranged genomes as was seen in the HNSCC sample (sample 13) (Supplementary Figure 1).Sample 16 was likely contaminated by normal tissue and has therefore been disregarded in this analysis.Low-grade dysplasia (sample 18) was characterized by an equally quiet genome as normal squamous mucosa (sample 15) (Supplementary Figure 1).For case 5, moderate dysplasia (samples 20 and 23) displayed several copy number changes more similar to high-grade dysplastic samples (samples 19 and 22) than compared to low-grade dysplasia (sample 24).Sample 21 illustrated the characteristically highly rearranged genome of an invasive carcinoma (Supplementary Figure 2). ",
"section_name": "Point mutations and copy number changes in primary tumors, premalignant lesions, and normal mucosa",
"section_num": "3.3"
},
{
"section_content": "A total of 36 samples representing various histological states of premalignancy and malignancy from seven patients were analyzed for alterations in gene expression in oncogenic immune signaling pathways.We examined gene expression patterns comparing all seven cases by selecting the most significantly, differentially expressed genes which showed a stepwise increase or decrease from benign to malignant phenotype and were observed in more Overview of somatic variants identified in HNSCC, HGD, MGD, LGD, hyperplasia and normal tissue from patients 4 and 5. (A) Oncoplot of the most frequently mutated genes.This shows a list of genes arranged based on the total number of variants in each gene with the percentage representing the ratio of samples with its genetic alteration to the total number of samples.Type of mutation, sample histology and anatomical localization are explained in the legend.The variant prevalence and spectrum of TP53 (B), NOTCH1 (C), and NOTCH3 (D) genes in this cohort.Green circles indicate missense mutations, black circles truncating mutations, and orange circles splice sites.HNSCC, Head and Neck Squamous Cell Carcinoma; HGD, high grade dysplasia; MGD, medium grade dysplasia; LGD, low grade dysplasia. than one patient (Supplementary Figure 3).Four significantly, differentially expressed genes are depicted in Figure 4.The highest ranked transcript was SPINK1 which was consistently downregulated in all cases with decreasing expression from lowgrade (-1.03-fold, p = 0.616) to moderate (-1.16-fold, p = 0.109) to high-grade dysplasia (-1.39-fold, p < 0.005), and lowest expression in HNSCC (-1.71-fold, p < 10 -4 ).Another transcript that showed a stepwise differential expression in three cases (cases 4, 5, and 6) was SPP1.Lowest expression of SPP1 was observed in hyperplastic samples, with increasing expression from low-grade (0.79-fold, p = 0.76) to high-grade dysplasia (1.24-fold, p < 10 -4 ) and highest expression seen in invasive carcinoma (1.6-fold, p < 10 -5 ).Other differentially expressed transcripts include S100A7, LCN2, CXCL14, and CD207 (decreasing expression from premalignant lesions to invasive carcinoma), as well as IDO1, IL6, IL8, THBS1, and FN1 (increasing expression from premalignant lesions to invasive carcinoma).Looking across all samples, other candidates that were among the top differentially expressed genes in moderate dysplasia, high-grade dysplasia, and HNSCC included LAG3, PSMB9, and PSMB10, among others (Figure 5).When Copy number plots from normal mucosa (31), hyperplasia (25,26) and HNSCC (28) with 2 recurrences (30, 32) from the same patient (case 6).Corresponding images of the tongue lesions as well as CT images with the tumor circled in red are shown on the right.Tumor volume is given in cm 3 .Diploid copies are shown in blue, gains in red and losses in green. investigating the top scoring 15 differentially expressed genes among different histological groups, we observed a clustering of hyperplastic with low-grade dysplastic samples and a clustering of moderate with high-grade dysplastic as well as invasive samples (Figure 6).Among the top downregulated genes with increasing malignancy were SPINK5, MAPK3, and MAPK14.LAG3, IFI35, IDO1, and CD7 were among those genes with increasing expression from hyperplasia to HNSCC.We then applied GSEA to identify functional gene sets that were correlated with increasing levels of malignancy.The most significantly upregulated signaling pathways were the interferon gamma and alpha response pathway (Figure 7 and Supplementary Table 11).Interestingly, an upregulation of these pathways could already be seen in moderate dysplasia.In HNSCC, IL2 STAT5 signaling was significantly enhanced (Figure 7 and Supplementary Figure 4).Compared with LGD and hyperplasia samples, an upregulation of TNFa signaling via NFkB was observed in moderate and high-grade dysplasia (Supplementary Figure 5). ",
"section_name": "Differential gene expression analysis in primary tumors, premalignant lesions, and normal mucosa",
"section_num": "3.4"
},
{
"section_content": "In this study, we have conducted a focused analysis of the genome and immune microenvironment from multiple, matched normal squamous tissues, premalignant lesions, primary, and recurrent tumors from seven patients with p16-negative HNSCC with the aim to identify novel molecular mechanisms that could drive tumor evolution in the upper aerodigestive tract.The unique aspect of this study is the comprehensive profiling of multiregionally and sequentially collected and well annotated samples at different disease stages, which also included a focus on the role of the immune microenvironment. Targeted analysis of genetic alterations revealed a characteristic mutational pattern in HNSCC, akin to findings reported in previous studies (39)(40)(41).The most prevalent genetic mutations observed in our cohort were in the TP53 and NOTCH1 genes, followed by mutations in CDKN2A, CREBBP, NOTCH3, CTNNB1, FGFR3, and FGFR4, among others.A notable proportion of mutations were exclusive to dysplasia and carcinoma, respectively, although approximately 20% were shared between these two stages.Gene mutations rated as (likely) pathogenic that were already identified in low-grade and moderate dysplastic samples included inactivating mutations in the tumor suppressor genes TP53, CDKN2A, and NOTCH1, implying a crucial role in stepwise carcinogenesis towards HNSCC.These results corroborate the empirical progression model of Califano et al., where TP53 is disrupted early (5,42).In that model, the earliest event of disease progression includes loss of chromosome arm 9p, which harbors CDKN2A (43), progressing to loss of chromosome arms 17p, where TP53 resides, and 3p.In our cohort, in earlier grades of dysplasia, individual genes were present at low prevalence (<15% VAF).This may support an early role in tumorigenesis as suggested by Leshchiner et al. who inferred genetic progression from exome sequencing of primary HNSCC tumors (44).While the role of TP53 as tumor suppressor gene in HNSCC is well established and different mutations could be linked to increased malignancy (45) and therapy-resistance (46), the functional significance of NOTCH1 mutations is less clear (47).NOTCH1 and NOTCH3 belong to the family of cell surface receptors that transduce juxtacrine signals of delta-like canonical Notch ligands and jagged canonical Notch ligands from adjacent cells, with significant roles in directing tissue commitment and cell differentiation (48).Loss of function of NOTCH1 has been linked to the acquisition of stem-like properties and a more aggressive phenotype.However, tumorsuppressing mechanisms of NOTCH1 signaling are not entirely understood (49).High expression of Notch3 on the other hand has been linked to poorer prognosis in SCC (50).Observed loss of 9p and therefore CDKN2A with subsequent cell cycle dysregulation as an early event in the carcinogenesis are in line with previous findings (51).The function of tumor suppressors is inherently difficult to restore.However, a recent study could show meaningful response rates to selective CDK4/6 inhibitors in CDKN2A-altered HNSCC, proving the clinical significance of CDKN2A mutations (52).Whether this approach could also target premalignant lesions, needs to be established.Recent work has demonstrated a role for chromosomal instability driving selection in cancer evolution with early somatic copy number alterations potentially being implicated in tumorigenesis (53).Our results also support an increasing genomic instability over time.Case 5 showed a high rate of chromosomal alterations even in moderate dysplasia, including loss of 3p and 9p, that was followed by multiple disease recurrences.Similarly, case 6 demonstrated progressive loss of 3p with subsequent local tumor recurrences.Previous studies have shown that dysplastic lesions that harbor 3p loss are 33 times more likely to progress to invasive carcinomas than those without (54) and that 3p deletion may be associated with poor disease outcomes (55).Furthermore, high-grade dysplasia showed a similarly rearranged genome as was seen in HNSCC, confirming its behavior like an invasive carcinoma.The existence of these multilocular distinct premalignant conditions therefore presents the need and opportunity to define which are at greatest risk for progression, including if, how, and when to intervene. A recent comprehensive study on early-stage untreated lung cancers from the TRACERx cohort demonstrated an interplay between the immune and genomic landscapes through copy number loss as a mechanism of subclonal immunoediting.Immunoediting mechanisms may impact on tumor evolution, affecting either antigen presentation or neo-antigenic mutations both at DNA and RNA level with chromosomal instability driving loss of neo-antigens (28).Other studies were able to establish a link between somatic copy number alterations and markers of immune evasion, with highly aneuploid tumors being less responsive to immunotherapy (56).In HNSCC, cumulative loss of chromosome Heatmap of all significantly (p<0.05),differentially expressed genes in HNSCC, HGD and MGD across all samples, using log2 fold change of normalised gene expression values.Samples are listed on the horizontal axis and genes are listed vertically.Red/orange indicates high scores; blue indicates low scores.HNSCC, Head and Neck Squamous Cell Carcinoma; HGD, high grade dysplasia; MGD, medium grade dysplasia; LGD, low grade dysplasia.arm 9p has been shown to be the strongest driver of immune evasion.Simultaneously, copy number-defined high-risk oral preinvasive and early invasive lesions were immunogenic, suggesting a possible clinical benefit of augmenting immune surveillance (57).Nevertheless, the exact role of the immune microenvironment in the early stages of cancer development remains largely unknown, and particularly how survival and clonal expansion of premalignant cells may be fostered by an immunosuppressive microenvironment that hinders immune surveillance remains elusive.In our study, pathways associated with the interferon alpha and gamma response exhibited heightened activity, even in moderate dysplastic lesions, with a progressively amplified expression in higher dysplasia grades and in carcinoma.Interferon gamma has been linked to antitumorimmune response and increased interferon gamma signaling has been proposed as a prediction marker for response to immune checkpoint therapy in HNSCC (58).In contrast, low-dose interferon gamma might lead to tumor progression (59).Early upregulation of this signaling pathway in dysplasia might indicate an increased, yet insufficient, immune response.Likewise, interferon alpha signaling contributes to antitumor activity in early stages and has been shown to promote tumor growth in equilibrium and escape phase of cancer development (60).It can therefore be speculated, whether immunomodulatory therapy targeting these pathways in dysplasia (in combination with immune checkpoint therapy) could have beneficial effects.Interestingly, a recent study could show that immune checkpoint inhibition can achieve regression in high-risk oral leukoplakia (61).Moreover, progression to invasive carcinoma could be linked to 9p21.3 loss, encompassing the type I interferon gene cluster and CDKN2A, pointing towards a crucial role in therapy resistance and carcinogenesis. Single gene expression analysis revealed a few markers differentially expressed in different grades of dysplasia compared to HNSCC.One candidate gene that was upregulated in moderate dysplastic compared to low-grade dysplastic samples with highest expression in HNSCC was secreted phosphoprotein 1 (SPP1), also known as osteopontin (OPN).SPP1, an integrin-binding glycophosphoprotein, has been shown to be associated with immune cell infiltration and found to be upregulated in multiple cancers, including HNSCC (62).High SPP1 expression has previously been shown to be associated with poor overall survival in colorectal cancer, with increasing expression from normal tissue to primary tumors and ultimately to metastases (63).Indoleamine 2,3-dioxygenase 1 (IDO1), another significantly differentially expressed gene that we found to be upregulated from benign to malignant phenotype, is a rate-limiting enzyme in the conversion of the essential amino acid tryptophan to kynurenine.IDO1 is immune regulation, inflammation, and invasion, has been shown to be upregulated in various cancer types and also predicts poor clinical outcomes in patients with HNSCC (71)(72)(73)(74).In our study, we could already show an increase in IL6 expression in moderate and high-grade dysplastic samples.In accordance with the literature, we also found lymphocyte activation gene-3 (LAG3) overexpressed in HNSCC samples.LAG3 is an immune checkpoint control protein that negatively regulates T-cell and immune response (75).Here, our results demonstrated that LAG3 upregulation could already be observed in moderate dysplasia.(76).In the case of serine peptidase inhibitor Kazal type 5 (SPINK5), its downregulation was even evident in low-grade dysplastic lesions, signifying an early deactivation event in the disease's evolutionary trajectory.SPINK5 encodes for a serine protease inhibitor, which plays a crucial role in regulating various cellular processes, including tissue remodeling and inflammation.In HNSCC, SPINK5 has emerged as a critical player, often serving as a tumor suppressor gene (77).This suggests that SPINK5's reduced expression may occur early in the development of HNSCC, potentially contributing to the initiation and progression of the disease. Our study has several limitations, primarily stemming from the relatively small sample size.We acknowledge that drawing comprehensive conclusions from a cohort of just seven patients is inherently limited.However, we believe that our study adds valuable insights to the limited body of research that has explored the relationship of the genetic landscape with immune microenvironmental changes in p16-negative HNSCC and matching premalignant and normal squamous mucosa.Additionally, the absence of sequencing data from matched peripheral blood lymphocytes is a constraint, as it hampers the ability to filter out variants originating from clonal hematopoiesis of indeterminate potential (78).Nonetheless, we made substantial efforts to address this issue through rigorous filtering methods, including cross-referencing with the dbSNP database.One notable strength of our study lies in the detailed clinical characterization of the enrolled patients, with extensive follow-up periods and a diverse array of available samples, encompassing, primary and recurrent tumor tissues, as well as different grades of dysplastic lesions and matched normal squamous mucosa.The utilization of archived FFPE tissue aligns with common clinical practice, making our approach more pragmatic for potential clinical implementation. In summary, findings from this study show that genetic changes and aberrations in immune gene expression become detectable in the early stages of upper aerodigestive tumor development, underscoring the prospect of intervening in the initial disease progression mechanisms.A better understanding of the concept that premalignant lesions may transform presumably with acquired mutations and copy number alterations to escape immune surveillance is crucial.It could provide scientists with new targets for intervention in those patients who are at risk of progression to HNSCC and clinicians with biomarkers for risk stratification and therapy decision-making in patients with high-risk premalignant lesions.In future, a deeper and more comprehensive assessment of epithelial, stromal, and immune heterogeneity in morphologically comparable grades of dysplasia in a cancerized field could potentially help predicting clinical outcomes and guide personalized therapies in patients with high-risk premalignancy.Ultimately, personalized diagnostics to guide treatment decisions, thereby avoiding unnecessary surgical procedures, their associated morbidities as well as reducing health economic costs may only be achieved by larger studies to confirm promising molecular biomarkers.and Dohme, Novartis, QuIP GmbH, Roche Pharma, Takeda, and Thermo Fisher. 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. ",
"section_name": "Discussion",
"section_num": "3"
}
] |
[
{
"section_content": "We thank all the patients who participated in this study.We thank Sabine Sagebiel-Kohler and Konstanze Schleich for technical support. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The author(s) declare financial support was received for the research, authorship, and/or publication of this article.The authors received funding from \"Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München e.V.\" and \"Friedrich-Baur-Stiftung\". ",
"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 studies involving humans were approved by Local ethics committee of the Ludwig-Maximilians-Universität (LMU) in .The studies were conducted in accordance with the local legislation and institutional requirements.The participants provided their written informed consent to participate in this study. AJ received honoraria for talks and Consulting or Advisory Board and reimbursement for travel, accommodation, and expenses from Amgen, AstraZeneca, Bayer Pharmaceuticals, BMS, Biocartis, Boehringer Ingelheim, Merck KGaA, Lilly Oncology, MSD Sharp 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/fonc.2024.1364958/full#supplementary-material ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "The studies involving humans were approved by Local ethics committee of the Ludwig-Maximilians-Universität (LMU) in .The studies were conducted in accordance with the local legislation and institutional requirements.The participants provided their written informed consent to participate in this study. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "AJ received honoraria for talks and Consulting or Advisory Board and reimbursement for travel, accommodation, and expenses from Amgen, AstraZeneca, Bayer Pharmaceuticals, BMS, Biocartis, Boehringer Ingelheim, Merck KGaA, Lilly Oncology, MSD Sharp ",
"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/fonc.2024.1364958/full#supplementary-material ",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.4321/s0365-66912007000500010
|
Edema bilateral del nervio óptico por leucemia linfocítica crónica
|
Caso clínico: Varón de 65 que consulta por pérdida de agudeza visual bilateral. Había sido diagnosticado de leucemia linfocítica crónica hacía 2 años y se encontraba sin tratamiento. Su agudeza visual era de 20/200 en el ojo derecho y 20/63 en el izquierdo y presentaba edema bilateral de papila. Se realizaron tomografía axial computerizada y resonancia magnética orbital y cerebral encontrándose una masa orbitaria con extensión al etmoides e infiltración de ambos nervios ópticos cuya biopsia reveló leucemia linfocítica crónica. Discusión: La infiltración leucémica del nervio óptico y del sistema nervioso central es extremadamente rara en la leucemia linfocítica crónica al contrario de lo que sucede en las formas agudas. La infiltración del nervio óptico es la manifestación oftalmológica leucémica que implica mayor trascendencia clínica y terapéutica por amenazar seriamente la función visual y ser tratable.
|
[
{
"section_content": "La afectación leucémica del nervio óptico (NO) reviste gran importancia por ser de una causa tratable de perdida irreversible de visión (1)(2)(3)(4); es por ello que es sumamente recomendable realizar un examen oftalmológico en el momento del diagnóstico a todos los casos de leucemia (2). Existen varios tipos de leucemia y no todos ellos son igualmente capaces de afectar al nervio óptico.Así, la leucemia linfocítica crónica (también llamada leucemia linfática crónica), es excepcional que afecte al NO, por lo que existen muy pocos casos publicados (1,5).En este artículo se presenta el caso de un paciente con leucemia linfocítica crónica e infiltración bilateral del NO. ",
"section_name": "INTRODUCCIÓN",
"section_num": null
},
{
"section_content": "Varón de 65 años que acude a la consulta de urgencias de oftalmología por perdida brusca bilateral de agudeza visual. Como antecedentes generales presentaba una leucemia linfocítica crónica diagnosticada hacía 2 años.En el momento del episodio oftalmológico se encontraba sin tratamiento y en seguimiento en el servicio de hematología de nuestro hospital. Presentaba una agudeza visual en visión lejana y con su corrección habitual de 20/200 que no mejoró con estenopeico en el ojo derecho (OD) y de 20/80 que mejoró a 20/63 con estenopeico en el ojo izquierdo (OI). La exploración de los reflejos pupilares reveló unas pupilas bradicóricas objetivándose un discreto defecto pupilar aferente relativo en el OD. La exploración biomicroscópica sólo reveló una pseudofaquia con lente intraocular en cámara posterior en ambos ojos. La exploración del fondo de ojo reveló una gran tumefacción papilar con pérdida total de los límites papilares en AO.Además en el OD existía una gran hemorragia peripapilar concéntrica a la papila, de unos 3 diámetros de disco y que temporalmente se extendía hasta la macula (figs. 1 y 2). Se realizó una tomografía axial computerizada (TAC) craneal y orbitaria de forma urgente encontrándose una masa infiltrativa que ocupaba ambos vértices orbitarios con extensión a hendiduras orbitarias superiores y senos cavernosos; la lesión se extendía hasta la cisterna prepontina englobando a arteria basilar y frontalmente, al etmoides (fig.3).También se realizó una resonancia magnética cerebral y orbitaria que confirmó los hallazgos de la TAC mostrando además, infiltración del nervio óptico a nivel de ambos vértices orbitarios (fig.4).Finalmente se completó la exploración sistémica con una TAC cervico-toraco-abdominal que no reveló cambios respecto a la situación previa del paciente. Se decidió junto con el servicio de otorrinolaringología realizar una biopsia de la infiltración etmoidal por entender que era esta la zona afectada más accesible.El estudio histopatologíco fue compatible con leucemia linfocítica crónica. Desde el punto de vista oftalmológico, se estableció el diagnóstico de edema de papila secundario a infiltración retrobulbar del nervio óptico. Ante la situación anatómica de la afectación leucémica y sus severas consecuencias visuales se instauró de inmediato tratamiento quimioterápico con fludarabina y ciclofosfamida así como radioterapia craneal y administración de corticoides sistémicos. La evolución fue buena, objetivándose 4 meses después del tratamiento, una regresión de la masa tumoral en las pruebas de imagen; asimismo la exploración funduscópica evidenció una resolución de la infiltración papilar encontrándose las papilas bien delimitadas con ligera palidez (figs.5 y 6).La agudeza visual en este momento mejoró hasta 20/50 en el ojo derecho y 20/32 en el ojo izquierdo. ",
"section_name": "CASO CLÍNICO",
"section_num": null
},
{
"section_content": "La infiltración del NO es una complicación extremadamente rara en la leucemia linfocítica crónica si bien existen anteriores casos descritos en la literatura (1,5).Cuando este u otro tipo de leucemia afectan al NO se distinguen dos tipos de afectación, la preliminar y retrolaminar.En los casos en los que se afecta la porción prelaminar del nervio óptico puede observarse directamente a la tumoración emer- giendo por la papila.En los casos en los que la afectación es retrolaminar la imagen funduscópica es la de un edema papilar (3,5).En nuestro caso, la infiltración del nervio óptico en los vértices orbitarios, produjo la imagen de papiledema descrita. Son precisamente los casos de afectación retrolaminar los que plantean un problema de diagnóstico diferencial con el papiledema (4); en estos casos la unilateralidad del proceso orienta hacia la afectación leucémica.La infiltración leucémica bilateral de NO es un cuadro atípico (4,5) y puede constituir un elemento de confusión que oriente el diagnóstico en el sentido de un proceso expansivo intracraneal al remedar un papiledema (4,5).En este caso, el diagnóstico previo de leucemia linfocítica crónica permitió sospechar la afectación leucémica y las pruebas de imagen determinar que la causa de la tumefacción papilar fue la infiltración retrolaminar del nervio óptico.El diagnóstico diferencial de la tumefacción de la cabeza del nervio óptico comprende, además del papiledema por hipertensión intracraneal, las neuritis anteriores, la neuropatía óptica isquémica anterior, las drusas del nervio óptico, el pseudopapiledema y la enfermedad de Leber. La infiltración del nervio óptico es la manifestación oftalmológica leucémica que implica mayor trascendencia clínica y terapéutica por amenazar seriamente la función visual y ser tratable (2)(3)(4)(5).Atendiendo a lo anterior, un examen oftalmológico completo debería formar parte de la exploración de todo paciente diagnosticado de leucemia o recidiva leucémica (3).Debe tenerse presente que la función visual puede estar preservada en las fases iniciales de la afectación leucémica del NO cuando ya existen signos oftalmoscópicos de afectación (4)(5).Ante una exploración oftalmológica compatible con papiledema es sumamente importante considerar el diagnóstico diferencial con una infiltración leucémica bilateral del NO; en estos casos, pruebas complementarias tales como el hemograma, la punción de médula ósea, la punción lumbar y las pruebas de imagen nos pueden orientar en un sentido u otro permitiéndonos instaurar el tratamiento oportuno para cada caso (4). ",
"section_name": "DISCUSIÓN",
"section_num": null
},
{
"section_content": "",
"section_name": "BIBLIOGRAFÍA",
"section_num": null
}
] |
[] |
10.3390/cancers11070896
|
Elevated Lactate Dehydrogenase Has Prognostic Relevance in Treatment-Naïve Patients Affected by Chronic Lymphocytic Leukemia with Trisomy 12
|
<jats:p>Chronic Lymphocytic Leukemia (CLL) patients with +12 have been reported to have specific clinical and biologic features. We performed an analysis of the association between demographic; clinical; laboratory; biologic features and outcome in CLL patients with +12 to identify parameters predictive of disease progression; time to treatment; and survival. The study included 487 treatment-naive CLL patients with +12 from 15 academic centers; diagnosed between January 2000 and July 2016; and 816 treatment-naïve patients with absence of Fluorescence In Situ Hybridization (FISH) abnormalities. A cohort of 250 patients with +12 CLL followed at a single US institution was used for external validation. In patients with +12; parameters associated with worse prognosis in the multivariate model were high Lactate DeHydrogenase (LDH) and β-2-microglobulin and unmutated immunoglobulin heavy-chain variable region gene (IGHV). CLL patients with +12 and high LDH levels showed a shorter Progression-Free-Survival (PFS) (30 months vs. 65 months; p < 0.001), Treatment-Free-Survival (TFS) (33 months vs. 69 months; p < 0.001), Overall Survival (OS) (131 months vs. 181 months; p < 0.001) and greater CLL-related mortality (29% vs. 11% at 10 years; p < 0.001) when compared with +12 CLL patients with normal LDH levels. The same differences were observed in the validation cohort. These data suggest that serum LDH levels can predict PFS; TFS; OS and CLL-specific survival in CLL patients with +12.</jats:p>
|
[
{
"section_content": "The clinical course of chronic lymphocytic leukemia (CLL) is very heterogeneous and the identification of prognostic and predictive factors for CLL is of great relevance and a field of active investigation [1,2].During the last decade several laboratory biomarkers have been identified as being correlated with outcomes for risk assessment [3][4][5][6].The analysis of aberrant chromosomal regions with specific DNA probes by fluorescence in situ hybridization (FISH) resulted in the detection of clonal aberrations and the main recurrent chromosomal abnormalities (del13q, +12, del11q and del17p) define different personal genetic profiling subgroups in CLL [7,8]. Trisomy 12 is the second most frequent cytogenetic abnormality identified by FISH in patients with CLL [7].It presents as an isolated aberration in about 70% of cases and when it is associated with additional chromosomal abnormalities portends a poor prognosis [7][8][9][10][11]. CLL patients with +12 have unique morphologic and immunophenotypic characteristics [12].CLL cells with +12 commonly have an atypical morphology, defined as the presence of cleaved nuclei and/or lymphoplasmacytoid features in more than 15% of cells [13][14][15][16].When analyzing their immunophenotype, cases with +12, in comparison with cases with a normal FISH, show a significantly higher expression of CD19, CD22, CD20, CD79b, CD24, CD27, CD38, CD49d, sIgM, sIgk and sIgλ and a lower expression of CD43 [9]. CLL patients with +12 show a greater percentage of unmutated immunoglobulin heavy-chain variable region gene (IGHV) cases (54%) versus those with del13q and normal karyotype (37% and 31%, respectively) [14][15][16][17].IGHV studies reported a significantly more frequent expression of stereotyped B-cell receptors compared to patients with CLL and no +12 (44% vs 27% respectively), with a higher prevalence of the IGHV 4-39 gene, particularly in cases that later developed Richter's syndrome (RS) [17][18][19].The role of the IGHV mutational status in predicting the clinical course of CLL patients with +12 has been well investigated by Bulian et al. [20]: it proved to be the sole prognostic factor able to stratify overall survival (OS) and time-to-first treatment (TTFT) in +12 CLL. CLL patients with +12 rarely have TP53 mutations or acquire them over time [6,21].On the contrary, NOTCH1 mutations are very frequent in +12 CLL patients and are detected in 30-40% of the cases [22][23][24]. In the literature there are few reports that describe in detail the clinical features of CLL cases with +12 [17,[25][26][27]: the two largest series were reported by Marin et al., (289 patients) [26] and Strati et al., (250 patients) [27].We now present a large series of treatment-naive +12 CLL patients and correlate the association between demographic, clinical, laboratory, and biologic features and clinical outcomes. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "Four-hundred and eighty-seven patients with CLL and +12 and 816 patients with negative FISH were included in the study.+12 patients had a median age at diagnosis of 65.5 years (range 33-90) with a male/female (M:F) ratio of 1.68 (305 males, 63%; and 182 females, 37%).Negative FISH patients had a median age at diagnosis of 63.0 years (range 27-93) and a male/female M:F ratio of 1.58 (500 males, 61%; and 316 females, 39%).All clinical, laboratory, and biologic characteristics at diagnosis are presented in Table 1.Patients with +12 had significantly higher levels of lactate dehydrogenase (LDH) and β-2-microglobulin; more frequently expressed ZAP70, CD38, and CD49d (p < 0.001); and more frequently had an unmutated IGHV status compared to the control group (p < 0.001). ",
"section_name": "Patients' Characteristics",
"section_num": "2.1."
},
{
"section_content": "2.2.1.Patients +12 CLL vs. Negative FISH CLL Among the 487 CLL patients with +12, 311 (64%) progressed, 298 received treatment (61%), and 125 (26%) died, resulting in a median progression-free-survival (PFS) of 51 months (confidence interval (CI) 95%: 44 to 64), a median treatment-free-survival (TFS) of 59 months (CI 95%: 47 to 65) and a median OS of 170 months (CI 95%: 147 to 182).We then analysed data regarding the 816 CLL patients with negative FISH: 374 progressed (46%), 328 received treatment (40%), and 145 died (18%), resulting in a median PFS of 100 months (CI 95%: 81 to 118), a median TFS of 141 months (CI 95%: 112 to 160), and a median OS of 199 months (CI 95%: 196 to 210).When comparing the curves, we noted significantly shorter survival of +12 patients than FISH negative in terms of PFS (p < 0.001; Figure 1A), TFS (p < 0.001; Figure 1B) and OS (p < 0.001; Figure 1C).No differences were noted in the occurrence of Richter transformation and second cancers between the two groups.We next performed analysis of all the investigated categorical and continuous variables to identify parameters associated with shorter survival in +12 CLL patients.In univariate analysis, Binet and Rai advanced stage, elevated LDH, and β-2-microglobulin levels and unmutated IGHV were associated with shorter PFS, TFS, and OS; ZAP70 positivity was associated with shorter TFS and OS; whereas older age and positivity for CD38 were associated only with shorter OS.In multivariate analysis, elevated LDH and β-2-microglobulin levels, unmutated IGHV, and Rai advanced stage maintained their association with PFS and TFS, whereas elevated LDH and β-2-microglobulin levels, unmutated IGHV, and older age showed significance for OS (Table S1). We noticed that elevated LDH levels at diagnosis were more common in CLL patients with +12 compared to negative FISH patients (31% versus 14%, p < 0.001).These levels, as a categorical variable in which each centre declared their ranges and upper limits, were considered elevated if stable over the time.All CLL patients included in the analysis did not show lymphadenomegaly or symptoms suggestive of RS or haemolytic anaemia at the onset of the disease that could cause high LDH levels. ",
"section_name": "Outcomes",
"section_num": "2.2."
},
{
"section_content": "Since LDH is an inexpensive and routinely performed laboratory parameter, we evaluated the frequency of elevated levels of LDH at diagnosis in +12 CLL patients.Among patients with +12, 142 (31.4%) patients showed elevated LDH levels and 310 (68.6%) patients showed levels within normal range.The characteristics of the two subgroups are summarized in Table 2.The patients with +12 and high LDH levels progressed in 72% of the cases with a median PFS of 30 months (CI 95% 20-42) and needed treatment in 70% of the cases with a median TFS of 33 months (CI 95% 24-45).In comparison, the patients with +12 and normal LDH levels progressed in 59% of the cases with a median PFS of 65 months (CI 95% 58-73) and needed treatment in 55% of cases with a median TFS of 69 months (CI 95% 62-89).Regarding OS, 113 patients died among the +12 CLL patients, 47 (33%) of those with high LDH and 66 (21%) of those with normal LDH.The median OS was 131 months (CI 95% 97-167) in the patients with high LDH and 181 months (CI 95% 166-199) in the patients with normal LDH.Comparing the outcomes between the two subgroups, statistically significant shorter PFS (p < 0.001; Figure 2A), TFS (p < 0.001; Figure 2B), and OS (p < 0.001; Figure 2C) were observed in the patients with high LDH levels.We then compared the causes of death in these patients.Thirty (64% of all the deaths) in the high LDH group and 23 (35%) in the normal LDH group were due to CLL.The median CLL-specific survival was 147 months in the high LDH group and 190 months in the normal LDH group with a different rate of CLL-related mortality at 10 years: 29% vs. 11% (Figure 3).When performing multivariate analysis in CLL patients with +12 according to CLL-specific survival, the role of LDH was confirmed (p < 0.001, hazard ratio (HR) 3.78, CI 95% 1.73-8.26;Table S1). We then investigated whether the negative prognostic role of LDH observed in +12 CLL could be extended to CLL patients with negative FISH.Analyzing the same variables and using the same univariate model followed by a multivariate model for the significant variables, LDH had a role for PFS and TFS in univariate analysis but it was not significant in multivariate analysis in which Binet stage, CD38, β-2-microglobulin, and the IGHV status showed a significant role.With respect to OS and CLL-specific survival, LDH was not significant even in univariate analysis (Table S2). Patients with +12 and normal LDH levels showed no significant differences in survival compared to patients with negative FISH (p = 0.22 for OS and p = 0.61 for CLL-specific survival; Figure 4A,B) indicating that the difference in outcomes is dependent on the patients with +12 and elevated LDH. ",
"section_name": "Characteristics of +12 and FISH-Negative CLL Patients Stratified according to LDH Levels",
"section_num": "2.2.2."
},
{
"section_content": "To validate the worse prognosis of +12 CLL patients with high LDH levels, the same analysis was performed on a population of 250 patients with +12 from a single US institution.Baseline patients' characteristics at presentation are shown in Table S3.This population was divided according to LDH levels available at diagnosis: the two subgroups of 104 and 145 patients with high LDH levels and normal LDH levels, respectively, are presented in Table S4.Differences in the outcomes were found also in these subgroups: patients with high LDH levels showed a shorter median PFS (24 months vs. 55 months in patients with normal LDH levels, p < 0.001; Figure 5A), shorter median TFS (25 months vs. 58 months, p < 0.001; Figure 5B), and higher death rate (22% vs. 11% from all causes and 65% vs. 12% for CLL-related mortality).Also OS (92 months vs. 103 months; p = 0.012; Figure 5C) and CLL-specific survival (99 months vs. 128 months; p < 0.001; Figure 5D) were significantly shorter in the high LDH subgroup.A univariate analysis was performed in this validation cohort, as in the multicenter cohort: Rai stage, LDH, ZAP70 and β-2-microglobulin resulted significant for PFS and TFS; age, LDH and β-2-microglobulin for OS; LDH and β-2-microglobulin for CLL-specific survival.When these variables were analysed in a multivariate model, LDH was the sole negative independent parameter for PFS, TFS, and CLL-specific survival; LDH and age were significant for OS (Table S5). ",
"section_name": "Validation Cohort",
"section_num": "2.2.3."
},
{
"section_content": "Our retrospective study confirmed that CLL patients showing +12 on FISH analysis have unique clinical and biologic features as reported in the literature [12,25].The higher expression of CD38 observed in +12 by Athanasiadou et al. [14] was confirmed by our data with a rate of 51%; also the rates of CD49d and ZAP70 were higher in +12 CLL vs. negative FISH CLL (79% and 54% vs. 27% and 36%, respectively).Finally, the higher prevalence of unmutated IGHV in our series of +12 CLL patients (57%) confirmed previously published data [17]. In our series, CLL patients with +12 had a worse prognosis compared to CLL patients with negative FISH disease.The biomarkers associated with shorter PFS, TFS, OS in multivariate analysis were high LDH, unmutated IGHV, and elevated β-2-microglobulin.It resulted that Rai stage was significant only for shorter PFS and shorter TFS, and age only for shorter OS. Data about the correlation between the IGHV status and outcome in CLL are well known from the literature, including +12 patients [20].Also in our cohort, high levels of β-2-microglobulin predicted a worse prognosis in terms of PFS, TFS, and OS, as reported in the literature.Advanced Rai stage had an impact on PFS and TFS and older age on OS, as expected. Analyses about clinicopathologic risk categorization of untreated CLL patients aimed to find predictive outcomes variables: a parameter such as LDH was identified in a study as an independent predictor of TFS independently from FISH categories and the impact of LDH was greater for patients with unmutated IGHV [28], in another case LDH was not included among the variables for a comprehensive prognostic index [29], but our report is the first one about biomarker approaches on the relationship between +12, high LDH levels, and outcome using both a discovery and a validation series of a great size. When we attempted to analyse the role of LDH by stratifying the +12 CLL cohort according to the LDH levels at diagnosis, patients with high LDH levels showed a significantly worse outcome in terms of PFS, TFS, OS, and CLL-specific survival both in univariate and multivariate analyses.None of the +12 CLL patients, including those with high LDH levels, showed signs or symptoms suggestive of RS at the onset of the disease. Overall, our data are consistent with previous reports that +12 CLL patients have an intermediate risk of progression compared to the other FISH-defined prognostic subsets [7].However, stratifying by LDH levels, it appeared clear that +12 CLL patients with high LDH levels showed a worse prognosis compared to +12 CLL patients with normal LDH levels, for whom prognosis was similar to that of patients with negative FISH. In a wide validation cohort of 250 patients, the parameters which resulted significant were comparable to the ones of the multicenter population, confirming the predictive role of LDH: it resulted that it was the sole negative independent biomarker for PFS, TFS, and CLL-specific survival and it was significant for OS together with age.These data reinforced the impact of LDH in the +12 CLL population, which is marked by unique clinical and biological features that could explain a high rate of LDH levels above the limit, possibly linked also to atypical morphological characteristics of their cells.So it is suggested that LDH, an easily available individualized tool capable of predicting a worse PFS, TFS, and OS, should be taken into account in daily clinical practice. It would be useful in future studies to investigate outcomes in the LDH-high and LDH-normal +12 CLL patients according to the treatment received, in particular after therapy with the novel targeted agents.Unfortunately, this was not possible in the current study because of the relatively small number of patients treated with targeted agents and short follow-up, precluding a meaningful statistical comparison.In addition, future studies should address the association between high LDH levels and NOTCH1 mutation, which has been detected in 30-40% of +12 cases.Such data were not available for our current study and may provide an explanation for the correlation between high LDH levels and poor prognosis in CLL patients with the +12 abnormality. ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "This is a retrospective observational study including treatment-naive CLL patients with +12 as an isolated aberration from 15 academic Italian centers, diagnosed between January 2000 and July 2016.A second cohort of patients who resulted negative for a FISH panel comprising the four common abnormalities, i.e., del13q, +12, del11q, and del17p, was collected from the same centers during the same study period.A previously published overlapping cohort of patients with +12 CLL followed at a single US institution was used for external validation but new and updated data were included [27]. CLL was diagnosed in all the patients according to the 2008 International Workshop on CLL (iwCLL) guidelines [30].Patients were also screened at baseline with a direct antiglobulin test and radiological examinations and if other possible causes of LDH elevation such as signs or symptoms suggestive of RS or haemolytic anaemia were noted, these patients were excluded from the analysis. Demographic, clinical, and laboratory data were collected for each patient.Older age was defined as ≥65 years or older, advanced stage as Rai stage III-IV and Binet stage C. IGHV somatic mutation status and expression of CD38, ZAP70, CD49d, were performed in a standardized fashion by all involved centers, as previously described [31,32].FISH analysis was performed on interphase nuclei of CLL cells of the peripheral blood and the panel included probes specific to TP53 (17p13.1),ATM (11q22.3),D13S319 (13q14.3),LAMP1 (13q34), and the centromeric region of chromosome 12 (12p11.1-q11)[33].Disease progression, treatment initiation, and death were recorded to calculate PFS, TFS, and OS. The study was approved by the Ethics Committee of the 'Fondazione Policlinico Gemelli' (Protocol No. 0028829/16; Date: 13th July 2016) and was conducted in accordance with the principles of the Declaration of Helsinki.The clinical and laboratory features were obtained by review of the medical records and all the data were centrally collected and analysed. PFS, TFS, and OS were calculated from the date of diagnosis to the date of progression, treatment, and death, respectively, or the date of last follow-up.We defined CLL-specific death as death secondary to progressive disease, RS, infections, and complications during the treatment or in patients with an active disease. Normality distributions of all variables were tested by the Shapiro-Wilk and Shapiro-Francia tests.Chi-square test or Fisher's exact test were used to compare categorical variables (age, gender, Binet and Rai stage, LDH and β-2-microglobulin levels, positivity for ZAP70, CD38 and CD49d, IGHV mutational status), while the Wilcoxon-Mann-Whitney test was applied for continuous variables (white blood and lymphocytes counts, haemoglobin and platelets levels, bone marrow infiltration).Data were summarized as medians, 25th and 75th percentiles.The Kaplan-Meier method was used for survival analyses, and the log-rank test was performed to compare patient subgroups.Univariate and multivariate Cox proportional hazards regression models were fit to assess associations between patients' characteristics and survival times.The proportional hazards assumption was assessed using the method of Grambsch et al. [34].Only variables in univariate Cox analysis with a p < 0.01 (or p < 0.05 for validation cohort) were added to the multivariate Cox regression model.p values lower than 0.05 were considered statistically significant and reported as two-sided.All statistics were carried out with the use of STATA/SE 12.0 for Windows. ",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "From our multicenter study on 487 patients with +12 and 816 patients with negative FISH and from the analysis of our validation cohort on 250 patients with +12, it emerged that (1) CLL patients with +12 have a significantly higher prevalence of elevated LDH compared to patients without ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Funding: This research received no external funding. The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Funding: This research 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": "FISH abnormalities, (2) +12 CLL patients have a worse prognosis than negative FISH CLL patients, (3) +12 CLL patients with normal LDH levels have the same prognosis as that of negative FISH patients, and (4) +12 CLL patients with high LDH levels show a worse PFS, TFS, and OS than +12 CLL patients with normal LDH levels or negative FISH patients independently of LDH levels. The following are available online at http://www.mdpi.com/2072-6694/11/7/896/s1,Table S1: Multivariate analysis in +12 CLL patients (Cox regression analysis); Table S2: Multivariate analysis in negative FISH CLL patients (Cox regression analysis); Table S3: Baseline characteristics of the validation cohort; Table S4: Baseline characteristics of the validation cohort CLL patients with +12 divided in two subgroups according to LDH levels; Table S5: Multivariate analysis in validation cohort patients (Cox regression analysis). ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "FISH abnormalities, (2) +12 CLL patients have a worse prognosis than negative FISH CLL patients, (3) +12 CLL patients with normal LDH levels have the same prognosis as that of negative FISH patients, and (4) +12 CLL patients with high LDH levels show a worse PFS, TFS, and OS than +12 CLL patients with normal LDH levels or negative FISH patients independently of LDH levels. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following are available online at http://www.mdpi.com/2072-6694/11/7/896/s1,Table S1: Multivariate analysis in +12 CLL patients (Cox regression analysis); Table S2: Multivariate analysis in negative FISH CLL patients (Cox regression analysis); Table S3: Baseline characteristics of the validation cohort; Table S4: Baseline characteristics of the validation cohort CLL patients with +12 divided in two subgroups according to LDH levels; Table S5: Multivariate analysis in validation cohort patients (Cox regression analysis). ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
10.7150/jca.12135
|
The Histone Deacetylase Inhibitor Vaproic Acid Induces Cell Growth Arrest in Hepatocellular Carcinoma Cells via Suppressing Notch Signaling
|
Hepatocellular carcinoma (HCC) is a type of malignant cancer. Notch signaling is aberrantly expressed in HCC tissues with more evidence showing that this signaling plays a critical role in HCCs. In the present study, we investigate the effects of the anti-convulsant drug valproic acid (VPA) in HCC cells and its involvement in modulating Notch signaling. We found that VPA, acting as a histone deacetylase (HDAC) inhibitor, induced a decrease in HDAC4 and an increase in acetylated histone 4 (AcH4) and suppressed HCC cell growth. VPA also induced down-regulation of Notch signaling via suppressing the expression of Notch1 and its target gene HES1, with an increase of tumor suppressor p21 and p63. Furthermore, Notch1 activation via overexpressing Notch1 active form ICN1 induced HCC cell proliferation and anti-apoptosis, indicating Notch signaling played an oncogenic role in HCC cells. Meanwhile, VPA could reverse Notch1-induced increase of cell proliferation. Interestingly, VPA was also observed to stimulate the expression of G protein-coupled somatostatin receptor type 2 (SSTR2) that has been used in receptor-targeting therapies. This discovery supports a combination therapy of VPA with the SSTR2-targeting agents. Our in vitro assay did show that the combination of VPA and the peptide-drug conjugate camptothecin-somatostatin (CPT-SST) displayed more potent anti-proliferative effects on HCC cells than did each alone. VPA may be a potential drug candidate in the development of anti-HCC drugs via targeting Notch signaling, especially in combination with receptor-targeting cytotoxic agents.
|
[
{
"section_content": "Hepatocellular carcinoma (HCC) is a type of malignant primary liver cancer with one of the lowest five-year survival rates (1).Conventional therapeutics, including chemotherapy and radiotherapy, have very limited efficacy with severe toxic side effects.Currently, sorafenib, a dual multiple kinase inhibitor of Raf kinase and VEGFR tyrosine kinase, is the only drug approved for treating advanced HCCs.Thus, there is an urgent need to develop novel and specific HCC-targeting drugs. Reportedly, HCC is strongly associated with hepatitis (2)(3)(4).However, various other signaling pathways like Hedgehog signaling, are also involved in HCC progression (5)(6)(7).Currently, the precise molecular mechanisms are not clearly known.Compared to other cancers, certain gene mutations in HCC cells such as PIK3CA/PTEN were less or undetectable (7).Conversely, some others such as Notch1/Notch3 are mutated more frequently in HCC cells (5,7).These mutations result in an aberrance of the associated molecular signaling.Many of these signaling pathways are involved in the pathogenesis of malignant HCC, including Wnt signaling, p53 signaling, chromatin remodelling, histone acetylation/deacetylation, Notch signaling, JAK signaling, Hedgehog signaling and multiple growth factors (5,(7)(8)(9).Notch signaling is frequently reported aberrantly activated in HCCs and various types of human cancers (10)(11)(12)(13) and is involved in carcinogenesis and cancer progression (12,14,15). Notch signaling, besides being involved in liver development, also plays a critical role in HCC carcinogenesis (16)(17)(18)(19)(20).Most HCC tissues have high Notch signatures, especially the overexpression of Notch1, Notch3 and Notch4 (12,(21)(22)(23).This accumulating evidence supports that Notch signaling may play an oncogenic role in HCC cells (5,16,21,23,24).Thus, it may be a potential therapeutic strategy for developing anti-HCC drugs to target Notch signaling pathways.However, there are other studies showing that Notch signaling is controversial (25)(26)(27).In the present study, we evaluate the effects of Notch signaling activation in HCC cells. Meanwhile, histone deacetylase (HDAC) inhibitors have been used to treat cancers and other diseases (28,29).Several of these HDAC inhibitors display effective anti-tumor efficacy by modulating multiple signaling pathways (15,30).The HDAC inhibitor VPA is currently being investigated for treating various cancers (30)(31)(32)(33).Our previous studies have demonstrated VPA's anti-cancer efficacy in cervical cancer cells, with the involvement of Notch signaling (13,34).VPA was also found to induce cell growth arrest in some other HCC cell lines (35)(36)(37).In the present study, we evaluated the role of Notch signaling and the effects of VPA in HCC HTB-52 cells having a high Notch signature.We attempt to understand whether Notch signaling is involved in VPA-mediated cell growth suppression.VPA also was evaluated for its effect on the expression of certain G protein-coupled receptors (GPCRs) and for its use in a combination treatment with the somatostatin receptor type 2 (SSTR2)-targeting peptide-drug conjugate camptothecin-somatostatin cytotoxin (CPT-SST). ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Valproic acid sodium salt (VPA) was purchased from Sigma (St. Louis, MO).Antibodies to SSTR2 (Cat.No.: sc-11609), p21 (sc-756), p63 (sc-8343), histone 4 (H4, sc-10810), HDAC4 (sc-11418), acetylation of histone 4 (AcH4, sc-8660-R), HES1 (sc-25392) and β-actin (sc-1616-HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), with Notch1 (Cat.No.: 4380) from Cell Signaling Technology (Danvers, MA).The plasmids are the kind gifts from Dr. Wu (University of Florida).The cytotoxic CPT-SST was synthesized in our laboratories (38). ",
"section_name": "Materials",
"section_num": null
},
{
"section_content": "Human HCC HTB-52 (SK-HEP-1) cells were purchased from ATCC (American Type Culture Collection, Manassas, VA) and maintained in MEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicilin/streptomycin and 0.5% kanamycin.For cell transfection, 500 µl of cells (2x10 5 cells/ml) were plated in each well of 24-well plates.Two µl of Lipofectamine TM 2000 (Lipo-2000) and 0.8 µg of DNA were added separately into a vial with 50 µl Opti-MEM transfection medium, and combined together after a 5-10 minute incubation.The DNA/Lipo2000 complexes were continuously incubated for 20-30 minutes and then added to each well.The transfected cells were incubated for different time based on different assays and then tested for their cell cycle and apoptosis, or for a cell proliferation as described below.The experiments were done separately three times. ",
"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 are as previously described (13,34).The PCR was regularly amplified with 35 cycles, with more or less cycles due to the difference in RNA abundance of these investigated genes.Primers for real-time PCR analyses were the same as described above.Real-time PCR assays were performed as described (13).β-actin was used as the internal control and results were calculated by applying 2 -∆∆ CT methods.The experiments were done separately three times. ",
"section_name": "RT-PCR and real-time PCR",
"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 minutes in boiling water.Supernatants were loaded to run on a 8-16% Tris-glycine gel after centrifugation at 12,000xg for 10 minutes.Protein was transferred to a nitrocellulose membrane and blocked with a 5% dried milk/TBST solution, then washed and incubated with primary antibody.The membrane was washed again and incubated with second antibody (Santa Cruz).Mem-branes were developed according to the ECL system protocol (Amersham Biosciences, England).The experiments were done separately three times. ",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Cells were plated on 6-well plates and incubated overnight.The test compounds were added.Cells were incubated overnight or as required and then were harvested for the assay.For the apoptosis assay, the Annexin V-FITC apoptosis detection kit (Cat.No.: APOAF) was purchased from Sigma (Saint Louis, MO).Into five hundred µl of cell suspension was added 5 µl of Annexin V-FITC conjugate and 10 µl of propidium iodide solution.The mixture was incubated for 10 minutes and assayed to determine fluorescence using a Beckman-Coulter Gallios analyzer.Data were analyzed with Gallios software.For the cell cycle assay, the Coulter DNA Prep reagents kit (Cat.No.: PN 6607055) from Beckman Coulter (Fullerton, CA) was used.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.Both experiments were repeated three times. ",
"section_name": "Cell apoptosis and cell cycle analysis",
"section_num": null
},
{
"section_content": "The cell proliferation assay (Promega, Madison, WI) was performed as described previously (38).Briefly, 50 µl aliquots of medium with different concentrations of compounds were added to 96-well plates.All compound concentrations were tested in triplicate.Fifty 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 assay",
"section_num": null
},
{
"section_content": "One ml of cells (1x10 5 cells/ml) were plated in 24-well plates and treated with or without the test compound (VPA).VPA-induced cell morphological changes were inspected and photographed under an inverted light microscope at 10x magnification.The cell attachment assay (ECM205) was used to study the effects of VPA on cell attachment components in-cluding fibronectin, vitronectin, laminin, collagen I and collagen IV (ECM205, Chemicon).The assay was performed after a 72-hour incubation according to the kit instructions.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 3-5 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 morphological change and cell attachment",
"section_num": null
},
{
"section_content": "VPA has demonstrated its cancer cell growth suppression in various types of cancer cells (13,31,32).In its anti-cancer effects, VPA serves as a HDAC inhibitor and is also believed to act as a Notch signaling regulator (13,32,33).Notch signaling is aberrant in HCC cells and is strongly associated with HCC progression.Thus, in the present study, we investigated for VPA's effects on HCC cell growth and the involvement of Notch signaling in VPA-meditated HCC cell growth arrest. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "VPA is believed to act as a HDAC inhibitor in its tumor suppression in various cancers (15,33).Thus, we first investigated the effects of VPA on histone acetylation/deacetylation in HCC HTB-52 cells.Western blot analysis showed that VPA reduced the expression of HDAC4 and induce the acetylation of histone H4 (AcH4), with no obvious change of histone H4 (Fig. 1B).We further did cell proliferation assay and found that VPA displayed anti-proliferative abilities in HCC HTB-52 cells.As shown in Fig. 1, VPA significantly induced cell proliferation suppression in a dose-dependent manner, with the suppressive rates being nearly 50% (VPA: 2.5 mM) and 70% (5 mM), respectively (Fig. 2A).VPA-induced decrease of the proliferation marker PCNA (0.35-fold) assayed by real-time PCR was observed as well (Fig. 1C).Another HDAC inhibitor trichostatin A (TSA), and the Notch signaling inhibitor dibenzazepine (DBZ) also displayed their suppressive effect on HTB-52 cell growth, with TSA at 1µM having more potent anti-proliferation and DBZ at 20 µM less (data not shown). ",
"section_name": "1.VPA acting as a HDAC inhibitor induces cell proliferation suppression",
"section_num": null
},
{
"section_content": "We also investigated the effects of VPA on cell apoptosis in HCC HTB-52 cells and found that VPA significantly induced cell apoptosis.VPA at 4 mM and a 72-hour incubation induced apoptosis, with an apoptotic rate (early apoptosis and necro-apoptosis together) of 31% compared to 20% in the control (Fig. 2B).There was also a decrease of the anti-apoptotic marker BCL-2 (0.6-fold), detected at the mRNA level by real-time PCR assay (Fig. 1C).Meanwhile, the HDAC inhibitor TSA at 1 µM markedly induced an apoptotic increase with the apoptotic rate of 68%, but Notch signaling inhibitor DBZ at 20 µM obviously have no effect, being similar to the DMSO control (data not shown). ",
"section_name": "VPA induces cell apoptosis",
"section_num": "2."
},
{
"section_content": "Besides its suppression of cell proliferation and induction of cell apoptosis as described above, VPA also was evaluated for its effects on the cell cycle.VPA at 4 mM and with an 18-hour incubation was found to induce cell cycle arrest at phase G2, with TSA at 1µM at G2, but DBZ did not cause arrest when compared to the effect of the DMSO control (data not shown).The time course analysis further showed the VPA-induced cell cycle arrest at phase G2 can be observed at an incubation of 18 (an increase of rate at phase G2: 10%) and 72 hours (13%) compared to control (5%), with less effect at a 3-hour incubation (7%) (Table 1).VPA also induced an increase of the tumor repressors p21 and p63 at the mRNA level as seen by RT-PCR and real-time PCR and at the protein level by Western blot analysis (Fig. 1A,1B,1C).These assays showed that the HDAC inhibitor VPA might suppress HCC cell growth via inducing cell cycle arrest.A, cell proliferation assay showed that VPA induced a cell growth suppression in a dose-dependent manner, with inhibitory rates being 48.4% (2.5 mM) and 68.9% (5 mM).Asterisk (*) shows a significant difference compared to control, with P value < 0.01 (One-way ANOVA analysis); B, Apoptosis assay showed that VPA obviously induced cell apoptosis.Assays from three experiments showed an increasing apoptotic rates at early apoptosis (VPA/Control: 23.6 ± 0.5% / 17.8 ± 2.9%) and necro-apoptosis (7.5 ± 0.6% / 2.0 ± 0.3%), with P value < 0.01 (t-test analysis). Table 1.VPA-induced cell growth arrest at phase G2 in HCC HTB-52 cells via cell cycle analysis. ",
"section_name": "VPA induces cell cycle arrest",
"section_num": "3."
},
{
"section_content": "Another major function of VPA is to induce cell morphological changes that are associated with differentiation and cell migration.Previously, we observed VPA-induced cell morphological change in many different cancer cells.Thus, VPA also was evaluated for its effects on cell morphological changes and on cell attachment in HTB-52 cells with epithelial morphology.VPA at 4mM and a 72-hour incubation was observed to induce cell morphological change (Fig. 3B).We further did attachment assay to evaluate the effects of VPA on cell attachment.In our previous study, we found that HCC HTB-52 cells more strongly attached to ECM component laminin, not to other components including fibronectin, vitronectin, collagen I and collagen IV.We further observed that VPA at 2 mM and 4 mM with a 72-hour incubation resulted in a significant suppression of cell-laminin attachment, with the inhibitory rates being 24 % (2 mM) and 51% (4 mM) (Fig. 3C).The observation was also acquired with the effects of VPA on MMP2 (cell invasive and metastasis marker) and E-cadherin (an epithelial cell marker) that are related to cell differentiation and migration (Fig. 1C).VPA was found to induce an increase of MMP2 (8-fold) and E-cadherin (7-fold) in HTB-52 cells.These findings support that VPA may inhibit HCC metastasis via blocking cell migration. ",
"section_name": "VPA induces cell morphological change and cell attachment suppression",
"section_num": "4."
},
{
"section_content": "Notch signaling is aberrantly expressed in HCC cells and plays a critical role in HCC progression (25,27,39).Thus, we evaluated the effects of Notch signaling in HCC HTB-52 cells.By investigating the expression profiles of four Notch receptor genes.We first found that Notch1 and 2 receptors were expressed at a higher level in HCC HTB-52 cells compared to undetected Notch3 and Notch4 (data not shown).We further investigated the effects of Notch1 activation on HCC cell growth.HCC HTB-52 cells were transiently transfected with the Notch1 active form ICN1 and assayed for the effects of ICN1 on cell proliferation.We found that ICN1 overexpression stimulated HCC HTB-52 cell proliferation in a dose-dependent manner, with an increase of 9% (ICN1: 200 ng), 18% (400 ng) and 67% (800 ng), respectively (Fig. 4A).We also observed that all the other three Notch active form ICN2, ICN3 and ICN4 stimulated HCC cell proliferation (data not shown). Also, ICN1 was investigated for its effects on HCC cell cycle and cell apoptosis.The cell cycle assay showed that ICN1 resulted in an significant accumulation at phase G1 with the rate over 80% compared to 68% of the control vector (transfected with pcDNA vector only) (Fig. 4B), different from VPA-induced cycle arrest at phase G2 mentioned above (Table 1).The apoptosis assay also showed that ICN1 overexpression induced anti-apoptosis, with the total apoptotic rate being 24 % compared to 48% in the control (data not shown).These findings indicate that Notch signaling stimulates cell growth by playing an oncogenic role in HCC HTB-52 cells. Fig. 4. The effects of Notch1 active form ICN1 on cell proliferation and cell cycle arrest.Cells (0.5 ml medium/per well) were plated in 24-well plates and cultured overnight, then transfected with ICN1 or pcDNA (control) and incubated for 5-6 hours.1.5 ml fresh medium were added and cell were cultured for different time (3 days for cell proliferation, overnight for cell cycle).A, the proliferation assay from three experiments showed that ICN1 induced cell proliferation, with an increase of 9% (ICN1: 200 ng), 18%(400 ng) and 67% (800 ng), respectively.One-way ANOVA showed an significant increase by the dose trend and the highest (Asterisk *) compared to control, with P < 0.01.B, the cell cycle analysis from three experiments showed that ICN1 overexpression induced an increase at phase G1 of cell cycle, with a rate 82.9 ± 0.8% compared to 67.3 ± 1.3% of control, a decrease at phase G2 with a rate 1.7 ± 0.1% (control: 3.9 ± 1.7% and phase S with a rate 15.4 ± 0.9 % (control: 28.8 ± 1.6%). ",
"section_name": "Notch signaling plays an oncogenic role",
"section_num": "5."
},
{
"section_content": "As described above, VPA acted as a tumor suppressor with Notch signaling activation playing an oncogenic role.This implies a connection between VPA and Notch signaling.We further evaluated the effects of VPA on the expression of Notch receptors in these cells.HCC cells were treated with VPA at a serial dose of 0, 1, 2, 4, and 8 mM.Further assays showed that VPA down-regulated the expression of Notch1 and Notch2 at the mRNA level as seen by RT-PCR (Fig. 1A) and real-time PCR (Fig. 1C).Western blot assays also confirmed VPA-induced suppression of Notch1 and the Notch downstream target gene HES1 at the protein level (Fig. 1B).We further co-treated HCC HTB-52 cells with ICN1 and VPA by first transfecting ICN1 for 4-5 hours and then adding VPA at 4 mM.After a 72-hour incubation, we found that Notch1-induced cell proliferation was reversed by VPA (Fig. 5), indicating that VPA functions as a tumor suppressor, possibly via blocking the oncogenic Notch signaling.Fig. 5. VPA reversed the stimulatory effects of Notch1 activation (ICN1) on cell proliferation.HCC cells were pre-plated in 24-well plates, transfected next day and incubated for 5-6 hours.1.5 ml fresh medium were added with/without VPA and cells were continuously cultured for 3 days.As shown, ICN1 significantly induced cell proliferation with an increase rate 26.6% and VPA significantly reduced cell proliferation with a decrease rate 41.8%.The combination of VPA and ICN1 showed that VPA reversed the stimulatory effects of Notch1 activation, with no significant difference from control.Asterisk (*) shows a significant difference compared to control, with P value < 0.01 (t-test analysis). ",
"section_name": "VPA suppresses Notch signaling and reverses Notch1-stimulated cell proliferation",
"section_num": "6."
},
{
"section_content": "Due to its effects on certain GPCR members, particularly SSTR2 which is aberrantly expressed in many cancer cells and has been used for receptor-targeting therapeutics, VPA was used in combination therapy with receptor-targeting peptide-drug conjugates (13,34).Thus, presently, VPA also was evaluated for its effects on some GPCRs in HCC HTB-52 cells.We found that VPA significantly increased the expression of SSTR2, bombesin receptor subtypes (GRPR and BRS3) at the mRNA level [Fig.1A], the increase of SSTR2 being confirmed by real-time PCR and Western blot assays [Fig.1B,1C].Thus, VPA in combination with receptor-targeting agents could be a potential strategy for HCC treatment.Our in vitro proliferation assay further identified that the combination of VPA and SSTR2-targeting CPT-SST could greatly enhance cell growth suppression compared to each alone [Fig.6].The in vivo assay is under preparation. ",
"section_name": "The application of VPA in combination with SSTR2-targeting cytotoxin",
"section_num": "7."
},
{
"section_content": "Traditional radio-and chemo-therapies, being severely toxic to the liver, are of limited use in HCC treatments except sorafenib, the only FDA-approved anti-HCC drug, being well-tolerated by HCC patients (40) (NCI website: http://www.cancer.gov).Thus, it is necessary to search and develop more effective anti-HCC drugs.VPA as an anti-convulsant drug and a HDAC inhibitor displayed wide anti-cancer activities in many different types of cancers (15,31,32).Our previous studies also showed that VPA suppressed cervical cancer cell growth and tumor growth (13,34).In this study, VPA was identified as a HDAC inhibitor to suppress HDAC4 and induce acetylated histone H4 in HCC HTB-52 cells.We also found that VPA induced cell proliferation suppression, cell cycle arrest and cell apoptosis, identical to other reports showing the suppressive effects of VPA on other HCC cells (35)(36)(37).VPA was also observed to induce cell attachment in HCC HTB-52 cells.These findings support that VPA may be a potential agent for HCC treatment. Accumulating evidence shows that Notch signaling may most likely be a HCC diagnostic marker.The activation of this signaling is frequently seen in human HCCs.There is a high Notch1 signature in most HCC tissues compared to the adjacent normal tissues (12,14,21).Dr. Bolondi reported high expression of Notch3 and Notch4 in most HCC tissues (22).In other reports, the high frequency of Notch1 and Notch3 were observed in HCC tissues (12,14,41). Moreover, HBV X protein (HBx), that is a critical factor in HCC carcinogenesis, is also found to up-regulate Notch signaling (5,12,42).These findings support that Notch signaling is critical for HCC development.However, different reports showed that Notch signaling may display controversial functions in HCC cells.The observations from several other studies show that Notch signaling may play a tumor-suppressive role (21,(25)(26)(27).Thus, the function of Notch signaling is not determined fully, with the precise Notch-mediated mechanisms remaining unclear. We evaluated the effects of Notch signaling activation in HCC cells.Our assays showed that Notch1 activation stimulated cell proliferation and reduced cell apoptosis, and conversely, its knockdown could suppress cell growth.We also found the positive effects of all other three Notch receptors (ICN2, ICN3, ICN4) on multiple HCC cells and identified the role of Notch signaling in serving as an oncogenic player in HCC cells, identical to the role of Notch1.Thus, the activation of Notch signaling in HCC cells plays an oncogenic role (5,8,23,24).it may be a promising anti-HCC approach to target oncogenic Notch signaling.In our present study, the HDAC inhibitor VPA was found to act as a Notch signaling inhibitor to suppress the expression of Notch1 and the Notch target gene HES1.Furthermore, VPA could reverse cell growth stimulated by Notch1 activation.Thus, the HDAC inhibitor VPA may possibly suppress HCC cell growth via acting as a Notch signaling inhibitor.Tumor suppressors like p53 and p27 also were reportedly associated with regulation of Notch signaling in HCC cells.In Dr. Giovannini's report, Notch3 knockdown increases the expression of the tumor suppressors p53 and p27, and enhances doxorubicin-induced apoptosis and DNA damage in HCC HepG2 cells (14).We also found that VPA induced an increase of tumor repressor p21 and p63, supporting VPA's suppressive effects in HCC cells. More importantly, VPA was found to significantly increase the expression of SSTR2 and GRPR in HCC HTB-52 cells.This provides a potentially novel opportunity for a receptor-targeted chemotherapy with VPA and a SSTR2-or GRPR-targeting peptide/antibody conjugate by taking advantage of VPA's dual functions of tumor suppression and receptor upregulation.Normally, due to its limited effects, VPA more frequently serves as an adjuvant for the combination treatments.VPA in combination with other anti-cancer agents such as sorafenib and acyclic retinoid was also reported to inhibit HCC cell growth (15,35,39,43).However, these combination treatments are not specific to HCC cells.Our new strategy showed that VPA in combination with receptor-targeting agents displayed a more effective syner-gic effect.In the present study, the combination of VPA and the SSTR2-targeting CPT-SST enhanced the suppression of HCC cell growth compared to each alone.Similar results were observed from our previous study in that the combination displayed synergic in vitro and in vivo anti-tumor effects on cervical cancer tumor growth (13,34).Due to receptor-targeting therapeutics could more specifically induce tumor suppression via binding with the specific receptor and increasing the internalization of the cytotoxic agents (44), VPA-induced increase of the target receptors can provide an enhanced synergic effect via a combination of VPA and receptor-targeting cytotoxins.This combination strategy is under preparation for in vivo evaluation. Conclusively, Notch signaling is highly expressed in HCC tissues, with more evidence showing it serving as a HCC oncogenic marker.The HDAC inhibitor VPA displayed its efficacy against HCC cell growth via targeting Notch signaling and tumor repressor. Particularly, the characteristics of VPA-mediated increase of receptors may provide us a novel and promising anti-HCC therapeutic approach with the combination of VPA and receptor-targeting cytotoxic agents. ",
"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), Financial supports from National Natural Science Foundation of China (Grant No. 81471777 and 81102409) and Tulane Peptide Research Fund. ",
"section_name": "Acknowledgement",
"section_num": null
},
{
"section_content": "VPA: valproic acid; HDAC: histone deacetylase; AcH4: acetylated histone 4; HCC: hepatocellular carcinoma; SSTR: somatostatin receptor; CPT-SST: camptothecin-somatostatin conjugate; TSA: trichostatin A; DBZ: dibenzazepine; GPCRs: G protein-coupled receptors. The authors have declared that no competing interest exists. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "VPA: valproic acid; HDAC: histone deacetylase; AcH4: acetylated histone 4; HCC: hepatocellular carcinoma; SSTR: somatostatin receptor; CPT-SST: camptothecin-somatostatin conjugate; TSA: trichostatin A; DBZ: dibenzazepine; GPCRs: G protein-coupled receptors. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interest exists. ",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.7150/jca.87128
|
Increased NOTCH1 expression is associated with low survival in moderate/ poor differentiated human oral squamous cell carcinoma patients
|
Notch deregulation has been reported in various types of cancers, including Oral squamous cell carcinomas (OSCCs). The role of Notch1 signaling in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, NOTCH1 was aberrantly expressed in human oral cancer tissues compared with that in normal marginal tissues and was associated with poor prognosis. The positive Notch 1 expression was significantly associated with poor tumor differentiation status. Kaplan-Meier survival curves revealed that elevated cytoplasmic NOTCH1 expression levels in OSCC patients were associated with poor overall survival. Moreover, multivariate COX proportional hazard models revealed that T N status, AJCC stage histological grade were independent prognostic factors for survival. Our result clearly demonstrates the oncogenic role of Notch1 in oral cancer and Notch1 may be a useful biomarker to target oral cancer patients.
|
[
{
"section_content": "Oral cancer is one of the most common cancers in the worldwide, according to the results shown by GLOBCAN 2012 (http://globocan.iarc.fr/)300,000new oral cancer cases and 145,000 oral cancer-related deaths were registered in 2012 [1,2].Despite of the improved diagnostic techniques and treatments, survival rate of oral cancer patients has not been improved [3].This could be improved by identifying the molecular biomarkers [4]; however, the developmental path towards a clinically suitable biomarker remains tremendously tough.Despite of the improved diagnostic techniques and treatments, survival rate of oral cancer patients has not been improved [3].This could be improved by identifying the molecular biomarkers [4], however, the developmental path towards a clinically suitable biomarker remains tremendously tough.Thus, it is essential to identify the novel molecular markers and develop effective approaches to treat OSCC patients. Notch signaling plays a significant role in cell proliferation, differentiation, and apoptosis [5,6].Notch families have four receptors (Notch1-4) and five ligands (Delta-like-1, Delta-like-3, Delta-like-4, Jagged1, and Jagged2) [7].NOTCH1 is one of the most commonly mutated tumor suppressor genes in Head and neck squamous cell carcinomas (HNSCCs) [8].Notch-1 expression was found to be elevated in salivary adenoid cystic carcinoma (SACC)tissues [9], and promotes proliferation of SACC and HNSCC [9,10].Several studies have reported tumor-suppressive ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "International Publisher role or oncogenic role of Notch signaling pathway in many cancer types [11][12][13][14] including HNSCCs [15][16][17].All these results suggest that the NOTCH1 pathway may have dual function in this tumor type.Thus, understanding NOTCH1 expression pattern and its function are likely to have therapeutic potential in OSCC [18,19]. In the present study, immunohistochemical (IHC) analysis was used to examine the protein expression of NOTCH1 in oral cancer patients.We also examined the relationship between NOTCH1 protein expression and OSCC clinicopathological variables and prognosis, and whether NOTCH1 could be a prognostic biomarker in these patients. ",
"section_name": "Ivyspring",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "To evaluate the association of NOTCH1 expression with clinical/pathological factors and patient survival, a total of 268 oral cancer tissues were obtained from 253 male and 15 female patients (age ranged from 30 to 90 years old) were obtained from Human Bio Bank, China Medical University and Hospital, Taiwan.The main treatment was tumor removal and radical neck dissection, including post operation irradiation as well as selective patients treated with 5-fluorouracil (5-FU) and cisplatin chemotherapy.The tumors were classified according to the International Union against Cancer TNM classification system [20].All samples were snap-frozen in liquid nitrogen and stored at -80 °C until use. ",
"section_name": "Patient samples",
"section_num": null
},
{
"section_content": "Tissue microarrays (TMAs) were created using the OSCC samples based on the methods outlined in previous reports [21].Two senior pathologists validated the morphology of the malignancy based on representative lesions revealed by hematoxylin and eosin (H&E) staining. Immunohistochemistry analysis was performed according to the standard protocol as previously described [22].Tissue sections were deparaffinized and rehydrated using routine techniques.Endogenous peroxidase activity was blocked with 3% H2O2 in methanol, hydrated with gradient alcohol and phosphate-buffered saline solution, and incubated in 10 mmol/L citrate buffer (pH 6.0).Tumor sections were incubated with NOTCH1 mouse monoclonal antibody (Catalog number: CF500248; 1:50 dilution; origene, Rockville, MD, USA) in room temperature for 20 mins.After washing three times with PBS, the sections were incubated with appropriate peroxidase-labelled secondary antibodies for 30 min at room temperature.The sections were washed three times with PBS and then labelled by diaminobenzidine and counterstained with Mayer's haematoxylin, dehydrated and mounted.Two experienced pathologists independently assessed the results of immunohistochemical staining, and a final agreement was obtained for each score at a discussion microscope. ",
"section_name": "Tissue microarray and Immunohistochemistry",
"section_num": null
},
{
"section_content": "All data were analyzed by the SAS 9.4 Software (SAS Institute, Inc.; Cary, NC, USA).Chi-square test was used to detect the importance of the clinicopathological variables of NOTCH1 protein expression and OSCC.The overall survival (OS) time, was estimated with the Kaplan-Meier method and compared using the Log rank test.Univariate and multivariate analysis was performed to confirm prognostic factors of OSCC using the Cox proportional hazard regression model [23].Statistically significant results were defined by a p value of < 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Earlier studies have found that Notch1 function as oncogene or tumor-suppressor [24][25][26][27].To better understand the role of the Notch pathway in oral cancer, we first analyzed for NOTCH-1 expression by immunohistochemical staining.Immunohistochemical examination revealed that Notch1 was accumulated in the cytoplasm or nuclei of human oral cancer samples (Figure 1a). To explore the potential application of determination of Notch1 levels for early diagnosis and prognosis in oral cancer, we analyzed the correlation between Notch1 expression and patient clinicopathological features and survival.Table 1 shows the demographic and clinicopathologic characteristics of OSCC patients.Specimens from 268 patients were included in this study (253 male and 15 female patients). Correlations between Notch1 expression and the clinicopathological characteristics of patients with oral cancer were investigated.As shown in Table 2, a significant positive correlation was observed between Notch1 expression and tumor differentiation (P<0.05),but not between Notch1 expression and TNM stage, AJCC stage and Death status (P>0.05).These results are consistent with the previous report by [28].The percentage of NOTCH1 positive cases in the poorly differentiated group was lower than that in the well/moderately differentiated group. ",
"section_name": "NOTCH1 protein expression in OSCC and clinicopathological variables",
"section_num": null
},
{
"section_content": "The effects of clinicopathologic factors and Notch1 expression on mortalityin the OSCC patient are shown in Table 3.The mortality density for patients with T III/IV, N2/N3, AJCC tumor stage (III/IV), Moderate/Poor tumor differentiation and Chemotherapy/Radiotherapy was 13.0, 24.2, 12.8, 9.5 and 13.0, respectively per 100 people-years.Higher mortality risk was observed to be related to Tumor size (III/IV), N stage (N2/N3), AJCC tumor stage (III/IV), moderate/poor tumor differentiation, and chemotherapy/radiotherapy was significantly associated with a higher mortality risk (aHR = 2.02, 3.06, 2.65, 2.73 and 3.42 respectively).The independent mortality risk for patients with negative Notch1 expression was non-significant as compared those with positive expression (95% CI, 0.76-1.64;P> 0.05).c Significant multiplicative-scale interaction between Tumor differentiation and the presence of NOTCH1 on mortality risk was identified (P for interaction, 0.0081). Because NOTCH1 expression was associated with tumor differentiation, we then evaluated the combined effect of NOTCH1 expression and tumor differentiation on mortality.As compared to patients with well differentiation and negative NOTCH1 expression, the mortality hazard risk was multiplicatively enhanced among patients with Moderate/Poor tumor differentiation and positive NOTCH1 expression (aHR = 2.54, 95% CI, 1.44-11.20;P = 0.0081 for multiplicative interaction, Table 3).Moreover, patients with a positive Notch1 expression had a shorter overall survival (OS) time than those with a negative Notch1 expression by Kaplan-Meier analysis (P=0.0009; Figure 1b). ",
"section_name": "Notch1 expression and survival",
"section_num": null
},
{
"section_content": "The role and clinical relevance of Notch1 in cancers have not been well illustrated.However, a few studies have suggested Notch1 as a tumor promoter in head and neck squamous cell carcinomas (HNSCCs) [27,29].Though, some studies have shown that Notch function as tumor suppressor [24].This study aimed to explores the expression of NOTCH1 in oral cancer tissues and its influence on prognosis. Notch1 is crucial in tumor progression and plays a dual role either as oncogene and tumor suppressor [30].In this present study Notch1 expression was seen in the cytoplasm of oral cancer cells, while a weak expression was found in normal tissues [4,[31][32][33].The relationship between Notch1 expression levels and certain clinicopathological parameters was evaluated.Our findings showed that high Notch1 protein level was associated with tumor differentiation, but not with TNM classification, tumor stage and death.Conversely, Tian J et al (2018) have found that the expression of Notch1 was positively correlated with distant metastasis (P=0.003) and tumor differentiation (P=0.031)[34]. The significant prognostic variables provide useful information for clinical treatment and it is beneficial for identifying patients who have a higher risk of disease recurrence or poor outcome.The significant prognostic variables for survival were the late-stage TN (III/IV) and Poor tumor differentiation, whereas NOTCH1 expression was not correlated with OSCC mortality.Therefore, we studied the combinatory effect of tumor differentiation and NOTCH1 expression on OSCC morality.Significant multiplicative-scale interaction between Tumor differentiation and the presence of NOTCH1 on mortality risk was identified (P=0.0081).Moreover, we found poor tumor differentiated patients with high NOTCH1 had a low survival rate at a 10 year follow up.Further research, i) using both in vitro and in vivo testing is required to support our data and assess the efficacy of NOTCH1 as a therapeutic target; ii) number of patients sample used in this study is relatively small and larger sampled are needed to validate these results. In conclusion, high NOTCH1 expression is observed in oral cancer and more likely to worsen survival in poorly differentiated oral cancer patients.These findings emphasize oncogenic role of NOTCH1 in poorly differentiated oral cancer patients. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by the University of Tabuk (0211-1443-S). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The data that support the findings of this study are available from the corresponding author, Z.M.M, upon reasonable request. ",
"section_name": "Data Availability Statement",
"section_num": null
},
{
"section_content": "Conceptualization, ZMM and MS.; methodology, ASA; validation, FMS, and MMJ; formal analysis, NHS.; investigation, ZMM; resources, ZMM; data curation, ASA; writing-original draft preparation, RB., CYH and BK.; writing-review and editing, RB, CYH and BK; supervision, ZM.; project administration, ZMM.; funding acquisition, ZMM. The authors have declared that no competing interest exists. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Conceptualization, ZMM and MS.; methodology, ASA; validation, FMS, and MMJ; formal analysis, NHS.; investigation, ZMM; resources, ZMM; data curation, ASA; writing-original draft preparation, RB., CYH and BK.; writing-review and editing, RB, CYH and BK; supervision, ZM.; project administration, ZMM.; funding acquisition, ZMM. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interest exists. ",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.1186/s12881-020-01035-9
|
Concurrent pathogenic variants in SLC6A1/NOTCH1/PRIMPOL genes in a Chinese patient with myoclonic-atonic epilepsy, mild aortic valve stenosis and high myopia
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Pathogenic <jats:italic>SLC6A1</jats:italic> variants have been reported in patients with myoclonic-atonic epilepsy (MAE). <jats:italic>NOTCH1</jats:italic>, encoding a member of the Notch family of proteins, is known to be associated with aortic valve disease. The <jats:italic>PRIMPOL</jats:italic> variant has only been identified in Chinese patients with high myopia. Exome sequencing analysis now allows the simultaneous detection of multiple genetic etiologies for patients with complicated clinical features. However, the presence of three <jats:italic>Mendelian</jats:italic> disorders in one patient supported by their respective pathogenic variants and clinical phenotypes is very rare.</jats:p> </jats:sec><jats:sec> <jats:title>Case presentation</jats:title> <jats:p>Here, we report a 4-year-old Chinese boy who presented with MAE, delayed language, borderline intellectual disability (ID), mildly impaired social skills and attention deficit hyperactivity disorder (ADHD). He also had mild aortic valve stenosis and high myopia. Using whole-exome sequencing (WES), we identified three variants: (1) <jats:italic>SLC6A1</jats:italic>, NM_003042.4: c.881-883del (p.Phe294del), (2) <jats:italic>NOTCH1</jats:italic>, NM_017617.5:c.1100-2A > G and (3) <jats:italic>PRIMPOL</jats:italic>, NM_152683.4:c.265 T > G (p.Tyr89Asp). Parental Sanger sequencing confirmed that <jats:italic>SLC6A1</jats:italic> and <jats:italic>NOTCH1</jats:italic> variants were de novo, whereas the <jats:italic>PRIMPOL</jats:italic> variant was inherited from the father who also had high myopia. Furthermore, the <jats:italic>PRIMPOL</jats:italic> variant was absent from the genomes of the paternal grandparents, and thus was also a de novo event in the family. All three variants are classified as pathogenic.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>The <jats:italic>SLC6A1</jats:italic> variant could explain the features of MAE, delayed language, borderline ID, impaired social skills and ADHD in this patient, whereas the features of aortic valve stenosis and high myopia of the patient may be explained by variants in <jats:italic>NOTCH1 and PRIMPOL</jats:italic>, respectively. This case demonstrated the utility of exome sequencing in uncovering the multiple pathogenic variants in a patient with complicated phenotypes due to the blending of three <jats:italic>Mendelian</jats:italic> disorders.</jats:p> </jats:sec>
|
[
{
"section_content": "SLC6A1 (MIM 137165) encodes a gamma-aminobutyric acid (GABA) transporter known to be crucial for the reuptake of GABA from the synaptic cleft [1].GABA is the main inhibitory neurotransmitter that counterbalances neuronal excitation in the brain, and disruption of this inhibitory balance can cause seizures.Previous reports have showed that pathogenic SLC6A1 variants are associated with MAE in patients by a loss-of-function mechanism in which SLC6A1 variants reduce or abolish the function of the GAT-1 GABA transporter [2][3][4][5][6][7].In addition, patients with MAE may also present with variable degrees of intellectual disability, language impairment and behavioral problems, such as aggressive behavior/irritability, ADHD and autistic features [2,[4][5][6]8]. NOTCH1 (MIM 190198) encodes a member of the Notch protein family, which includes NOTCH1, NOTCH2, NOTCH3 and NOTCH4 receptors [9].Notch proteins belong to single-pass transmembrane receptors that regulate cell fate determinations during development, ensure crosstalk between different types of cells and their physiological differentiation, and are particularly critical in the development of cardiac valvulogenesis.Evidence suggests that pathogenic variants in this gene are related to aortic valve disease [10][11][12][13][14]. PRIMPOL (MIM 615421) encodes a primasepolymerase protein that is ubiquitous expressed in the eye and 26 other tissues [15].Zhao et al. (2013) identified a heterozygous missense variant (c.265 T > G, p.Y89D) in PRIMPOL in affected members of a 4generation Chinese family with high myopia and in 4 sporadic Chinese patients with myopia.The PRIMPOL variant (p.Y89D) segregated with the disease and was absent in 270 Chinese controls, which implicated it in high myopia [16]. Here, we report three concurrent pathogenic variants in SLC6A1/NOTCH1/PRIMPOL genes in a Chinese boy with complicated clinical phenotypes, including MAE, delayed language, borderline ID, behavioral problems, aortic valve stenosis and high myopia. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "The proband, a 4-year-old Chinese boy, was the firstborn to a nonconsanguineous couple and had a healthy younger brother.He was born at 38 weeks' gestation with a weight, height, and head circumference well within the normal ranges.His family history was unremarkable.He sat independently at 6 months, spoke his first word at 7 months, and walked without assistance at 13 months.At the age of 2 years 6 months, his language development showed a gradual delay.Seizures occurred at 2 years 8 months of age, characterized by initial cessation of activity and then progression to a fall sometimes followed by myoclonic movements.The event lasted for 10 s each time with spontaneous remission.The patient was conscious throughout the seizures.Seizures were induced by fatigue, strong light and overexcitement.Seizure types observed included absences and myoclonicastatic, absence and partial seizures.Brain magnetic resonance imaging was normal at this age, but the electroencephalogram (EEG) was obviously abnormal due to paroxysms of bilateral independent and generalized 2.0-3.0Hz frontally dominant high-voltage rhythmic spikes/ polyspikes-and-slow waves.After valproate therapy was initiated, his myoclonic seizures were controlled to an average of one episode per 2 weeks.He displayed borderline ID and mildly impaired language and had behavioral problems, including obvious ADHD, irritability, mildly impaired social reciprocity and poor eye contact. Then, he was referred for a comprehensive clinical examination.Mild aortic valve stenosis was revealed by echocardiography.High myopia was also diagnosed, which was probably an inherited event because his father was also affected by high myopia during his early childhood without other suspected ocular diseases.However, his paternal grandparents had totally normal vision. ",
"section_name": "Case presentation",
"section_num": null
},
{
"section_content": "This study was approved by the Committee on Ethics of the Dongguan Maternal and Child Health Care Hospital.DNA of family members was extracted from peripheral blood lymphocytes using standard methods and the sample from the proband was sent for whole exome sequencing.The parental DNA was used to confirm suspected variants by Sanger sequencing.WES was operated with an Illumina HiSeq 2500 system (Illumina).The bcl2fastq2 Conversion Software (v2.20) was employed for extracting Fastq files.BWA (v0.2.10) was used for genome alignments and variant detection.Clinic Sequence Analyzer (CSA) software was employed for biological analysis and interpretation.The pathogenicity of the sequence variants was interpreted in accordance with the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines [17]. Using WES, a de novo inframe deletion variant at chr3:11067490-11,067,492 (hg19) in SLC6A1 was identified in our patient and was not observed in his parents or younger brother.The SLC6A1 change, c.881-883del (p.Phe294del), is predicted to cause the deletion of Phe (Fig. 1a).The Phe294 residue is highly conserved among different species.This inframe deletion variant is not present in either the 1000 genome databases or Exome Aggregation Consortium.Furthermore, this variant was previously reported to be a de novo event in patient 13, who was affected by seizures, described by Johannesen et al. (2018) [4].Thus, this variant was categorized as clinically likely pathogenic according to ACMG/AMP guidelines (PS2 + PM1 + PM2) (PS: strong; PM: pathogenic moderate). Then, the patient was given a comprehensive clinical examination.Unexpectedly, mild aortic valve stenosis and high myopia were revealed.Thus, WES data were reanalyzed to search for potential pathogenic variants causally linked with these clinical features.A de novo variant (c.1100-2A > G) in the NOTCH1 gene was identified in our patient, and his younger brother did not carry the variant (Fig. 1b).The variant alters the canonical splice acceptor site of intron 6.In silico analysis using Human Splicing Finder v3.0 (Aix Marseille University and Inserm, Marseille, France) predicted that this variant would eliminate the acceptor site, which was speculated to cause exon 7 to be incorrectly spliced out of the RNA transcript.Thus, this variant was classified as clinically pathogenic (PVS1 + PS2) (PVS1: pathogenic very strong).Finally, the variant (c.265 T > G, p.Tyr89Asp) in PRIMPOL was identified in the proband and his younger brother, who had not undergone an ophthalmic evaluation for his age, and was inherited from the affected father with high myopia.Further segregation analysis was performed and showed that the variant arose de novo in the father and was not seen in the paternal grandparents (Fig. 1c), moreover, the paternal grandparents had totally normal vision.Furthermore, functional studies have been established to support a damaging effect of the variant on PRIMPOL [18].According to the ACMG/AMP guidelines, the variant was classified as pathogenic (PS2 + PS3 + PP1).The pedigree of our patient with three Mendelian disorders is depicted in Fig. 2. ",
"section_name": "Whole-exome sequencing (WES)",
"section_num": null
},
{
"section_content": "Exome sequencing has been recommended as the first-tier clinical diagnostic test for individuals with neurodevelopmental disorders and provides opportunities for gaining insights into the relationships between specific multilocus genomic variations and disorders [19,20].However, the presence of three Mendelian disorders in one patient supported by their respective pathogenic variants and clinical phenotypes is very rare. In this study, we reported a 4-year-old Chinese boy who presented with MAE, borderline ID, delayed language, behavioral problems, mild aortic valve stenosis and high myopia.The three concurrent pathogenic variants were identified in our patient and were responsible for the complicated clinical phenotypes.Initially, the patient was referred to the clinic for MAE as the primary symptom at 2 years and 8 months of age.WES was performed for the patient and identified a de novo inframe deletion variant (c.881-883del, p.Phe294del) in SLC6A1 that has been reported in patients with MAE, but few SLC6A1 variant cases have been reported in Chinese patients.Previous reports indicated that individuals with MAE responded effectively to a ketogenic diet or valproic acid [2,4,5].Thus, valproate therapy was recommended and greatly reduced his absence seizures to an average of one episode per 2 weeks.Individuals with MAE commonly display intellectual disability, language impairment and behavioral problems, including aggressive behavior/irritability, ADHD and autistic features [2,[4][5][6]8].Then, neurodevelopmental assessments were performed for the patient.As a result, he was diagnosed with borderline ID, mild delayed language, and behavioral problems, including obvious ADHD, irritability, mild impaired social reciprocity and poor eye contact.The severity of the clinical presentation may differ among patients carrying SLC6A1 variants, even between patients with the same mutation [2,4].Our patient Fig. 1 WES identified three concurrent pathogenic variants in our patient.a A de novo variant (c.881-883del, p.Phe294del) was identified in SLC6A1.b A de novo variant (c.1100-2A > G) was identified in NOTCH1.c A pathogenic variant (c.265 T > G, p. Tyr89Asp) in PRIMPOL was identified in our patient and his younger brother and was inherited from the affected father.Furthermore, the variant was absent from the genomes of the paternal grandparents and thus was also a de novo change in the family.Red arrows, mutant bases carried the same mutation as patient 13 (Johannesen et al., 2018) [4].patient displayed borderline ID, whereas patient 13 had mild ID before epilepsy onset and moderate ID after epilepsy.Our patient had irritability, whereas patient 13 did not show this feature, but both presented with ADHD.Patient 13 had mild ataxia, whereas our patient did not.Patient 13 became seizurefree with the combined treatment of lamotrigine and ethosuximide, whereas our patient nearly became seizure-free with valproic acid being the most effective drug.Thus, both the severity and prognosis of our patient's clinical manifestations were better than those of the patient 13.However, cognitive competence and language development should be persistently followed for our patient. Mild aortic valve stenosis and high myopia as incidental clinical features were revealed for our patient through a comprehensive clinical exam.Then, WES data reanalysis was initiated to search for probable pathogenic variants causally linked with these clinical phenotypes.In return, a de novo variant (c.1100-2A > G) in NOTCH1 and a pathogenic variant (c.265 T > G, p.Tyr89Asp) in PRIMPOL inherited from his father with high myopia were identified, which were missed in the initial data analysis.The main reasons for the initial negative result were due to incomplete recognition of the patient's phenotypes.Therefore, the improvement in access to more detailed phenotypic data of the patients and the possibility of estimating the changing phenotype over time would contribute to the increased diagnostic yield [21]. Echocardiography revealed mild aortic valve stenosis, which may be a consequence of the de novo variant (c.1100-2A > G) in NOTCH1 [10][11][12][13][14].At present, mild aortic valve stenosis does not have an impact on his daily life, but it needs to be regularly examined. The variant (c.265 T > G, p.Tyr89Asp) in PRIMPOL was originally identified in affected members of a 4generation Chinese family with high myopia and in 4 sporadic Chinese patients with myopia.The evidence that the variant segregated with disease and was absent in 270 Chinese controls indicated that the variant (p.Y89D) in PRIMPOL was associated with high myopia [16].Furthermore, Keen et al. (2014) performed a functional study for the variant, unequivocally establishing that this variant has a significant impact on the function of this enzyme [18].However, Li and Zhang (2015) provided contradictory evidence that the variant was identified in two of 407 patients with high myopia, 13 of 813 patients with other ocular diseases, and seven of 384 normal controls [22].Thus, further work is required to establish the relationship between PRIMPOL and high myopia [22,23].There is no doubt that myopia is a complex genetic trait.However, our patient was only a 4-year-old boy with high myopia, who was obviously little affected by environmental factors.Furthermore, WES also excluded other known genetic causes responsible for ocular disorders to the greatest degree possible.Next, we performed further segregation analysis in the members of the family and showed that the variant was inherited from the father who had been affected by high myopia since early childhood without other suspected Fig. 2 Pedigree of our proband with the variants in SLC6A1, NOTCH1 and PRIMPOL.The two variants in SLC6A1 and NOTCH1 were de novo events, and the PRIMPOL variant was inherited from the affected father, which was also a de novo change since the variant was absent from the paternal grandparents.Proband is marked with an arrow.dn: de novo ocular diseases, which was a de novo event in that it was not present in the paternal grandparents who had totally vision.The evidence for cosegregation in our family may support a role for PRIMPOL in high myopia. WES has allowed clinicians to better understand the pathogenesis of patients with complicated clinical features [19][20][21].The improvement of clinical phenotypes is very important for the interpretation or reanalysis of WES data, and the incidental findings of WES analysis will also promote the revelation of relevant phenotypes, as both complement each other and ultimately form a closed loop for accurate diagnosis of genetic diseases. ",
"section_name": "Discussion and conclusions",
"section_num": null
}
] |
[
{
"section_content": "We would like to express our sincere gratitude to our patient and his family for their cooperation. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This study was supported by the Dongguan Social Development Project (No. 201950715007158 to Yanhui Liu).The funding body participated in the design of the project and interpretation of whole-exome sequencing data. The data used and/or analyzed in the present report was deposited in the NCBI BioProject database.The data is accessible via the accession number: PRJNA627312; or via the links: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA627312 ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This study was supported by the Dongguan Social Development Project (No. 201950715007158 to Yanhui Liu).The funding body participated in the design of the project and interpretation of whole-exome sequencing data. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The data used and/or analyzed in the present report was deposited in the NCBI BioProject database.The data is accessible via the accession number: PRJNA627312; or via the links: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA627312 ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Abbreviations MAE: Myoclonic-atonic epilepsy; ID: Intellectual disability; ADHD: Attention deficit hyperactivity disorder; WES: Whole-exome sequencing; GABA: Gamma-aminobutyric acid Authors' contributions HMY drafted the first versions of the manuscript.HMY and YHL were responsible for the design of the project, data analysis, and manuscript writing.QMW, YFL, SXC and JXL assisted in the literature review and participated in the experiments, data entry, and data analysis phases of the project.All authors have read and approved the final version of the manuscript. All procedures in this study were approved by the Committee on Ethics of the Dongguan Maternal and Child Health Care Hospital.Detailed written informed consent was obtained from all participants.In the case of children or those cognitively impaired and unable, written informed consent for participation in this study was obtained from the patients' parents or guardians. Written informed consent for publication of clinical and genetic data was obtained from all participants.The parents /legal guardian of the children (under the age of 18) has signed written informed consent for publication of clinical and genetic data. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Abbreviations MAE: Myoclonic-atonic epilepsy; ID: Intellectual disability; ADHD: Attention deficit hyperactivity disorder; WES: Whole-exome sequencing; GABA: Gamma-aminobutyric acid Authors' contributions HMY drafted the first versions of the manuscript.HMY and YHL were responsible for the design of the project, data analysis, and manuscript writing.QMW, YFL, SXC and JXL assisted in the literature review and participated in the experiments, data entry, and data analysis phases of the project.All authors have read and approved the final version of the manuscript. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All procedures in this study were approved by the Committee on Ethics of the Dongguan Maternal and Child Health Care Hospital.Detailed written informed consent was obtained from all participants.In the case of children or those cognitively impaired and unable, written informed consent for participation in this study was obtained from the patients' parents or guardians. ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Written informed consent for publication of clinical and genetic data was obtained from all participants.The parents /legal guardian of the children (under the age of 18) has signed written informed consent for publication of clinical and genetic data. ",
"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/s13045-022-01333-0
|
Thrombotic and bleeding complications in patients with chronic lymphocytic leukemia and COVID-19: A study of ERIC, the European Research Initiative on CLL
|
<jats:title>Abstract</jats:title> <jats:p><jats:underline>Background:</jats:underline> Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to COVID-19 related poor outcomes, including thrombosis and death, due to the advanced age, the presence of comorbidities, and the disease and treatment-related immune deficiency. In this retrospective multicenter study, conducted by ERIC, the European Research Initiative on CLL, we assessed the risk of thrombosis and bleeding in patients with CLL affected by severe COVID-19.<jats:underline>Methods:</jats:underline> The study included patients from 79 centers across 22 countries. Data collection was conducted between April and May 2021.<jats:underline>Results:</jats:underline> A total of 793 patients from 79 centers were included in the study with 593 being hospitalized (74.8%). Among these, 518 were defined as having severe COVID: 162 were admitted to the ICU while 356 received oxygen supplementation outside the ICU. Most patients (90%) were receiving thromboprophylaxis. During COVID-19 treatment, 8.8% developed a thromboembolic event, while 4.8% experienced bleeding. Thrombosis developed in 20.5% of patients who were not receiving thromboprophylaxis, but only in 8.1% of patients who were on thromboprophylaxis. Bleeding episodes were more frequent in patients receiving intermediate/therapeutic versus prophylactic doses of low-molecular-weight heparin (LWMH) (11.1% vs. 4.2%, respectively) and in elderly. In multivariate analysis, peak D-dimer level was a poor prognostic factor for thrombosis occurrence (OR=1.020, 95%CI 1.006‒1.033), while thromboprophylaxis use was protective (OR=0.194, 95%CI 0.061‒0.614). Age and LMWH intermediate/therapeutic dose administration were prognostic factors in multivariate model for bleeding (OR=1.055, 95%CI 1.013-1.103 and OR=2.490, 95%CI 1.044-5.935, respectively). <jats:underline>Conclusions:</jats:underline> Patients with CLL affected by severe COVID-19 are at a high risk of thrombosis if thromboprophylaxis is not used, but also at increased risk of bleeding under the LMWH intermediate/therapeutic dose administration. <jats:bold> </jats:bold></jats:p>
|
[
{
"section_content": "High rates of venous thromboembolism (VTE), predominantly pulmonary embolism (PE), have been documented in patients with coronavirus disease 2019 (COVID- 19), particularly in critically ill patients admitted to the intensive care unit (ICU) [1,2].Despite the use of prophylactic or even therapeutic doses of anticoagulation therapy, thromboembolic complications have developed in many patients, implying that the risk of thrombotic complications remains high despite treatment, while also prompting the use of higher than usual doses of anticoagulants in hospital settings [3,4].The pathophysiology of this prothrombotic state is multifactorial and not yet completely elucidated.However, immune dysregulation [5], endotheliopathy [6] and coagulopathy [7] are distinctive elements of COVID-19 that have a major impact on thrombosis development. The use of anticoagulation therapy, particularly at intermediate and therapeutic doses, is associated with an increased risk of haemorrhagic events [8].Initial reports revealed limited evidence of COVID-19 therapy-related bleeding, but more data concerning the risk of bleeding are accumulating, particularly as regards the use of therapeutic doses of anticoagulation therapy [9].Considering the ongoing pandemic and its impact on vulnerable groups of patients, it is of immense importance to assess the actual rate of both thrombotic and bleeding events in specific patient populations. Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the western world [10].Patients with CLL may be more susceptible to COVID-19-related poor outcomes, such as thrombosis and death [11].Due to advanced age, the presence of various comorbidities, and the inherent immune deficiency of patients with CLL, there is a need for a robust analysis of the effects of patient and CLL-related characteristics, and thromboprophylactic therapy to define the optimal management of these patients during the COVID-19 pandemic. In this retrospective international multicenter study, we assessed the risk of thrombosis as well as the risk of bleeding due to the administration of thromboprophylaxis in severely ill patients with CLL and COVID-19 and sought to identify potential predictors of thrombosis. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "This is a retrospective multicenter study conducted by ERIC, the European Research Initiative on CLL, including patients from 79 centers across 22 countries.Data collection was conducted between April and May 2021.The study was approved by the ethics committees of the collaborating institutions.This cohort of CLL patients represents a subgroup of recently published ERIC and Campus CLL study [12]. In adherence to the international standard of practice, the criteria for COVID-19 diagnosis were positive real-time polymerase chain reaction (RT-PCR) assay for SARS-CoV-2 on nasal or pharyngeal swabs.Patients whose radiological or clinical assessments were suspicious of COVID-19, but had a negative swab test, were not included in the study. The CLL diagnostic procedures, patient assessment, clinical decisions, and actual treatment were performed by local hematology teams following international CLL guidelines [13,14]. The following patient clinical characteristics and laboratory data were obtained in the survey: baseline demographics; CLL diagnosis date; treatment status; presence, number, and type of comorbidities [cumulative illness rating scale (CIRS)], date of COVID-19 diagnosis; symptoms, treatment, and outcome of COVID-19; need for and duration of hospitalization; type of ward (intensive care unit (ICU) vs. non-ICU ward); peak absolute lymphocyte count (ALC); peak C-reactive protein (CRP); nadir albumin level; peak D-dimer level; use, type, and dosage of thromboprophylaxis; development and type of thrombotic events, presence and severity of bleeding complications during the hospitalization for COVID-19.Dosage of low-molecular weight heparin (LMWH) was defined as: prophylactic dose 50 IU/kg s.c.daily, intermediate dose 100 IU/kg s.c.daily and therapeutic dose 200 IU/kg s.c.daily.The use of extended thromboprophylaxis after discharge from hospitalization was defined as prophylactic dosage of anticoagulation administered to patients at high risk for VTE for up to 39 days postdischarge [15].Thrombotic events were classified as: pulmonary embolism (PE), deep vein thrombosis (DVT), stroke, myocardial infarction (MI), line associated thrombosis, extracorporeal circuit clotting in haemodialysis or ECMO lines and pernio-like skin lesions.Bleeding events have been classified as major using the International Society on Thrombosis and Haemostasis (ISTH) definition, whereas all non-major bleeding events were classified as minor [16]. To eliminate collection bias, we restricted our analysis to the group of patients who were considered to have severe COVID-19.Severe COVID-19 was defined as hospitalization and need of oxygen or admission into ICU while nonsevere/mild COVID-19 was defined as confinement at home or hospitalization without need of oxygen [12]. In order to enhance reporting, we used the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) checklist, which is an evidence-based, minimum set of recommendations for reporting observational studies in biomedical sciences [17]. ",
"section_name": "Data collection",
"section_num": null
},
{
"section_content": "Numerical data were presented as means with standard deviation or with median with 25-75th percentile.Categorical variables are summarized as absolute numbers with percentages or rates with corresponding 95% confidence intervals (CIs).The Kolmogorov-Smirnov test was used to assess the normality of data distribution.Student's t-test for independent samples or the Mann-Whitney U test was applied for numerical variables according to the data distribution.For categorical variables, Pearson's chi square analysis and Fisher's exact test were used.Predictors of thrombosis and bleeding occurrence during treatment were identified using univariate and multivariate logistic regression analyses, and presented with odds ratios (ORs) and corresponding 95% CIs.Variables were selected based on their associations with increased risk for thrombosis and bleeding (p < 0.10; univariate analysis) or known relevance, and were included in the variable pool for a stepwise-regression model.No imputation methods were used in analysis.If an outcome was missing, the patient data was excluded from the analysis.Receiver operating characteristic (ROC) curve analysis was used to test the model's discrimination performance based on sensitivity and specificity.Statistical analysis was performed using IBM SPSS statistical software (SPSS for Windows, release 25.0, SPSS, Chicago, IL, USA). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "We collected data from a total of 793 patients with SARS-CoV-2 infection (Fig. 1).Most patients (742; 93.6%) were diagnosed with CLL, while 36 (4.5%) and 15 (1.9%) were diagnosed with small lymphocytic lymphoma (SLL) and monoclonal B-cell lymphocytosis (MBL), respectively.The patients were predominantly men (69.5%), with a median age of 69 years (25th-75th percentile: 61-77 years).Five hundred and ninety-three (74.8%) patients were admitted to the hospital.Among these, 349 needed oxygen supplementation outside the ICU, while 162 were admitted to the ICU.Further analysis was restricted to this group of patients (n = 511) who were considered to have severe COVID-19.Median followup time i.e., duration of hospitalization for CLL patients with severe COVID-19 was 16 days (25-75th percentile, 10-26 days). CLL patients with severe COVID-19 were predominantly male (69.5%), with a median age of 70 years (25th-75th percentile, 63-79 years).Most cases had a significant burden of two or more comorbidities (62.9%), with hypertension (49.9%), diabetes (22.2%), coronary artery disease (12.2%), arrhythmias (9.8%), and other cardiovascular comorbidities and non-hematological malignancy (8.8% and 7.5%, respectively) being the most common.The reported median CIRS score was 4 (25th-75th percentile, 2-7).Forty-five percent were treatment naive (\"watch and wait\"), while 55% had received at least one line of CLL therapy (median, 1; range 1-5).At the time of COVID-19 diagnosis, 34.3% of patients were receiving active CLL therapy, most commonly Bruton tyrosine kinase inhibitors (BTKi's) (54.9%). Out of 511 CLL patients with severe COVID-19, data regarding thromboembolic events were available for 460, while data regarding bleeding were available for 468 patients.In this cohort of severe COVID-19 patients with CLL, 11.1% of patients (51/460, 95%CI 8-14%) developed thromboembolic events during treatment for COVID-19: 37 patients developed PE (8.0%), 7 patients deep vein thrombosis (1.5%), 5 patients stroke (1.1%), 2 myocardial infarction (0.4%), one patient developed line associated thrombosis and one developed pernio-like skin lesions.There were no extracorporeal circuit clotting in haemodialysis or ECMO lines.A total of 4.1% (19/460) of deaths were suspected to be related to thrombosis (Table 1).Twenty-three patients (23/468, 4.9%, 95%CI 3-7%) experienced bleeding during COVID-19 treatment (12 major bleeding; 11 non-major bleeding cases).Detailed information about patient characteristics according to thrombosis and bleeding status is presented in Table 2.There were no differences in baseline patient characteristics between patients who developed thrombosis during COVID-19 treatment versus those who did not develop thrombosis, with the exception of the presence of other cardiovascular diseases.Patients who experienced bleeding were significantly older than patients who did not experience bleeding. Patients with CLL and severe COVID-19 presented with fever (82.6%), and respiratory symptoms, including dyspnea (60.6%) and cough (53.8%).Other symptoms included fatigue (22.1%), headache (5.7%), myalgias/ arthralgias (9.5%), anosmia/ageusia (4.9%), and gastrointestinal symptoms (10.1%).Other symptoms were observed in 15.6% patients.Data regarding specific symptoms manifested during COVID-19 are presented according to thrombosis and bleeding status in Table 3. There was no statistically significant difference in symptoms between the groups.One hundred and seventy five (34.3%) patients were receiving active CLL-directed therapy while ill with COVID-19 though, 140 (80.5%)stopped the CLL treatment after the infection.BTK inhibitors (n = 95) were the most common therapy used (54.9% of patients receiving CLL therapy).Neither continuation nor discontinuation of BTKi in CLL patients with COVID-19 infection impacted thrombosis and bleeding occurrence in patients with CLL (Table 4).Venetoclax was administered as monotherapy in 21 patients, and in combination with anti-CD20 monoclonal antibodies in 12 patients.A minority of patients received other therapies, including anti-CD20 monoclonal antibody monotherapy (n = 5) and phosphatidylinositol-3-kinase (PI3K) inhibitors monotherapy (n = 5), while a combination of anti-CD20 monoclonal antibodies and PI3K inhibitors received one patient.Fifteen patients received either chemotherapy or chemoimmunotherapy.Corticosteroids for CLL were administered to 12.0%. Pharmacological treatment for COVID-19 included antivirals (45.6%), azithromycin (40.5%), hydroxychloroquine or similar drugs (37.9%), anti-IL6 or anti-IL6R The biochemical characteristics of the patients according to thrombosis and bleeding status are shown in Table 5. Peak D-dimer level was significantly higher in patients who developed thrombosis in contrast to patients who did not develop thrombosis, as well as in patients who experienced bleeding in contrast to patients who did not experience bleeding. Most patients (90.5%) were receiving thromboprophylaxis for COVID-19: 85.9% received LMWH, 3.6% received direct oral anticoagulants (DOACs), and 1.1% aspirin.Five patients treated with ECMO and three patients on haemodialysis were switched to unfractionated heparin (UFH) after initial LMWH approach.Thrombosis developed in 21.6% of patients who were not receiving thromboprophylaxis in contrast to 10.6% of patients who were on thromboprophylaxis (p = 0.043).Prophylactic dose was administered to 68.1%, intermediate to 14.3% and therapeutic to 17.7% of patients who received LMWH.Patients receiving intermediate/ therapeutic doses of LMWH experienced more frequent thrombosis than patients who received prophylactic doses (22/126, 17.5% vs. 18/261, 6.9%, respectively) (p = 0.001), and experienced more frequent bleeding (10/124, 8.1% vs. 10/262, 3.8%, respectively) (p = 0.079).Extended thromboprophylaxis was administered to 26.8% of patients. In univariate logistic regression analysis, admission to ICU, anti-IL6 or anti-IL6R treatment and steroids use for COVID-19 were predictive of thrombosis occurrence, (p < 0.001, OR = 3.086, 95%CI 1.707-5.578;p < 0.001, OR = 3.744, 95%CI 1.942-7.215and p = 0.026, OR = 5.141, 95%CI 1.220-21.665,respectively).High C-reactive protein to albumin ratio and D-dimer values were also predictive of thrombosis occurrence (p = 0.009, OR = 1.030, 95%CI 1.007-1.052and p = 0.002, OR = 1.016, 95%CI 1.006-1.027,respectively).Thromboprophylaxis was protective factor for thrombosis occurrence (p = 0.049, OR = 0.428, 95%CI 0.184-0.996).Presence of other cardiovascular diseases was of borderline significance (p = 0.050, OR = 2.316, 95%CI 1.000-5.366).In multivariate analysis, peak D-dimer level, high C-reactive protein to albumin ratio and anti-IL6 or anti-IL6R treatment were poor prognostic factors for thrombosis occurrence (p = 0.005, OR = 1.022, 95%CI 1.007-1.038;p = 0.042, OR = 1.025, 95%CI 1.001-1.051and p = 0.018, OR = 2.654, 95%CI 1.182-5.958), in contrast to thromboprophylaxis use that was protective (p = 0.007, OR = 0.199, 95%CI 0.061-0.645)(Table 6).In univariate logistic regression analysis, age (p = 0.012, OR = 1.055, 95%CI 1.012-1.100)and convalescent hyperimmune plasma (p = 0.017, OR = 3.821, 95%CI 1.275-11.450)were predictive of bleeding, while use and LMWH intermediate/therapeutic dose use was of borderline significance (p = 0.078, OR = 2.150, 95%CI 0.917-5.041).In multivariate analysis, age (p = 0.007, ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "CLL is the most prevalent leukemia in the western world, hence the need for improved understanding of COVID-19 in this group of patients is essential, particularly since patients with CLL are at higher risk of adverse outcomes of COVID-19 [11].Against this background, data about the risk for TE events and bleeding complications in CLL patients with COVID-19 is scarce. In the present cohort, the rate of thromboembolic events in CLL patients with severe COVID-19 was 11.1% (51/460), with PE being the most frequent (8.0%, 37/460).No significant differences were observed in CLL patients with or without thrombosis in terms of baseline patient characteristics, comorbidities and COVID-19-related symptoms.The published data by Chatzikonstantinou et al. [12] reported the VTE rate of 6.2% in the study that included 941 CLL patients with COVID-19.The study of 124 patients with various hematological malignancies [18], of whom 21 were patients with CLL, reported the rate of VTE of 8%, while the rate of composite thrombotic events (arterial and venous) was 13.4%.Besides the limitation of small patient numbers, direct comparison of this study and ours is of questionable relevance because the higher rate of cumulative thrombotic events in the former could be due to inclusion of particular hematological malignancies with well-established higher risk for thrombosis (e.g., plasma cell dyscrasia and myeloproliferative neoplasms).Comparisons with the general population with COVID-19 are also hindered by various confounding factors, not least of which is the fact that the rate of thrombosis depends largely on disease severity and, consequently, the hospital department (ICU vs. general ward): indeed, the disclosed rates of VTE in critically ill patients in ICU vary between 25 and 69%, in contrast to 7% in general wards [19].In a systematic review and meta-analysis, the prevalence of VTE in non-ICU and ICU patients were 7.9% and 22.7%, respectively, while the prevalence of PE in non-ICU and ICU patients were 3.5% and 13.7%, respectively [20]. When diagnosed with COVID-19, 175 (34.3%) patients of the present cohort were receiving active CLL-directed therapy.BTK inhibitors were the most frequent CLL-directed therapies, followed by venetoclax.There were no significant differences regarding the type and (dis)continuation of CLL treatment between CLL patients with or without thromboembolic events related to COVID-19, including BTKi.Several possible reasons could account for this finding.First, no CLL-directed specific treatment was associated with an increased risk of thromboembolic events.Second, the treatment was stopped in the majority of CLL patients (80.5%) after the COVID-19 diagnosis was established.Third, the potential beneficial effect of BTK inhibitors on the amelioration of the COVID-19 clinical course [21] was principally due to the modulation of immunological response [22,23], other than through the notable platelet inhibition effect [24] of BTK inhibitors.Lastly, a small number of CLL patients included in the study were treated with therapeutic options other than BTK inhibitors, which somehow limited the statistical analysis. Focusing on the potential impact of pharmacological treatment for COVID-19 on the occurrence of thrombosis in the present cohort, patients who were administered corticosteroid therapy and anti-IL6 or anti-IL6R monoclonal antibody were significantly more often diagnosed with thromboembolism.Further, in univariate logistic regression analysis, admission to ICU and use of anti-IL6/anti-IL6R and corticosteroids were predictive of thrombosis occurrence.Anti-IL6 or anti-IL6R monoclonal antibodies have been extensively used in order to ameliorate the hyperinflammatory state.A previous report [25] pointed out a transient surge in D-dimer levels and an increased risk of death secondary to thromboembolism.The limitations of that study were the small number of patients (n = 24), the retrospective nature of the study, and the non-specified severity of COVID-19.Overall, further investigation is warranted regarding the possible relationship between the use of anti-IL6 or anti-IL6R monoclonal antibodies and thrombotic risk thoroughly.In our study, the use of convalescent hyperimmune plasma was more common among patients who experienced bleeding, in contrast to patients who did not experience bleeding.Coagulation profile of human COVID-19 convalescent plasma was found to be impoverished with coagulation factors and, consequently, has prolonged coagulation time [26].Such a profile might contribute to hemostasis impairment and higher incidence of bleeding events.D-dimer levels have been extensively studied in COVID-19.It was recognized as a marker of adverse outcome of infection [27] and as an indicator of VTE.Our analysis showed that high CAR and D-dimer values were predictive of thrombosis occurrence also in the context of CLL.Evidently, coagulopathy in COVID-19 infection, coupled with malignancy related coagulopathy, results in state of highly elevated risk of thrombosis development [28,29].A higher D-dimer cut-off level in our cohort of COVID-19 CLL patients corresponded to the higher D-dimer levels found in cancer patients with COVID-19 [30], emphasizing the need for strict followup of this specific group of patients.Albumin level, as an acute phase reactant, has been associated with both the adverse outcome of COVID-19 and the development of thrombotic events during COVID-19.Hypoalbuminemia as a consequence of acute or chronic inflammation or increased albuminuria can contribute to the development of thrombosis, because of albumins anticoagulant and antiplatelet characteristics [31][32][33]. Similar to the general population, the admission to ICU was found to be predictive of thrombosis occurrence.This finding was one of the initial hallmark observations of COVID-19 infection, which has been later extensively confirmed [34][35][36].The combination of COVID-19 disease severity of patients in ICU, long list of risk factors related to ICU conditions and treatment and solely patients' characteristics (including malignancies and comorbidities), lead to a detrimental combination for thrombosis development. Most CLL patients included in the study were administered thromboprophylaxis (90.5%).In keeping with the literature [37], thromboembolic events were significantly more frequent among CLL patients without thromboprophylaxis than those with thromboprophylaxis.The rate of thromboembolism was lower in patients who were administered prophylactic anticoagulation, in comparison with intermediate and therapeutic anticoagulation.However, this latter finding should be cautiously interpreted considering that higher dosages of anticoagulation therapy were probably administered to the patients with more severe clinical course of COVID-19.In addition, it was shown that the use of thromboprophylaxis is associated with lower mortality rate in severely ill COVID-19 patients [38]. Higher doses of anticoagulation were universally recognized as major drivers of bleeding complications [9,39,40].In our study, 5.0% (23/468) had bleeding events, of which more than 50% were classified as major.Patients treated with intermediate/therapeutic doses of LMWH had a higher rate of bleeding than those treated with prophylactic doses of anticoagulation (8.1% and 3.8%, respectively).That said, the risk of bleeding in these patients depends on numerous factors besides the dosage of anticoagulation therapy, including age, CLL disease status (watch and wait or active disease), severity of COVID-19, comorbidities, and inherited or acquired coagulation abnormalities.Of note, we did not identify any association between CLL-specific treatment with BTK inhibitors and the occurrence of a bleeding episode. The COVID-19 pandemic has raised questions regarding the changes to therapy for the CLL patients being treated, who tested positive for SARS-CoV2.In our study continuation vs. discontinuation of BTKi was not predictive of thrombosis or bleeding occurrence in the patients with CLL who were receiving BTKi at the time of severe COVID-19 infection.Based on this finding and recently published reports suggesting a possible benefit from the BTKis in the setting of severe COVID-19 infection, and the fact that stopping ibrutinib can result in a disease flare-up in patients with CLL, we may recommend that BTKis therapy should be administered until the risks outweigh the therapy benefits [21,22]. Our study had several limitations particularly stemming from its retrospective nature, including heterogeneity in the treatment approaches for COVID-19.Additionally, we restricted the analysis to patients with severe COVID-19, thus patients with mild or asymptomatic SARS-CoV-2 infection were not studied. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, patients with CLL diagnosed with COVID-19 are at a high risk of thrombosis if thromboprophylaxis is not used, but also at increased risk of bleeding under the LMWH intermediate/therapeutic dose administration.More collaborative studies are needed to define the optimal anticoagulation treatment strategy that will provide sufficient benefit, without harm, for severely ill CLL patients hospitalized with COVID-19.Age, serum CRP, albumin level, and D-dimer are simple, easily accessible parameters and may be good candidates for defining subgroups of CLL patients who are at increased risk for thrombosis and bleeding during COVID-19. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The datasets used and analysed during the current study are available from the corresponding author on reasonable request. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Author contributions DA, VO-designed research, performed research; DA, VO, NM-wrote the paper; NM, NR-statistical analysis; TC, LS, PG, KS, MC-revision, additional ideas, upgrade the paper; all others-data collection.All authors reviewed the manuscript.All authors read and approved the final manuscript. Mentioned in the manuscript. Not applicable. L.S. received advisory boards fees from AbbVie and Janssen; educational activity AstraZeneca.DA received funding to attend symposia from Gilead and Bayer.MA received advisory boards fees from AbbVie, AstraZeneca, Janssen-Cilag; travel support from AbbVie and Novartis.RC received speaker fees from Roche, Janssen, AbbVie, AstraZeneca, Celgene, BMS, Kite, and Takeda; advisory board fees from Janssen, AbbVie, Celgene, BMS, Kite, Takeda, Incyte, Kyowa-Kirin, and ADCT; travel and accommodation expenses from Roche, Janssen, AbbVie, Celgene, BMS, Kite, Takeda, and Pfizer; research grant from Pfizer.MC received honoraria from AbbVie, Gilead, Janssen, and AstraZeneca.LS received advisory boards fees from AbbVie, BeiGene and Janssen; educational activity AstraZeneca.BE received consulting or advisory boards fees from Janssen, Roche, Novartis, AbbVie, Gilead, Celgene, ArQule, AstraZeneca, Oxford Biomedica (UK), and BeiGene; speaker / speaker's bureau fees from Janssen, Gilead, Roche, AbbVie, Novartis, Celgene, Adaptive Biotechnologies, BioGene, and AstraZeneca; research support / research funding from Janssen, Gilead, Roche, AbbVie, BeiGene, and AstraZeneca; travel, accommodations, expenses from Janssen, Roche, Novartis, AbbVie, Gilead, and Celgene.MF received honoraria from Abbvie, Janssen, Gilead.JAG-M received honoraria for advisory board and speaker´s bureau from Mundipharma, Glaxo, AbbVie, Roche, Gilead, AstraZeneca, and Janssen; research support from Hoffman-La Roche, AbbVie, and Janssen.RG-S received educational grants from AbbVie, Janssen, and Novartis.EG received travel grants, honoraria as a consultant and/or speaker bureau for Janssen-Cilag, Roche, and AbbVie.MGDS received honoraria for consultancy/advisory boards with Roche, Janssen Cilag, Gilead, AbbVie, and BMS; research Grant from Gilead, and AstraZeneca.YH received honoraria from AbbVie, Janssen, AstraZeneca, Roche and Medison, outside the submitted work.JAH received honoraria and advisory boards fees from Janssen, AbbVie, AstraZeneca, Roche, Beigene, Gilead, and BMS-Celgene.OJ received honoraria from AbbVie, Janssen, and Roche.AK received honoraria from Janssen, BMS, Astra Zeneca, Roche/Genentech; research money from Janssen, BMS, AstraZeneca, and Roche/Genentech.SK received Travel grant from Celgene; research funding from Janssen, Roche/Genentech, and AbbVie.LL received honoraria for advisory board and lecturing from Janssen, Gilead, AbbVie, Roche, and AstraZeneca.M-DL received advisory board and travel expenses from AbbVie, Janssen, Takeda, and Roche.AG received speaker's • 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 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Author contributions DA, VO-designed research, performed research; DA, VO, NM-wrote the paper; NM, NR-statistical analysis; TC, LS, PG, KS, MC-revision, additional ideas, upgrade the paper; all others-data collection.All authors reviewed the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Mentioned in the manuscript. ",
"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": "L.S. received advisory boards fees from AbbVie and Janssen; educational activity AstraZeneca.DA received funding to attend symposia from Gilead and Bayer.MA received advisory boards fees from AbbVie, AstraZeneca, Janssen-Cilag; travel support from AbbVie and Novartis.RC received speaker fees from Roche, Janssen, AbbVie, AstraZeneca, Celgene, BMS, Kite, and Takeda; advisory board fees from Janssen, AbbVie, Celgene, BMS, Kite, Takeda, Incyte, Kyowa-Kirin, and ADCT; travel and accommodation expenses from Roche, Janssen, AbbVie, Celgene, BMS, Kite, Takeda, and Pfizer; research grant from Pfizer.MC received honoraria from AbbVie, Gilead, Janssen, and AstraZeneca.LS received advisory boards fees from AbbVie, BeiGene and Janssen; educational activity AstraZeneca.BE received consulting or advisory boards fees from Janssen, Roche, Novartis, AbbVie, Gilead, Celgene, ArQule, AstraZeneca, Oxford Biomedica (UK), and BeiGene; speaker / speaker's bureau fees from Janssen, Gilead, Roche, AbbVie, Novartis, Celgene, Adaptive Biotechnologies, BioGene, and AstraZeneca; research support / research funding from Janssen, Gilead, Roche, AbbVie, BeiGene, and AstraZeneca; travel, accommodations, expenses from Janssen, Roche, Novartis, AbbVie, Gilead, and Celgene.MF received honoraria from Abbvie, Janssen, Gilead.JAG-M received honoraria for advisory board and speaker´s bureau from Mundipharma, Glaxo, AbbVie, Roche, Gilead, AstraZeneca, and Janssen; research support from Hoffman-La Roche, AbbVie, and Janssen.RG-S received educational grants from AbbVie, Janssen, and Novartis.EG received travel grants, honoraria as a consultant and/or speaker bureau for Janssen-Cilag, Roche, and AbbVie.MGDS received honoraria for consultancy/advisory boards with Roche, Janssen Cilag, Gilead, AbbVie, and BMS; research Grant from Gilead, and AstraZeneca.YH received honoraria from AbbVie, Janssen, AstraZeneca, Roche and Medison, outside the submitted work.JAH received honoraria and advisory boards fees from Janssen, AbbVie, AstraZeneca, Roche, Beigene, Gilead, and BMS-Celgene.OJ received honoraria from AbbVie, Janssen, and Roche.AK received honoraria from Janssen, BMS, Astra Zeneca, Roche/Genentech; research money from Janssen, BMS, AstraZeneca, and Roche/Genentech.SK received Travel grant from Celgene; research funding from Janssen, Roche/Genentech, and AbbVie.LL received honoraria for advisory board and lecturing from Janssen, Gilead, AbbVie, Roche, and AstraZeneca.M-DL received advisory board and travel expenses from AbbVie, Janssen, Takeda, and Roche.AG received speaker's • 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": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.7759/cureus.3084
|
Not the Typical Pneumonia: An Unusual Case of Rasburicase-induced Methomoglobinemia
|
We present a rare case of rasburicase-induced methemoglobinemia and hemolytic anemia in the setting of presumed glucose-6-phosphate dehydrogenase (G6PD) deficiency. A 78-year-old male with a known history of chronic lymphocytic leukemia presented to the clinic with fever of unknown origin. Laboratory results were significant for hyperuricemia. He was empirically started on levofloxacin and rasburicase. He then presented to the emergency department with shortness of breath and syncope. Physical examination was remarkable for a fever of 102.8 °F, conjunctival pallor, and scleral icterus. An infiltrate was observed on his computed tomography (CT) angiogram of the chest. Arterial blood gas on 50% fraction of inspired oxygen was significant for an arterial oxygen level of 222 millimeters mercury and oxyhemoglobin of 85.9%. Co-oximetry was then obtained and methemoglobin level was 13.4%. Laboratory results were noteworthy for a drop-in hemoglobin, indirect hyperbilirubinemia, low haptoglobin and elevated lactate dehydrogenase; depicting hemolytic anemia. The patient received two units of packed red blood cells, intravenous broad-spectrum antibiotics and he clinically improved.
|
[
{
"section_content": "Tumor lysis syndrome (TLS) is an oncological emergency referring to the constellation of metabolic disturbances resulting from the rapid destruction of tumor cells leading to the release of intracellular ions and nucleic acids, eventually metabolized to uric acid, into the blood stream [1].The metabolic disturbances associated with TLS include hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, and acute kidney injury (AKI) [2].TLS can be precipitated either following the administration of cytotoxic chemotherapeutic agents or spontaneously in the setting of hematological malignancies [3]. In TLS, the main prophylactic methods include intravenous hydration, allopurinol and rasburicase administration [4].For patients at high risk for TLS or in those where allopurinol is contraindicated, rasburicase is currently recommended by the Guidelines for the Management of Pediatric and Adult Tumor lysis syndrome published by the American Society of Clinical Oncology in 2008 [4].Rasburicase is a recombinant urate-oxidase enzyme approved by the Food and Drug Administration (FDA) for the treatment and/or prevention of hyperuricemia and TLS.It converts uric acid to allantoin (an inactive and soluble metabolite of uric acid) facilitating the renal clearance of uric acid and decreasing its precipitation in the kidneys [5].Multiple studies demonstrated a greater reduction in uric acid levels with a lower risk of AKI, compared to allopurinol [5][6][7]. Rasburicase is usually well tolerated; however, several side effects are of concern.Methemoglobinemia is an infrequent (<1%) but serious complication associated with the administration of rasburicase [8].In addition, the drug is contraindicated in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency as hydrogen peroxide (a by-product of uric acid catabolism) can present an oxidative stress thus, precipitating hemolysis in this setting [9].There are United States (US) boxed warnings for both these side effects and advice to screen all African, South-East Asian, Middle Eastern, and Mediterranean patients for G6PD prior to rasburicase administration [10].In the following, we illustrate the morbidity and management of rasburicase-induced methemoglobinemia and hemolysis in an African-American male patient with presumed G6PD. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "A 78-year-old African-American man weighing 53 kilograms with a past medical history significant for chronic lymphocytic leukemia (CLL), hyperuricemia with chronic gouty arthropathy, chronic kidney disease (CKD) stage 3 presented to the Emergency Department (ED) with the chief complaint of shortness of breath and fatigue for one day.His outpatient medication included ibrutinib 420 milligrams (mg) and allopurinol 300 mg daily.He was diagnosed with CLL two years prior to presentation.Initially, he was started on bendamustine with an appropriate response.However, an escalation in his lymphocyte count was appreciated one month prior to presentation and ibrutinib was initiated for CLL progression.One day prior to admission to the hospital, the patient was evaluated by his primary oncologist at an outpatient visit.He was febrile with a temperature of 101.6 degrees fahrenheit (°F) but otherwise asymptomatic.Blood and urine cultures were obtained and was started on oral levofloxacin empirically.His labs were significant for worsening hyperuricemia with a uric acid level of 13.0 milligram per deciliter (mg/dl).A single dose of intravenous rasburicase 3 mg was then administered.The following day, he presented to the ED complaining of significant fatigue associated with dry nonproductive cough of one day duration.Examination was remarkable for a fever of 102.8°F, an oxygen saturation (SPO2) of 85% on room air, conjunctival pallor and scleral icterus.A left lower lobe infiltrate was observed on his computed tomography (CT) angiogram of the chest (Figure 1).Laboratory results were noteworthy for a drop-in hemoglobin (4.9 mg/dL), indirect hyperbilirubinemia (7.2 mg/dL), low haptoglobin (<10 mg/dL) and elevated lactate dehydrogenase of 1611 International Units per liter (IU/L) compared to (756 IU/L) the day before; depicting acute hemolytic anemia.SPO2 persisted at 85% despite the use of nasal cannula, nonrebreather and non-invasive positive pressure ventilation.The patient was admitted to the intensive care unit (ICU).Arterial blood gas on 50% fraction of inspired oxygen (FiO2) was significant for an arterial oxygen level (PO2) of 222 millimeters mercury (mm Hg) and an oxyhemoglobin of 85.9%.Co-oximetry was then obtained and methemoglobin level was 13.4%.The patient was started on vancomycin and cefepime and transfused with three units of packed red blood cells.Methylene blue was not administered as the patient was assumed to have G6PD deficiency given his race and this episode of hemolysis. After an ICU stay of two days, the patient clinically improved, SpO2 normalized, his hemoglobin levels improved, methemoglobin levels trended down and was transferred back to the floor (Table 1).A G6PD level sent during the acute attack yielded a result within the normal range.In addition, mycoplasma serum antibodies were negative.Despite broad spectrum antibiotic coverage, the patient continued to spike fevers as high as 102.7°F from day one to day four.He subsequently started complaining of right knee pain associated with right knee swelling and tenderness on examination.Arthrocentesis revealed monosodium urate crystals and was started on colchicine and prednisone.Fever, knee pain, swelling and tenderness resolved.The patient was discharged to a skilled nursing facility on prednisone taper, allopurinol and colchicine.He was continued on ibrutinib to date (five months later) and his white blood cell count is currently within normal limits.A repeat CT chest performed three monthts later documented resolution of the previously seen left lower lobe pulmonary inifiltrate. ",
"section_name": "Case Presentation",
"section_num": null
},
{
"section_content": "",
"section_name": "FIGURE 1: Computed tomography (CT) of the chest demonstrating a left lower lobe infiltrate",
"section_num": null
},
{
"section_content": "We present a rare case of rasburicase-induced methemoglobinemia and hemolytic anemia in the setting of G6PD deficiency.According to an international TLS expert consensus panel developed by Cairo et al., our patient was at high risk for developing TLS confirming the appropriate use of rasburicase in this scenario [11].As previously mentioned, there is a US boxed warning advising to screen all African, South-East Asian, Middle Eastern and Mediterranean patients for G6PD prior to rasburicase administration [10].Due to the urgency of the situation and the patient's worsening uric acid levels, despite being on allopurinol, our patient was not screened for G6PD prior to administration.The benefits of treating this oncological emergency outweighed the risks of administering rasburicase in a patient whose G6PD status is unknown.An important aspect in testing G6PD enzymatic activity is the timing.During acute hemolysis, the RBCs with severely reduced G6PD activity will hemolyze.So, the measured assay will reflect the activity of G6PD in the remaining RBCs which carry enzymes with a relatively normal activity, resulting in a false negative result. Methemoglobinemia occurs when the ferrous ions incorporated in hemoglobin are oxidized to ferric ions [12].Ferric ions are unable to reversibly bind to oxygen; thus, increasing the affinity of the remaining ferrous hemoglobin to oxygen, shifting the oxygen dissociation curve to the left and therefore decreasing the release of oxygen to the tissue inducing tissue hypoxia [12].One of the cellular reactions involved in reducing methemoglobin to hemoglobin in the body utilizes nicotinamide adenine dinucleotide phosphate (NADPH) generated by the G6PD in the hexose monophosphate shunt.This reaction is not physiologically functional and requires activation by electron carriers such as methylene blue or riboflavin [13].Therefore, the first line of therapy for the management of methemoglobinemia, in addition to avoiding the precipitating chemical, involves the use of methylene blue to facilitate the reduction of ferric to ferrous ions [14].In patients with G6PD deficiency, NADPH generation is reduced thus this reaction is deemed useless and methylene blue is ineffective in such patients.Furthermore, methylene blue has oxidant potential which may induce hemolysis in G6PD deficient patients [15].In these patients, ascorbic acid is recommended [16].Ascorbic acid is slow in onset and requires up to 24 hours and therefore is a poor choice in such emergencies [16]. Reviewing the literature, only one case was successfully managed using ascorbic acid alone [9].Other alternative therapies include simple or exchange blood transfusions and/or hyperbaric oxygen therapy.Given the combination of hemolysis and mild methemoglobinemia <20% in our patient, we opted for a simple blood transfusion. As per our knowledge, the number of rasburicase-induced methemoglobinemia reported cases is limited. Having both methemoglobinemia and acute hemolysis makes this case exceedingly rare.We suggest that simple transfusion alone is an effective alternative in the management of rasburicase-induced methemoglobinemia and hemolytic anemia, particularly in mild to moderate cases with methemoglobin levels less than 20%.In such scenarios, simple transfusion serves two purposes: correcting the anemia and counteracting methemoglobinemia by supplying normal hemoglobin species preventing tissue hypoxia. ( ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Rasburicase-induced methemoglobinemia and hemolytic anemia have been identified as a direct cause for morbidity and mortality in high-risk individuals.Methylene blue is strongly discouraged for rasburicaseinduced methemoglobinemia even if the G6PD status is unknown.Management with simple blood transfusions in mild to moderate severity cases is a relatively safe and effective strategy.Spreading awareness and using order sets to remind physicians of risks associated with rasburicase and methylene blue use might be helpful.It is enouraged to report such cases and highlight the management to help set up the stage to formulate a unified consensus for treatment. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "Human subjects: Consent was obtained by all participants in this study.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 Disclosures",
"section_num": null
}
] |
10.3390/cancers10050129
|
EBV Positive Diffuse Large B Cell Lymphoma and Chronic Lymphocytic Leukemia Patients Exhibit Increased Anti-dUTPase Antibodies
|
<jats:p>The Epstein-Barr virus (EBV), which is a ubiquitous γ-herpesvirus, establishes a latent infection in more than 90% of the global adult population. EBV-associated malignancies have increased by 14.6% over the last 20 years, and account for approximately 1.5% of all cancers worldwide and 1.8% of all cancer deaths. However, the potential involvement/contribution of lytic proteins to the pathophysiology of EBV-associated cancers is not well understood. We have previously demonstrated that the EBV-deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate and adaptive immune responses by engaging the Toll-Like Receptor 2 (TLR2), which leads to the modulation of downstream genes involved in oncogenesis, chronic inflammation, and in effector T-cell function. Furthermore, examination of serum samples from diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia patients revealed the presence of increased levels of anti-dUTPase antibodies in both cohorts compared to controls with the highest levels (3.67-fold increase) observed in DLBCL female cases and the lowest (2.12-fold increase) in DLBCL males. Using computer-generated algorithms, dUTPase amino acid sequence alignments, and functional studies of BLLF3 mutants, we identified a putative amino acid motif involved with TLR2 interaction. These findings suggest that the EBV-dUTPase: TLR2 interaction is a potential molecular target that could be used for developing novel therapeutics (small molecules/vaccines).</jats:p>
|
[
{
"section_content": "Epstein-Barr virus, which is a γ herpesvirus, is a ubiquitous virus that establishes a latent infection in over 90% of the global adult population.In addition to being the etiological agent of infectious mononucleosis (IM), it is implicated in several human malignancies including Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), classical Hodgkin lymphoma (cHL), gastric cancer, and diffuse large B cell lymphoma (DLBCL) [1].DLBCL is a heterogeneous disease that is classified based on micro-array-based gene expression profiling as germinal center B-cell like (GCB) DLBCL or activated B-cell-like (ABC) DLBCL [2].A small percentage (10-20%) of DLBCLs are EBV-genome positive and such tumors are usually classified in the ABC DLBCL group [3].While EBV-genome positive tumors were originally discovered in older (>50 years of age) immunocompetent individuals [4][5][6][7][8], they have recently been reclassified to EBV + DLBCL-NOS due to the increased occurrence of EBV + DLBCL in younger immunocompetent patients [9].EBV is an independent factor that adversely affects risk and/or survival among patients with DLBCL [5,7].Conversely, while EBV is not generally associated with developing chronic lymphocytic leukemia (CLL), which is the most common leukemia in adults in western countries [10], it is associated with Richter Syndrome (RS).RS, which occurs in 10% to 15% of patients with CLL, is a histological transformation to DLBCL resulting in a more aggressive lymphoma with a poorer prognosis [11][12][13][14].While several studies have implicated EBV in RS [15,16], a mechanistic relationship has not been determined.However, a recent study demonstrated that therapy related to immunosuppression in patients with CLL resulted in EBV reactivation, which drove RS and the formation of the ABC subtype DLBCL in some patients [17].This study as well as a recent study of DLBCL [18] has suggested that products of lytic EBV may contribute to the development of these malignancies, but additional studies need to be performed. Studies to examine the roles of EBV-encoded proteins in cellular transformation have focused primarily on those proteins and RNAs expressed during latency.These studies have demonstrated unequivocally the roles of the latent membrane proteins LMP1 and LMP2A in the transformation process and the immunological response of the host to these proteins [19,20].Until recently, there have been very few studies directed toward determining the role(s) of proteins expressed during the lytic replication of EBV in immune modulation or in transformation despite the fact that low levels of EBV reactivation and expression of genes associated with lytic replication are typically observed in a small number of cells in many tumors [21][22][23][24][25][26][27][28].However, recent studies have demonstrated the expression of a large number of lytic genes in cell lines and, more importantly, in biopsy tissue [29][30][31].It has been suggested that proteins encoded by these genes may contribute to EBV oncogenesis by modulating the tumor microenvironment through the release of growth factors and/or immunosuppressive cytokines [32] or more directly by inducing genomic instability [28].Additional data obtained from studies using SCID and humanized mouse models support this premise [33][34][35][36]. The EBV gene BLLF3 encodes for a deoxyuridine triphosphate nucleotidohydrolase (dUTPase), which is expressed during lytic/abortive lytic replication of the virus.While it has been difficult to quantify the amount of EBV-dUTPase present in tissue or serum because of the lack of sensitive assays, Ersing et al. [37] recently examined virus-host interactions during lytic replication using systemic proteomic quantitative analysis with tandem mass tags and mass spectrometry and estimated that the concentration of the EBV-dUTPase was 6000 nM and 7500 nM, respectively, in Akata and P3HR1 cells.There is indirect evidence to support the premise that EBV-encoded dUTPase is expressed and released from cells in vivo by following lytic and/or abortive replication.We have demonstrated, using quantitative real-time PCR, the expression of BLLF3 in tumors (9/10) obtained from SCID mice injected with C666-1 cells, which is an EBV-genome positive NPC cell line [38].Zhang et al. [39], using microarray technology, demonstrated the expression of BLLF3 in PBMCs from a patient with acute phase IM and in EBV genome positive tumor cell lines established from patients with nasal NK/T-cell lymphoma.In addition, the EBV-encoded dUTPase protein has been detected using immuno-histochemical techniques in the upper epithelial layers of oral hairy leukoplakia (HL) lesions and the expression pattern was the same for BZLF-1 [40].Similar results were obtained with lymphoid cells in tonsils from patients with IM and in NPC tissue [40,41].Furthermore, we recently demonstrated by using immunohistochemistry the presence of the EBV-dUTPase in kidney biopsies from class III/IV Lupus nephritis (LN) patients.The EBV-dUTPase localized in infiltrating plasma-cell aggregates near glomeruli where neighboring cells expressing increased toll-like receptor 2 (TLR2) and IL-17 protein levels were observed, which suggests that EBV-dUTPase may exacerbate the immunopathologies in some LN patients [42].We, as well as others, have demonstrated the presence of specific anti-EBV-encoded dUTPase antibodies in the sera of patients with IM, in reactivated and chronic EBV infections, in immunocompromised patients with HIV infections, and in immunocompetent patients with EBV genome positive diffuse large B-cell lymphoma, chronic lymphocytic leukemia and NPC [43][44][45], and unpublished data. We have demonstrated that the dUTPases encoded by the human herpesviruses represent a new class of pathogen-associated molecular pattern (PAMP) proteins that have novel immuno-regulatory and neuro-regulatory functions, which may contribute to the pathophysiology of diseases caused by these viruses.Using the EBV-dUTPase as the prototype, our studies have demonstrated that it possesses novel functions independent of its enzymatic activity.Among them, the EBV-dUTPase acts as a trigger for TLR2, which leads to the activation of NF-κB and subsequent modulation of downstream genes involved in chronic inflammation and oncogenesis [46].We have also demonstrated that these viral dUTPases are capable of differentially inducing the secretion of the pro-inflammatory T H 1/T H 17 cytokines IL-1β, IL-6, IL-8, IL-12p70, TNF-α, CCL20, and IFN-γ as well as the anti-inflammatory cytokine IL-10 in human primary immune cells [47][48][49][50][51].Not only is CCL20 reported to promote cellular proliferation and differentiation of numerous cell types including malignant cells but IL-6, which is a positive regulator of CCL20, also functions as an autocrine growth factor for EBV-immortalized B-cells [52][53][54]. Since the interaction of EBV-dUTPase with TLR2 is the critical step for initiating the signaling cascade that leads to the establishment of a microenvironment that may support the survival and proliferation of EBV-transformed cells, the purpose of the present study was to elucidate the amino acid residues in the EBV-dUTPase important for interacting with TLR2. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "The EBV-encoded dUTPase is composed of 278 amino acids and, while it is the smallest of the human herpesviruses' dUTPases, it contains all five motifs characteristic of dUTPases [55] as well as a unique motif (motif 6) found in herpesviruses' dUTPases (see Figure 1) [56]. Cancers 2018, 10, x 3 of 15 possesses novel functions independent of its enzymatic activity.Among them, the EBV-dUTPase acts as a trigger for TLR2, which leads to the activation of NF-κB and subsequent modulation of downstream genes involved in chronic inflammation and oncogenesis [46].We have also demonstrated that these viral dUTPases are capable of differentially inducing the secretion of the pro-inflammatory TH1/TH17 cytokines IL-1β, IL-6, IL-8, IL-12p70, TNF-α, CCL20, and IFN-γ as well as the anti-inflammatory cytokine IL-10 in human primary immune cells [47][48][49][50][51].Not only is CCL20 reported to promote cellular proliferation and differentiation of numerous cell types including malignant cells but IL-6, which is a positive regulator of CCL20, also functions as an autocrine growth factor for EBV-immortalized B-cells [52][53][54]. Since the interaction of EBV-dUTPase with TLR2 is the critical step for initiating the signaling cascade that leads to the establishment of a microenvironment that may support the survival and proliferation of EBV-transformed cells, the purpose of the present study was to elucidate the amino acid residues in the EBV-dUTPase important for interacting with TLR2. ",
"section_name": "Identification of a Putative TLR2 Binding Motif within the EBV-dUTPase",
"section_num": "2.1."
},
{
"section_content": "The EBV-encoded dUTPase is composed of 278 amino acids and, while it is the smallest of the human herpesviruses' dUTPases, it contains all five motifs characteristic of dUTPases [55] as well as a unique motif (motif 6) found in herpesviruses' dUTPases (see Figure 1) [56].Using computer-generated algorithms (hydrophilicity, flexibility, mobility, solvent exposure, amphiphilicity, reverse turns, α-helical properties, and protrusion) to predict amino acid sequences that have the potential to interact with other proteins, we identified five sequences, which were then computer-ranked based upon their respective algorithms [51,57].The amino acid sequences were 83-103 (rank 2), 109-140 (rank 1 contains the entire conserved motif 1), 174-194 (rank 4 contains most of motif 6, which is restricted to herpesviruses' dUTPases), 210-237 (rank 5 contains conserved motif 4), and 253-276 (rank 3 contains conserved motif 5). Since amino acid residues 83 to 103 were identified by computer-generated algorithms as the only non-conserved motif containing sequences with a high likelihood for interacting with other proteins and amino acid sequence 174 to 194 contains most of the unique motif 6 of unknown function, we next constructed synthetic peptides corresponding to amino acid residues leucine 83 through lysine 103 (L83-K103) as well as leucine 174 through serine 194 (L174-S194) and tested whether or not these peptides could induce NF-κB activation by engaging TLR2.As shown in Figure 2, only the L83-K103 peptide induced the transcriptional activation of the NF-κB reporter gene by 34fold, which is approximately 50% of that exhibited by the full-length EBV-dUTPase protein (74-fold) while L174-S194 and corresponding scrambled control peptides did not cause a significant activation of NF-κB in human embryonic kidney 293 cells (HEK293)-stably expressing TLR2.Using computer-generated algorithms (hydrophilicity, flexibility, mobility, solvent exposure, amphiphilicity, reverse turns, α-helical properties, and protrusion) to predict amino acid sequences that have the potential to interact with other proteins, we identified five sequences, which were then computer-ranked based upon their respective algorithms [51,57].The amino acid sequences were 83-103 (rank 2), 109-140 (rank 1 contains the entire conserved motif 1), 174-194 (rank 4 contains most of motif 6, which is restricted to herpesviruses' dUTPases), 210-237 (rank 5 contains conserved motif 4), and 253-276 (rank 3 contains conserved motif 5). Since amino acid residues 83 to 103 were identified by computer-generated algorithms as the only non-conserved motif containing sequences with a high likelihood for interacting with other proteins and amino acid sequence 174 to 194 contains most of the unique motif 6 of unknown function, we next constructed synthetic peptides corresponding to amino acid residues leucine 83 through lysine 103 (L83-K103) as well as leucine 174 through serine 194 (L174-S194) and tested whether or not these peptides could induce NF-κB activation by engaging TLR2.As shown in Figure 2, only the L83-K103 peptide induced the transcriptional activation of the NF-κB reporter gene by 34-fold, which is approximately 50% of that exhibited by the full-length EBV-dUTPase protein (74-fold) while L174-S194 and corresponding scrambled control peptides did not cause a significant activation of NF-κB in human embryonic kidney 293 cells (HEK293)-stably expressing TLR2.Further cytokine analysis of synthetic peptides in human PBMCs revealed that stimulation of cells with the EBV-dUTPase synthetic peptide L83-K103 but not scrambled peptide resulted in an increased production of IL-6 (9-fold increase over scrambled control peptide), IL-8 (2.5-fold increase), TNF-α (5-fold increase), IL-10 (4.8-fold increase), and IL-1β (3.3-fold increase) cytokines compared to untreated control cells.However, the cytokine response induced by L83-K103 peptide was not as strong as that observed in cells stimulated with the full-length EBV-dUTPase protein especially for IL-1β, TNFα, and IL-10 (see Table 1).While the size and sequence homologies of the dUTPases encoded by members of the Herpesviridae Family vary considerably, a common feature in the dUTPases encoded by EBV, HSV-2, HHV-6, HHV-8, and VZV is their ability to trigger the activation of TLR2 [40,43,44].Blast analyses of the amino acid sequences of the herpesviruses dUTPases as well as the human nuclear dUTPase demonstrated that the herpesviruses contained a sequence that was somewhat divergent especially with the β-herpesviruses that may represent a conserved sequence (see Table 2).Further cytokine analysis of synthetic peptides in human PBMCs revealed that stimulation of cells with the EBV-dUTPase synthetic peptide L83-K103 but not scrambled peptide resulted in an increased production of IL-6 (9-fold increase over scrambled control peptide), IL-8 (2.5-fold increase), TNF-α (5-fold increase), IL-10 (4.8-fold increase), and IL-1β (3.3-fold increase) cytokines compared to untreated control cells.However, the cytokine response induced by L83-K103 peptide was not as strong as that observed in cells stimulated with the full-length EBV-dUTPase protein especially for IL-1β, TNFα, and IL-10 (see Table 1).While the size and sequence homologies of the dUTPases encoded by members of the Herpesviridae Family vary considerably, a common feature in the dUTPases encoded by EBV, HSV-2, HHV-6, HHV-8, and VZV is their ability to trigger the activation of TLR2 [40,43,44].Blast analyses of the amino acid sequences of the herpesviruses dUTPases as well as the human nuclear dUTPase demonstrated that the herpesviruses contained a sequence that was somewhat divergent especially with the β-herpesviruses that may represent a conserved sequence (see Table 2).This TLR2 putative interactive motif is located in the β6 strand structure adjacent to motif 3, which is part of the catalytic site.This motif contains eight amino acid residues, which are included in the computer-generated algorithm sequence 83-103.The possibility of this motif being part of the TLR2 interactive domain is further supported by the studies described above, which demonstrate that a synthetic peptide corresponding to amino acids L83-K103 of the EBV-dUTPase induced NF-κB activation (see Figure 1) in TLR2-HEK293 cells and stimulated the secretion of pro-inflammatory cytokines in human PBMCs (see Table 2).To further confirm the importance of this motif for triggering TLR2 signaling, we generated a recombinant EBV-dUTPase protein containing a triple mutation ( 82 ELR 84 to 82 GGG 84 ; EBVdUTPase/TMutTLR2BD) and demonstrated that it significantly reduced NF-κB activation by 83% to 12.31-fold compared to the wild-type EBV-dUTPase protein (75-fold increase) (see Figure 3).Decreased cytokine secretion by stimulated PBMCs was also observed (data not shown), which highlights the importance of amino acid residues glutamate (E) 82, leucine (L) 83, and arginine (R) 84 in the activation of TLR2 signaling and further supporting this motif as a TLR2 putative interactive motif.This TLR2 putative interactive motif is located in the β6 strand structure adjacent to motif 3, which is part of the catalytic site.This motif contains eight amino acid residues, which are included in the computer-generated algorithm sequence 83-103.The possibility of this motif being part of the TLR2 interactive domain is further supported by the studies described above, which demonstrate that a synthetic peptide corresponding to amino acids L83-K103 of the EBV-dUTPase induced NF-κB activation (see Figure 1) in TLR2-HEK293 cells and stimulated the secretion of pro-inflammatory cytokines in human PBMCs (see Table 2).To further confirm the importance of this motif for triggering TLR2 signaling, we generated a recombinant EBV-dUTPase protein containing a triple mutation ( 82 ELR 84 to 82 GGG 84 ; EBVdUTPase/TMutTLR2BD) and demonstrated that it significantly reduced NF-κB activation by 83% to 12.31-fold compared to the wild-type EBV-dUTPase protein (75fold increase) (see Figure 3).Decreased cytokine secretion by stimulated PBMCs was also observed (data not shown), which highlights the importance of amino acid residues glutamate (E) 82, leucine (L) 83, and arginine (R) 84 in the activation of TLR2 signaling and further supporting this motif as a TLR2 putative interactive motif. Figure 3. Site-directed mutagenesis of the putative TLR2 binding motif in the EBV-dUTPase inhibits NF-κB activation.TLR2-HEK293 cells were transiently transfected with NF-κB luciferase reporter plasmid as we have described [40,43,44].After 24-36 h, cells were treated with wild-type EBV-dUTPase, a triple mutant ( 82 ELR 84 to 82 GGG 84 ) of the EBV-dUTPase TLR2 putative binding motif (EBVdUTPase/TMutTLR2BD), EBV-dUTPase peptide L83-K103, scrambled peptide L83-K103 (10 μg/mL) or left untreated for 8 h and luciferase reporter gene activity was measured.Values represent the mean fold induction (FI) ± SD relative to control (n = 3).* p < 0.05 (Groups compared: dUTPase treated vs. untreated and dUTPase triple mutant vs. wild-type dUTPase). ",
"section_name": "Identification of a Putative TLR2 Binding Motif within the EBV-dUTPase",
"section_num": "2.1."
},
{
"section_content": "A better understanding of the diversity in the humoral response to EBV-dUTPase in health and disease states may enable us to identify EBV-dUTPase antibody patterns that could be used as markers for early diagnosis and/or to monitor treatment.Using our standard neutralization assay [44], we next conducted a pilot study to examine the humoral response to the EBV-dUTPase in Figure 3. Site-directed mutagenesis of the putative TLR2 binding motif in the EBV-dUTPase inhibits NF-κB activation.TLR2-HEK293 cells were transiently transfected with NF-κB luciferase reporter plasmid as we have described [40,43,44].After 24-36 h, cells were treated with wild-type EBV-dUTPase, a triple mutant ( 82 ELR 84 to 82 GGG 84 ) of the EBV-dUTPase TLR2 putative binding motif (EBVdUTPase/TMutTLR2BD), EBV-dUTPase peptide L83-K103, scrambled peptide L83-K103 (10 µg/mL) or left untreated for 8 h and luciferase reporter gene activity was measured.Values represent the mean fold induction (FI) ± SD relative to control (n = 3).* p < 0.05 (Groups compared: dUTPase treated vs. untreated and dUTPase triple mutant vs. wild-type dUTPase). ",
"section_name": "Anti-EBV-Encoded dUTPase Antibody in Patients with DLBCL and Chronic Lymphocytic Leukemia (CLL)",
"section_num": "2.2."
},
{
"section_content": "A better understanding of the diversity in the humoral response to EBV-dUTPase in health and disease states may enable us to identify EBV-dUTPase antibody patterns that could be used as markers for early diagnosis and/or to monitor treatment.Using our standard neutralization assay [44], we next conducted a pilot study to examine the humoral response to the EBV-dUTPase in healthy EBV carriers (n = 89) and in the B cell malignancies DLBCL (n = 36) and CLL (n = 66) sera samples from the European EPILYMPH case-control published study [58] exhibiting either a normal or abnormal/reactive antibody pattern to EBV, which was determined by de Sanjose et al. [58] 75, respectively, were tested for the presence of anti-EBV dUTPase neutralizing antibodies.This study revealed an overall increase in neutralizing antibodies specific to the EBV-dUTPase in the case cohorts (36.11% and 39.39% for DLBCL and CLL, respectively) compared to the controls (12.76%)(see Table 3).Data analysis by gender shows a difference in the prevalence of dUTPase neutralizing antibodies between females and males within each cohort and across disease type relative to the controls.Within the DLBCL sera samples, there was a higher prevalence of dUTPase neutralizing antibodies in females than males (42.86% versus 26.66% in males).Additionally, the opposite was observed in the CLL cohort with males exhibiting a higher prevalence of dUTPase neutralizing antibodies than females (34.37% versus 44.12% in males).No differences were found within the control cohort between females and males, which suggests that the prevalence of dUTPase neutralizing antibodies in this group is independent of gender.More importantly, when comparing dUTPase antibody prevalence across disease type, it was found that the highest increase was observed in CLL male (44.12% versus 13.98% in controls) and DLBCL female cases (42.86% versus 11.67% in controls) (a 3.16-fold and 3.67-fold increase respectively), which were followed by the CLL female (34.37%, 2.95-fold increase) and DLBCL male cases (29.67%, 2.12-fold).a dUTPase neutralizing assays were performed as described previously [44].Values in parentheses represent the number of positive sera in either cases or controls/total sera.The total number of control sera (n = 431) include 89 samples from the EPILYMPH case-control study [58] as well as 352 samples from other published studies [44,45,48].b Individuals' sera exhibiting an abnormal reactive Ab pattern to EBV (ARP_EBV) was determined by de Sanjose et al., as part of the EPILYMPH case-control previously published study [58].Values represent the percentage of sera samples exhibiting EBV-IgG reactivity to combined immuno-dominant epitopes of EBNA1 and VCA-p18-based ELISA assays and abnormal reactivity/intensity score on immunoblots to EBV antigens (ex: EAd-p47/54, EAd-p138) other than/besides EBNA1, VCA-p40, VAC-p18, and ZEBRA predominantly recognized by healthy EBV immunocompetent individuals [58].Values in parentheses represent the number of individuals exhibiting increased/abnormal Ab responses to EBV proteins in either cases or controls sera/total sera.c Values represent the percentage of sera samples that were positive for anti-EBV-dUTPase antibodies and also had increased/abnormal EBV reactivity.Values in parentheses represent the number of positive sera in either case or controls/total sera. ",
"section_name": "Anti-EBV-Encoded dUTPase Antibody in Patients with DLBCL and Chronic Lymphocytic Leukemia (CLL)",
"section_num": "2.2."
},
{
"section_content": "EBV-associated malignancies are reported to account for approximately 1.5% of all cancers worldwide and represent 1.8% of all cancer deaths [59].While most studies have focused on the contribution of EBV latency proteins such as LMP-1 and LMP-2A in oncogenesis, few studies have addressed the role, if any, that EBV proteins produced during lytic/abortive lytic replication may have in this process. In the current study, we demonstrate the presence of increased neutralizing antibodies against the EBV-dUTPase in the sera of DLBCL and CCL patients from the EPYLYMPH study, which examined abnormal humoral responses to EBV [58].In the EPYLYMPH study, the investigators reported that patients with aberrant EBV activity were identified by a broad immuno-reactive profile including antibodies to several peptides from proteins composing the Early Antigen diffuse (EA-D) complex (BMRF1, BALF2, BGLF5, and BXLF1) as well as EBNA1, VCA, and BZLF1 while uncomplicated carriers and sera from some patients with lymphomas exhibited a more restricted antibody pattern (EBNA1, VCA, and BZLF1).The dUTPase, which is encoded by the BLLF3 gene, is an early protein that forms part of the EA-D complex.An important finding of our study is the observation that CLL male cases exhibited the highest prevalence of dUTPase neutralizing antibodies (44.12% versus 13.98% in controls) of all cases examined and had the lowest prevalence of an abnormal reactive antibody pattern to EBV.By contrast, CLL females had the highest increase in abnormal reactive antibody patterns to EBV (53.12% versus 22.22% in controls as determined by de Sanjose et al. [58]) but had the second lowest prevalence in dUTPase antibodies.Furthermore, within the DLBCL cohort, 67% (6/9) of female and all male sera samples (4) that tested positive for the presence of dUTPase neutralizing antibodies also expressed a normal/non-reactive antibody pattern to EBV.Overall, an increased prevalence of dUTPase neutralizing antibodies was consistently observed in sera of DLBCL and CLL patients who exhibited a normal/non-reactive antibody pattern to EBV.A recent study demonstrated that immediate early and early EBV proteins expressed during lytic replication of EBV are expressed in some tumor cells in patients with EBV + DLBCL and that antibodies against these proteins are detected in patients' sera.This led the investigators to propose that products from lytic/abortive lytic replication may contribute to tumor growth and survival [18].Our previous studies have demonstrated that the EBV-dUTPase protein induces IL-6 in primary dendritic cells and PBMCs [46][47][48].IL-6 is a growth factor for EBV-immortalized B cells and IL-6 over-expression has been shown to enhance growth of EBV-transformed lymphoblastoid cell lines (LCL's) in SCID mice [60][61][62][63].In addition, we have shown that EBV-dUTPase up-regulates the expression of CCL20 (335-fold) [49], which, in turn, may increase migration and trafficking of regulatory T cells (Tregs) into the tumor environment.Therefore, this dampens the immune response to EBV [54].EBV-dUTPase also up-regulates the expression of BIC/miR155 [38], which is associated with aberrant inflammatory responses and oncogenesis, enhanced B-cell transformation, and the development of Tregs [64].In all, these data support the premise that the dUTPase could modify the tumor microenvironment [32,38,65] and that the presence of antibodies directed against the dUTPase may be a useful marker for detecting aberrant virus replication in a subset of patients with DLBCL. While several studies have demonstrated increases in EBV viral load, EBV miRNA and anti-EBV-antibodies in the sera of patients with CLL [66][67][68][69][70], the potential role of EBV in the development of CLL still remains poorly understood.Another study by de Sanjose et al. [58] reported that CLL samples exhibited the highest prevalence of abnormal anti-EBV antibody reactivity (40%) of any lymphomas examined.This finding was also observed in our analyses of anti-EBV dUTPase antibodies in the same samples.Two independent studies have recently demonstrated that a subgroup (53-59%) of patients presenting with CLL had significantly higher EBV-DNA copy numbers [69,70].These patients required early treatment [69] and exhibited shorter survival rates [69,70].It is also well documented that a small percentage (10-15%) of patients with CLL will undergo histological transformation into an aggressive form of DLBCL [11][12][13][14] referred to as Ritchter syndrome (RS).Recently, it was reported that RS might occur following aggressive therapy during CLL that results in the reactivation of EBV [17].Therefore, it is possible that the CLL patients tested in this study and the study by de Sanjose et al. [58] represent two subgroups one in which EBV is a negative prognostic factor for patients with CLL and a second subgroup in which EBV contributes to RS transformation.While additional studies are necessary to delineate additional markers to distinguish these subgroups, the results of this study as well as that of de Sanjose et al. [58] suggest that antibodies against EBV-dUTPase and EA-D, which the dUTPase is a component of, could be useful markers for initially identifying such patients. There is a growing body of evidence demonstrating that the reactivation of latent herpesviruses, as indicated by higher antibody titers to proteins expressed during lytic or abortive-lytic replication, occurs when the immune system is compromised [45,48,[71][72][73][74].Several studies on EBV have established that reactivation of the virus usually results in abortive-lytic replication in which only immediate early and early genes are expressed and, therefore, no new virus is produced [75][76][77].Since the dUTPase is expressed as an early protein, this would suggest that abortive and/or lytic replication occurs in a subset of patients with DLBCL and CLL. We have previously demonstrated that the EBV-dUTPase triggers NF-κB activation by engaging TLR2 homodimers [46] while the dUTPases encoded by HSV-2, HHV-6, HHV-8, and VZV require ligation of the TLR2/1 heterodimer complex to activate NF-κB [50].Follow-up studies demonstrated that these viral dUTPases are capable of differentially inducing the secretion of the pro-inflammatory T H 1/T H 17 cytokines IL-1β, IL-6, IL-8, IL-12p70, TNF-α, and IFN-γ as well as the anti-inflammatory cytokine IL-10 in human primary immune cells [46][47][48][49][50][51].This suggests that they can modulate the cellular microenvironment [32,38,65].While sequence analyses have demonstrated that the α and γ herpesvirus members contain the five highly conserved motifs characteristic of the homotrimeric and monomeric dUTPases, members of the β herpesvirus group do not.However, all the human herpesviruses contain an additional conserved motif (domain 6) that is absent in the homotrimeric dUTPases [56].It has been suggested that this novel herpesvirus-specific domain may contribute to some unknown novel function.With the exception of the EBV-dUTPase [55], no crystal structure data is available for the other human herpesviruses' dUTPases.The results shown in this study demonstrate that amino acid residues between 81 G to 103 K of the EBV-dUTPase are important for binding to and activating TLR2 signaling.This TLR2 putative interactive motif is located in the β6 strand structure adjacent to motif 3, which is part of the catalytic site. The lack of control of EBV abortive/lytic replication may reflect a variety of physiological processes including stress and aging, which affect T-cell function and, therefore, the role of EBV in oncogenesis may or may not be a direct effect.However, our results suggest that lytic/abortive lytic replication occurs in patients with DLBCL as well as CLL.The EBV-dUTPase may alter the tumor microenvironment [32,38,65] by providing a selective advantage (growth/survival) to the malignant cell.Future studies using a larger cohort of patients will be necessary to determine whether there is a possible relationship between EBV-dUTPase expression and malignant progression as well as whether or not the presence of anti-EBV-dUTPase antibodies could be useful for diagnostic purposes.While additional experiments involving crystal structures of TLR2: EBV-dUTPase complexes are needed to confirm the specific amino acid residues of the EBV-dUTPase that interacts with TLR2, the results from the current study support the premise that the EBV-dUTPase-TLR2 interaction could be used as a target for developing novel therapeutics specifically small molecules and/or vaccines. ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "EBV-dUTPase containing a triple point mutation E82G, L83G, and R84G ( 82 ELR 84 to 82 GGG 84 ) was generated by site-directed mutagenesis using the QuikChange Lightning Mutagenesis system (Stratagene, Santa Clara, CA, USA), which was previously described [78] and the primer set: Forward: 5 -CCGGTCACGTCTCATGTTGGCATCATCGATCCCGGCTACACG-3 ; Reverse: 5 -CGTGTAGCCGGGATCGATGATGCCAACATGAGACGTGACCGG-3 .The PCR conditions used include one cycle at 95 • C for 2 min, which was followed by 18 cycles of 95 • C for 20 s, 60 • C for 10 s, and 68 • C for 2.5 min, and one cycle at 68 • C for 5 min.Amplified products were DpnI digested and screened for the β-galactosidase (β-gal+) phenotype.DNA was then purified and the amino acid changes E82G, L83G, and R84G were verified by using sequence analysis. ",
"section_name": "Construction of EBV-dUTPase Triple Mutant",
"section_num": "4.1."
},
{
"section_content": "EBV-dUTPase peptides 83 LRLILQNQRRYNSTLRPSELK 103 , 174 LAMQGILVKPCRWRRGGVDVS 194 as well as the respective scrambled controls peptides 83 ELQPKRTLQSRLYRINLSNRL 103 and 174 KRLGVCIQWVGLPRDVMRSAG 194 were synthesized in house in the Peptide Protein Engineering Laboratory at the Tzagrournis Medical Research Facility at The Ohio State University.Peptide synthesis was performed on a Milligen/Biosearch 9600 solid-phase peptide synthesizer (Bedford, MA, USA) using Fmoc/t-But chemistry.The C-terminal amino acid loaded on CLEAR ACID resin (0.32 mmol/gm) (e.g., in case of peptide WILL 83-103, Fmoc-Ile-CLEAR ACID Resin (Peptides International, Louisville, KY, USA) was used for the synthesis.All peptides were cleaved from the resin using cleavage reagent B (Trifluoroacteic acid:Phenol:Water:Triisopropyl silane 90:4:4:2) and crude peptides were purified on preparative Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC) using Vydac C-4 column and the acetonitrile-water (0.1% TFA) gradient system.All fractions were analyzed on analytical RP-HPLC and characterized by using Matrix Assisted Laser Desorption Ionization mass spectroscopy (MALDI) at The Ohio State University Campus Chemical Instrumentation Center.RP-HPLC fractions showing the same mass spectrum peak were pooled together and lyophilized.The respective scrambled peptides were synthesized using the number of amino acids present in natural sequences and were scrambled manually.All pure peptides were further characterized using MALDI mass spectroscopy analysis to confirm the calculated and observed molecular weight, which include L83-K103 (M+H+) Cal/Obs 2598.49 ",
"section_name": "Peptide Synthesis",
"section_num": "4.2."
},
{
"section_content": "Sub-cloning and purification of recombinant EBV-dUTPase mutant and wild-type proteins were performed as previously described [40,41,44].All recombinant dUTPase protein preparations were tested for the presence of contaminants, which was described previously [40,41,44], and were free of detectable levels of LPS, peptidoglycan (SLP-HS), DNA, or RNA.Protein concentration was determined using the Qubit fluorimeter (Invitrogen, Carlsbad, CA, USA).The purified recombinant dUTPase proteins used in these studies were stored at -80 • C until further use. ",
"section_name": "Purification of Recombinant EBV-dUTPase Protein",
"section_num": "4.3."
},
{
"section_content": "Neutralization assays for the EBV dUTPase were performed as previously described [44].In brief, 5 µL of human serum were mixed with 5 µL of purified EBV-dUTPase (3-5 units of enzyme) for 30 min at room temperature prior to assaying for enzymatic activity.EBV-dUTPase activity was determined as described previously [44].For positive controls, assays were performed in the presence of human serum that lacked detectable antibodies to the EBV.Negative controls were also performed in the absence of the enzyme preparation.A unit of EBV-dUTPase activity was defined as the amount of enzyme required to convert 1 nmole of dUTPase to dUMP and pyrophosphate/min/mL of enzyme at 37 • C. Units of enzymatic activity neutralized per mL of serum were obtained as follows: (Ucontrol-Userum).Serum with neutralizing units greater than or equal to two standard deviations from the control were considered \"positive\" for dUTPase neutralizing antibodies. ",
"section_name": "EBV-dUTPase Neutralization Assays",
"section_num": "4.4."
},
{
"section_content": "The patient samples in this study were collected from 1988-2003 as part of the EPILYMPH case-control study carried out in six European countries by de Sanjose et al. [58].Cases were defined as all consecutive patients having their initial diagnosis of lymphoid malignancy during the study period.The diagnosis of lymphoma was verified by histology and 99% of cases were supplemented by immunohistochemistry tests and flow cytometry.The cases were categorized according to the World Health Organization (WHO) Classification for Neoplastic Diseases of the Lymphoid Tissues and included all B-cell, T-cell, and NK-cell neoplasms as well as Hodgkin's lymphoma [79].Additionally, 20% of all diagnosed cases in each country were externally reviewed by a panel of international pathologists.The panel diagnosis is the one used in this analysis in the rare circumstance of disagreement between the local and the panel pathologists.Subjects with a diagnosis of uncertain malignant potential such as post-transplant lymphoproliferative disorder or monoclonal gammopathies of undetermined significance were excluded.Immunosuppressed patients were excluded from the analysis. ",
"section_name": "Patients",
"section_num": "4.5."
},
{
"section_content": "Human embryonic kidney (HEK293) cells stably expressing human TLR2 (TLR2-HEK293; Invivogen, San Diego, CA, USA) were maintained in DMEM-supplemented medium, as recommended by the manufacturer [46,49,50,78].Human peripheral blood mononuclear cells (PBMCs) from healthy subjects were obtained from Astarte Biologics (Bothell, WA, USA). ",
"section_name": "Cell Culture",
"section_num": "4.6."
},
{
"section_content": "HEK293 cells (2.5 × 10 5 ) were seeded into 12-well plates and 24 h later transiently transfected with pNFκB-Luc, pRL-TK reporter vectors (Promega, Madison, WI, USA), or with empty vectors as described [46,49,50,78].About 24 h to 36 h after transfection, cells were treated with recombinant wild-type or truncated EBV dUTPase proteins (10 µg/mL), zymosan (10 µg/mL; positive control for TLR2 activation) for 8 h or left untreated.After treatment, cell lysates were prepared and reporter gene activities were measured using the dual-luciferase reporter assay system (Promega).Data were normalized for transfection efficiency by measuring Renilla luciferase activity and expressed as mean relative stimulation ± SD. ",
"section_name": "Luciferase Reporter Gene Assays",
"section_num": "4.7."
},
{
"section_content": "PBMCs were seeded at a density of 2.5 × 10 5 in 24-well plates and cultured in AIM-V serum-free medium supplemented with L-glutamine (2 mM), streptomycin (50 µg/mL), and gentamycin (10 µg/mL).The next day, cells were stimulated with wild-type or truncated dUTPases (10 µg/mL), EBV-dUTPase peptide L83-K103, scrambled peptide L83-K103 or left untreated for 24 h.Following treatment, cell culture supernatants were collected and cytokine levels were measured by using ELISA (MSD Multi-array and Multi-spot human cytokine kit), which we described previously [46,49,50,78].Concentrations are expressed as pg/mL and represent the mean ± SD of an n of 3. ",
"section_name": "Cytokine Profile Induced by Herpesviruses-Encoded dUTPases",
"section_num": "4.8."
},
{
"section_content": "Statistical analyses were performed using a paired two-sample t-test for the means and p values were reported when displaying a significant value (p < 0.05).Values represent the mean ± SD of at least three independent experiments. ",
"section_name": "Statistical Analysis",
"section_num": "4.9."
},
{
"section_content": "Examination of serum samples from diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) patients revealed the presence of increased anti-dUTPase neutralizing antibodies in both cohorts compared to controls with the highest levels (3.67-fold increase) observed in DLBCL female cases and the lowest (2.12-fold increase) in the DLBCL males.Furthermore, using computer-generated algorithms, dUTPase amino acid sequence alignments, and functional studies of BLLF3 mutants, we identified a putative amino acid motif involved with TLR2 interaction and demonstrated that amino acid residues between 81 G to 103 K of the EBV-dUTPase are important for binding to and activating TLR2 signaling.These findings suggest that the EBV-dUTPase: TLR2 interaction is a potential molecular target that could be used for developing novel therapeutics (small molecules/vaccines). ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "We would like to thank the European sample collectors in the EPYLYMPH study as well as Silvia de Sanjose and Yolanda Benavente enabling us to obtain the case and control sera samples used in this study via the NIH cancer repository. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was supported by the National Institutes of Health grant R01 A1084898 to M.W. and M.E.A., and by funds from the University of South Carolina Centenary plan initiative to M.E.A. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Author Contributions: Maria Eugenia Ariza and Marshall Williams conceived, designed, and performed the experiments, analyzed the data, and wrote the paper. The authors declare no conflict of interest.The funding sponsors 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 following abbreviations are used in this manuscript: ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Author Contributions: Maria Eugenia Ariza and Marshall Williams conceived, designed, and performed the experiments, analyzed the data, and wrote the paper. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest.The funding sponsors 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 following abbreviations are used in this manuscript: ",
"section_name": "Abbreviations",
"section_num": null
}
] |
10.1186/s12955-020-01341-z
|
Validation of the Chinese EORTC chronic lymphocytic leukaemia module – application of classical test theory and item response theory
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Purpose</jats:title> <jats:p>The association of chronic lymphocytic leukemia (CLL) with health-related quality of life (HRQoL) is rarely studied globally. This study evaluated the psychometric properties of the EORTC-Chronic Lymphocytic Leukaemia (CLL17 [phase III]) module, a newly developed assessment on CLL patients’ HRQoL, among Chinese CLL patients.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>The Chinese CLL17, comprised of three subscales (symptom burden [SB], physical condition [PC] and worries/fears [WF]), was provided by the developer team through EORTC. A cross-sectional online survey was conducted to collect data. The classical traditional theory (CTT) and the item response theory (IRT) were used to evaluate the psychometric properties of CLL17. Internal consistency reliability was determined by the Cronbach’s alpha and item-total correlation. Dimensionality was verified through confirmatory factor analysis (CFA). Convergent validity was also assessed. The generalized partial credit model was used for the IRT. The difficulty, discrimination, item fit, and differential item functioning (DIF) were calculated to assess the instrument’s psychometric properties.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>In all, 318 patients, aged between 26 and 82 years, completed the questionnaire. A good level of internal reliability was achieved (Cronbach’s alpha = 0.92). The item-total correlation coefficient ranged from 0.46 to 0.72. There was a mid-to-high correlation between CLL17 and domains of EQ-5D and QLQ-C30. The IRT model showed a satisfactory homogeneity, item fit and good discrimination of items, except for item 4, 6 and 16 (< 1.0). low information provided by item 16 and 17. SB and PC provided more information with theta > 0, whereas WF provided more information with theta < 0. Item 17 perform inconsistently for respondents from different age groups (DIF).</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>The EORTC-CLL17 Chinese version shows acceptable reliability and validity, making it a valuable instrument to evaluate the impact on the HRQoL of Chinese CLL patients.</jats:p> </jats:sec>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults, affecting the white blood cells that develop slowly over time [1].In the United States, the National Cancer Institute reported that the number of new cases of CLL was 4.9 per 100,000 that annually caused 1.2 per 100,000 deaths [2].Cancer Research United Kingdom stated that between 2014 and 2016, nearly 1% of the total cancer cases reported in the UK included people living with CLL [3].In Asian countries, including China, although the CLL was less common than western countries, the prevalence is increasing dramatically in recent years.According to the latest research reported by China's National Cancer Centre, lymphoma was found to be one of the top ten causes of cancer death in cities, and one of the top ten most common cancers among men.CLL, as one of the most common lymphomas, is estimated to affect an increasing number of the Chinese population [4].Since the incidence rate of CLL rises with age, and aging is becoming an increasingly significant problem in China, the morbidity and mortality of CLL are expected to rise rapidly in the next few decades [5].Hence, even though the prevalence of CLL is not \"high\" for now, it still deserves attention and research from academia. In the last two decades, the treatment of CLL has dramatically improved and significantly extended patients' life expectancy [1,6].The National Institute of Health reported that from 2009 to 2015, the five-year survival rate of CLL was 85.1%, which is higher than most other cancers for both males and females in the U.S. [2].Compared with the flourishing studies on treatments of CLL disorders, research assessing the health-related quality of life (HRQoL) of CLL patients is scarce.Although some have encouraged the adoption of cancer-specific measures, such as EORTC QLQ-C30 [7], to assess CLL patients' HRQoL in previous studies, others have argued that these may be unsuitable for measuring the effectiveness of interventions or policies focusing on CLL care due to the bias and uncertainties brought up by non-CLL specific measures [8].Recently, the application of FACT-Lymphoma (FACT-L), an instrument developed to assess the HRQoL of patients living with all types of lymphoma, has increased [9][10][11].However, lymphoma has many different subtypes, such as Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, and so on.Each subtype is unique with its own causes, symptoms, and methods of treatment, and thus its impact on patients' HRQoL may also differ.Whereas the FACT-L can assess the overall HRQoL of lymphoma patients, the nuances in differences among patients with different subtypes may not be detected.It is, therefore, imperative to develop a set of scales to assess a disease-specific HRQoL among patients with different subtypes of lymphoma. Recently, a growing number of targeted therapies (e.g., ibrutinib, idelalisib, venetoclax, obinutuzumab), used as a monotherapy or in combination, have been introduced for CLL treatment.As the 2020 updated recommendations on CLL diagnosis and treatment suggest, there is an \"impressive and historically unique chance\" for the long-term control of the disease.To achieve this, CLL management is also critical besides clinical trials [12].Accordingly, the assessment of CLL patients' HRQoL by using a disease-specific measure seems imperative.Moreover, it has also been argued that the use of general measures, such as the SF-12, may limit a full appraisal of outcome difference between arms in clinical trials [13].With a standardized and validated HRQoL scale, the sensitivity to detect functional limitations and symptoms in future trials on CLL patients is expected to increase. In 2018, the Quality of Life Group of the European Organisation for Research and Treatment of Cancer (EORTC) developed a series of lymphoma-specific measures to assess the HRQoL of patients with different types of lymphoma [13].Among them, EORTC QLQ-CLL17 (CLL17), a CLLspecific scale, was introduced.Although the scale is in phase III of development, which means that it can be used for validation within different social and cultural contexts but should not be regarded as the final version of the scale, it has been reported to have good reliability (the Cronbach's alpha ranges between 0.85 and 0.88) and validity when using a sample of 86 participants from five Western countries [13].The new scale provides an advanced assessment of the HRQoL, including both the physical and mental dimensions of patients living with CLL.In China, although the population of patients affected by CLL has expanded in recent years [5], studies on the impact of CLL on patient's HRQoL are still nascent.It is important to provide a valid, reliable, and responsive measure to evaluate the impact of CLL and associated interventions on the HRQoL changes.Therefore, this study evaluated the psychometric properties of CLL17 among Chinese CLL patients using the methods based on both classical traditional theory (CTT) and item response theory (IRT). ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Method",
"section_num": null
},
{
"section_content": "A cross-sectional study was conducted to survey the medical, social, and economic status of people affected by rare diseases in China, which was approved by the Chinese University of Hong Kong Institutional Review Board committee (Ref No.: SBRE-18-268).In all, 4225 patients with lymphoma participated in the survey and completed the questionnaire.Of these, data from 318 patients who reported having CLL were used in this study.The inclusion criteria were patients 1) who were ≥ 18 years old, 2) with a confirmed clinical diagnosis of CLL, 3) who could complete the questionnaire independently, and 4) were native Chinese speakers.Patient with cognitive problems were excluded from the survey.Respondents belonged to 30 of the total 34 provinces of China and were aged between 26 and 82 years.All the CLL patients were recruited via House086, the largest national lymphoma patient organization in China, between May and July 2019.House086, established in 2011, aims to help patients with all types of lymphomas in China and currently has more than 40,000 registered members (lymphoma patients and their families only).The survey was conducted online.All participants were asked to read the consent form presented on the first page of the online survey.If they agreed to participate, they clicked \"next page\" and were directed to the main body of the questionnaire. The questionnaire comprised seven parts, which included demographics, HRQoL, social support, and others.Respondents resided in 30 provinces across China, and the three largest groups were from Jiangsu (29 respondents), Henan (24 respondents), and Zhejiang (23 respondents).All responses were recorded anonymously and treated with strict confidentiality.The sample size was calculated based on the assumption of conducting psychometric evaluation [14,15].Considering that CLL is listed as a rare disease in China, a minimum sample of 200 was needed. ",
"section_name": "Study design and participants",
"section_num": null
},
{
"section_content": "The CLL17 module is a supplementary questionnaire module to be employed in conjunction with the EORTC-C30 to evaluate the HRQoL of patients living with CLL.The CLL17 comprises 17 items divided into three subscales: 'Symptom burden' (SB, 6 items, 1-6), 'Physical condition/ fatigue' (PC, 4 items, 7-10), and 'Worries/fears about the health and functioning' (WF, 5/7 items, 11-17, the last two items are required only when the patients' conditions are applicable).The questions are answered using a 4-point response scale (1 = Not at all, 2 = A little, 3 = Quite a bit, 4 = Very much) to indicate the extent of the problem experienced by the respondents for each item during the past week(s) [13].The scores of each subscale are calculated based on the EORTC's protocol and then transformed to a 0-100 scale.The first step is calculating a raw score for each subscale (Raw score = {(I 1 + I 2 + … I n )/n}, I 1 is the item 1, n is the number of the item in that subscale).The second step is linear transformation to obtain the standardized score S (0-100, S = {Raw score -1}/range} *100).The simplified Chinese version of CLL17 (CLL17-C) was provided by the developer team through EORTC.It was translated by a professional translation service provider, specializing in the translation of patient-reported outcome measures.They followed the standard EORTC translation process as described in the EORTC's Translation Manual (The Manual is available under request).Translators were selected following ISO requirements. ",
"section_name": "The instrument -EORTC CLL17 Chinese version",
"section_num": null
},
{
"section_content": "In this study, data were analyzed based on the methods from both CTT and IRT using R (R foundation, Austria).The p-value was set at ≤0.05.The demographic data are reported as percentages, means, standard deviations (SD), and ranges.The feasibility of the questionnaire was evaluated based on the calculation of the percentage of missing data and the time to complete the questionnaire.The ceiling and floor effects, which reflect the majority of the values occurring at the upper or bottom limit of the scale, were calculated (< 15% is acceptable [16]). For the CTT methods, the internal consistency reliability was reported, which included 1) Cronbach's alpha coefficient (α), α > 0.7 was considered as acceptable [14], and 2) the item-total correlation coefficient, wherein an item with a low corrected item-total correlation (r < 0.3) was considered to be removed from the scale [17].Confirmatory factor analysis (CFA) was used to evaluate the dimensionality of CLL-C.The comparative fit index (> 0.90), Tucker-Lewis index (> 0.90), standardized root mean square residual (< 0.08), and root mean square error of approximation (< 0.08) [14] were used to evaluate the performance of the scale.The EuroQol five-dimension fivelevel (EQ-5D-5 L) and the EORTC QLQ-C30 were used to evaluate the convergent validity of CLL-C.The EQ-5D is the most commonly used generic instrument to describe and value health worldwide.It has five dimensions (Mobility, Self-care, Usual activities, Pain/Discomfort, and Anxiety/depression), and each dimension has three (3 L) or five (5 L) levels.The combinations of the dimensions and levels could generate a single summary index score for health status used in the economic evaluation of health care interventions [18].The EORTC QLQ-C30 is a questionnaire developed to specifically assess the quality of life of cancer patients [19].It has five functional scales (Physical, Role, Cognitive, Emotional, and Social Functioning), three symptom scales (Fatigue, Pain, and Nausea/Vomiting), a Global Health Status scale, and six single items (Constipation, Diarrhea, Insomnia, et al.).Scores for each scale range from 0 to 100.For functional scales and Global Health Status, a higher score indicates better functioning or HRQoL.However, for symptom scales and single items, a higher score indicates worse status. For the IRT evaluation, a multidimensional extension of the generalized partial credit model (GPCM) was applied [20], which means the discrimination of all the items were estimated separately.The key assumptions, i.e., monotonicity (item-category characteristic curves, ICCs) and local dependence (discrimination< 4), were confirmed with the analysis.The indicators of difficulty (b), discrimination (a), and item fit (the p-value of S-χ 2 < 0.05) were calculated for each item, along with the item information curves (IICs) and test information curve (TIC) [21].Furthermore, differential item functioning (DIF) was also checked for possible item bias caused by responses from different subgroups (sex and age group) in the sample, followed by the Monte Carlo simulated empirical criteria [17].The detailed information of DIF detection can be found in Choi et al. [22]. ",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Table 1 presents the demographics of the respondents.In total, 318 respondents completed the questionnaire with a mean age of 55 years (26~82 years).About 61.9% were male, 45.6% received a tertiary or above educational certificate, and 40% were retired.Moreover, the average duration of the lymphoma was 3.91 years (SD = 3.05). ",
"section_name": "Demographics",
"section_num": null
},
{
"section_content": "The percentage of the missing data at the item level was zero.Given that the CLL-C was embedded in a survey to investigate the health conditions of the lymphocytic patients, estimating the exact time required to complete the CLL-C was impossible.However, based on our pilot study, and the responses from some participants, the time to complete the CLL-C was less than 5 min, which is entirely acceptable. ",
"section_name": "Feasibility",
"section_num": null
},
{
"section_content": "Table 2 shows the item statistics and internal consistency reliability of CLL-C.Item 15 (WF subscale) had the highest average score (2.94), whereas item 7 (PC subscale) had the lowest average score (1.66).There were, overall, 10 items with an average score greater than 2 points [1][2][3][4].The Cronbach's alpha for the overall was 0.92.For SB, PC, and WF, α was 0.81, 0.85, and 0.91, respectively.Additionally, 12/17 items had α ≥ 0.85.The value of the item-total correlation coefficient ranged between 0.46 and 0.72, and all reached the predefined criteria.No item violated the assumption that α would be smaller without a particular item.The ceiling effect for items ranged from 1.88 to 34.27% (6/17 presented ceiling effect), whereas the floor effect ranged from 5.35 to 41.82% (12/17 presented floor effect). ",
"section_name": "Item statistics and reliability",
"section_num": null
},
{
"section_content": "The result of CFA supports the three-subscale construct of CLL-C.However, the model of 15 items (without items 16 and 17) showed a better performance than the model of 17 items (Table 3).Table 4 presents the results of known-group validity.We identified that patients receiving treatment were prone to report a higher score (low HRQoL) on all three subscales.The scores of SB and PC correlated significantly with treatment for CLL patients. ",
"section_name": "Construct validity",
"section_num": null
},
{
"section_content": "Compared with EQ-5D-5 L, there was a strong correlation between WF and anxiety/depression of EQ-5D-5 L (r = 0.573), followed by the correlation between SB and pain/discomfort (r = 0.473).Compared with QLQ-C30, the PF of CLL-C correlated strongly with fatigue (r = 0.713), and SB and PF of CLL-C seem to have a closer relationship with domains of QLQ-C30 than the WF.All the correlation coefficients were statistically significant (Table 5). ",
"section_name": "Convergent validity",
"section_num": null
},
{
"section_content": "Table 6 indicates the discrimination and difficulty parameters for CLL-C.The discrimination of 17 items ranged between 0.761 and 3.877.The value of items 2, 3, 8, 10, 11, 12, 13, and 15 fell within the range greater than 3.0, which indicates that these items can distinguish individuals with either lower or higher HRQoL, corresponding with the latent trait sensitively.The p-value of S-χ 2 indicates that all the items fit the scale, except for item 7 (p-value = 0.05), which might have a minor misfit. ",
"section_name": "IRT analysis -model fit",
"section_num": null
},
{
"section_content": "Figure 1 graphically presents the item-category characteristic curves of items 6, 9, and 15, showing how the items relate to each latent trait.The selected three items reflected the different (high, moderate, and low) levels of discrimination of all items.Item 15 had the highest discrimination (a = 3.877), item 9 was moderate (a = 2.603), and item 6 had the lowest discrimination (a = 0.761).Although the order of categories' thresholds for most items (e.g., item 9 and item 15) was good (the interaction of 'harder' options had a higher theta value than the interaction of 'easier' options), the thresholds for very few items (e.g., item 6) had an overlap among the response categories, which led to those categories offering little in terms of placing a respondent on the scale.For the location parameter, the response category for item 15 was endorsed at lower levels of the latent trait (WF), but items 9 and 6 were endorsed at higher levels (PC and SB).All the Item-category characteristic curves are shown in the supplementary file. ",
"section_name": "IRT analysis -item-category characteristic curves",
"section_num": null
},
{
"section_content": "Figures 2, 3, 4 show item and test information curves for three subscales separately.Information function curves demonstrate the precision and information provided by each item [23].These curves graphically present the information functions of all the items on a scale, demonstrating the precision of a scale across different levels of the targeted latent trait [15].Items 2 and 3 in the SB subscale, and items 8 and 10 in the PC subscale provided more information than the other items.For WF, however, items 16 and 17 seem to provide the least information.The test information curves were sufficiently informative for the middle level of the latent trait with the range of theta from 1 to 2.5, 1.5 to 2.5, and -0.5 to 0.5 for SB, PC, and WF, respectively.For SB and PC, the overall distribution of information revealed that the subscales provided more information alongside the trait region associated with theta ranges between zero and six; however, for WF, more than half of the information was provided alongside the trait region associated with theta ranges between -6 and zero. ",
"section_name": "IRT analysis -information function curves",
"section_num": null
},
{
"section_content": "Item 17 showed a uniform DIF when considering the respondents from different age groups.It revealed that the concerns about continuing to work or study improve with increased age for CLL patients even when they report the same levels of WF.However, when looking at differences in pseudo-R 2 values, the magnitude of R 2 is smaller than 0.13, suggesting that the effect size of DIF is very negligible [24]. The figure of DIF is presented in the supplementary file. ",
"section_name": "DIF analysis",
"section_num": null
},
{
"section_content": "CLL-C is a new and valid instrument developed by EORTC to assess the HRQoL in patients with CLL [13]. Our study used its simplified Chinese version and evaluated its psychometric properties.Our results indicate that the reported mean scores of SB and PC were similar to the mean scores reported by patients using the original English version [13].For the WF (emotional subscale), however, the mean score was much higher than the scores reported in the original study for both with and without items 16 and 17.Previous studies identified CLL as one of the most common forms of leukemia in Western countries, accounting for 25% of all leukemia in adults.However, in Asian countries, including China, CLL is rare [25].Regarded as a rare disease in China, patients diagnosed with CLL are prone to experience more emotional burden or depression than their Western counterparts.Discussions about such relationships may go beyond the purpose of this study.Therefore, further investigations are encouraged. Our psychometric evaluation demonstrates that the multi-trait scaling structure of CLL17-C is generally similar to that of its original English version [13].The CLL17-C has good internal consistency reliability, as supported by both Cronbach's alpha and item-total correlation coefficient.Moreover, statistically significant correlations between the subscales of CLL17-C, the EQ-5D-5 L, and QLQ-C30 suggest that the subscales of CLL17-C could assess the distinct components of the construct of HRQoL.For the known-group validity, the results also meet our expectations that the patients who received clinical interventions got higher scores on CLL17-C (worse status).The findings suggest that CLL17-C is capable of distinguishing patients based on the severity of their health status. Based on the results of the IRT analysis, using the polytomous extension of the S-χ2 analysis, none of the items of CLL17-C were identified as a misfit at p-value < 0.05 after controlling the type I error rates [26].The slope estimates of the 17 items ranged from 0.76 to 3.877 (12/17 greater than 1.5), indicating a modest variation in item discrimination.As expected, the higher categories have higher item locations, indicating the endorsement of severe HRQoL problems.For SB, b1 ranged between -1.317 and -0.286, b2 ranged between 0.634 and 2.634, and b3 ranged between 1.697 and 2.515.For PC, b1 ranged between -1.226 and -0.290, b2 ranged between 0.797 and 1.979, and b3 ranged between 0.727 and 2.340.For WF, b1 ranged between -1.707 and -1.143, b2 ranged between -0.374, and 1.043 and b3 ranged between 0.329 and 1.268.However, WF items were much 'easier' to answer than the items of SB or PC.In other words, the CLL patients in our sample experienced emotional burdens more than physical discomfort.Moreover, although the discrepancy was not large, the thresholds of categories between b2 and b3 for items 1, 4, and 6 (SB sub-scale) were disordered.This might suggest that the response category \"quite a bit\" did not work consistently as intended in SB.However, considering our limited sample size, the different mechanism of parameter estimation using different IRT models [27], and no similar comparisons could be found around the world.Currently, it would be incorrect to conclude that the SB subscale of CLL17-C is not psychometrically sound.Further revisions are needed through more explorations. The item information provided by SB, PC, and WF ranged from 2.24 to 9.49, 4.53 to 10.96, and 2.28 to 11.64, respectively.Based on Nunnally's suggestion that values of information from 3.3 to 10 correspond to the Cronbach's alpha ranges from 0.70 to 0.90 [28].Therefore, the majority of the items indicate acceptable reliability.Items 4 (information: i = 2.58, α = 0.62), 6 (i = 2.24, α = 0.56), 16 (i = 2.28, α = 0.57), and 17 (i = 3.05, α = 0.68) may have some room for improvement.The TIC indicate that SB and PC had relatively smaller standard errors (SEs) at the high endpoint of theta than at the low endpoint of theta.However, for WF, the SEs were larger at both endpoints of theta, which mightindicate that when assessing the CLL, the WF subscale does not have a very reliable performance for patients with either high or low scores on emotional burdens related to CLL. In this study, it is noteworthy that SB and PC items show a heavy ceiling effect, while there is an existence of a floor effect in the WF items.Moreover, the last two items of WF (items 16 and 17), which are not compulsory items, show both moderate ceiling and floor effects.Previous studies indicate that both the ceiling and floor effects are common in the HRQoL data [29][30][31][32].A series of studies using SF-6D report the floor effects in a wide variety of clinical settings, whereas a strong correlation between the ceiling effect [33,34] and the EQ-5D data have been identified [29,31].The floor effect might weaken the subscales' ability (SB and PC) to capture the gains when patients have moderate or severe physical conditions or suffering.The magnitude of change experienced by CLL patients might be underestimated when using these two subscales, and the bias caused by the underestimation could potentially affect the evaluation of the effectiveness and efficiency of such interventions or policies.Similarly, the ceiling effect of items potentially threatens the responsiveness of WF, and the impacts are exactly the opposite of the floor effect.Currently, no other publications have reported the floor or ceiling effect of CLL17 in other language versions, and more information should be provided. Item invariance is an important component of a good scale.If an item performed differently in subgroups after controlling for ability, the item is considered to have DIF [15].To measure item invariance across different subsamples of the CLL17-C, we set the two variables, sex and age, as the anchor items for all three subscales.The results indicate that the items in the three subscales performed consistently on assessing the HRQoL in terms of sex.However, item 17 of WF demonstrated a minor DIF in terms of the age group (≤50, 51-60, ≥61), which indicates less discrimination (flatter slope) for respondents younger than 50 than respondents older than 61.In other words, when patients worry or have fears about their health and functioning, patients of different ages do not give consistent answers to item 17.Nguyen et al. indicated that the possible reason for detecting DIF was how the item was administered [15].Given that the data for this study came from an online self-report survey, it is unclear whether the process of data collection created some bias compared with a traditional paper-based questionnaire.However, until now, no DIF analysis was reported based on CLL17, and no other information could be provided at this moment.We explain this finding with caution. As discussed above, the performance of item 17 in the CLL17-C appeared controversial, which seemingly leads to some negative effects, especially using the IRT model, on the estimation of the HRQoL of CLL patients.First, although the CFA suggests that the structure of CLL17-C is generally similar to its original English version, the 15item (without items 16 and 17) version performed better than the full 17-item version.Secondly, the discrimination of item 17 was small (a = 1.022), indicating that item 17 may not differentiate patients with either higher or lower concerns about their work or education raised by CLL.Thirdly, the IIC demonstrates that item 17 provides the least information amongst all the items of CLL17-C. Moreover, despite its negligible magnitude, item 17 shows a DIF in terms of age.The patients with CLL were usually older than patients reportedly living with other lymphomas.In our sample, the average age was 55 years.Whereas the participants' average age in the study on the development of the CLL original version was 69 years [13].Other studies of CLL also reported a similar demographics of their study sample, for example, 64 years in a study conducted in HK and Singapore [25], and 65 years reported by another UK study [35].Thus, item 17, evaluating the impact of work and education on the HRQoL, may not provide useful information for CLL patients.However, since the CLL17 scale is still under the development of phase III, its final stage of development should include an improved version. This study has some limitations.First, CLL patients with severe physical or psychological conditions might be underrepresented in our study.The absence of their information could lead to some bias in our results.Second, there is no golden standard or magical numbers that can be proposed to conduct the IRT analysis.The minimum requirement to test the questionnaire with polytomous response format was suggested to be 250 [36]; however, larger sample sizes, such as ≥500, are recommended considering different test purposes and IRT model selection [37,38].Our potentially insufficient sample size may have brought some bias in examining the fit of the model and over-or under-estimations of the parameters.A larger sample will be approached in the future to obtain a more precise measurement of CLL17-C item characteristics.Third, a high proportion of our participants came from economically developed areas in China, which might have led to potential selection bias.Lastly, we could not approach the patients with low willingness to participate in the survey since the data came from a self-report online survey, possibly leading to some selection bias. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Although the treatments of CLL are fast developing, their effects on patients' HRQoL in long-term care are still ambiguous.Our findings expand the understanding of HRQoL among CLL patients using a specific patient self-report outcome measure.This study provided comprehensive insights on the psychometric properties of CLL17-C using CTT analysis supplemented with IRT methods in a sample of Chinese patients.Both approaches showed that the three subscales had acceptable reliability and validity.However, given the limited sample size and the potential selection bias, further assessment of the measurement's precision is encouraged to maximize the application of CLL17 regionally and internationally. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "Not applicable ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "No funding supports ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Note applicable ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1186/s12955-020-01341-z. Additional file 1: Table S1 The study was approved by the Chinese University of Hong Kong Institutional Review Board committee (Ref No.: SBRE-18-268). 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/s12955-020-01341-z. Additional file 1: Table S1 ",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "The study was approved by the Chinese University of Hong Kong Institutional Review Board committee (Ref No.: SBRE-18-268). ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "",
"section_name": "Not applicable",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1186/s13148-019-0695-0
|
Clustered protocadherins methylation alterations in cancer
|
Clustered protocadherins (PCDHs) map in tandem at human chromosome 5q31 and comprise three multi-genes clusters: α-, β- and γ-PCDH. The expression of this cluster consists of a complex mechanism involving DNA hub formation through DNA-CCTC binding factor (CTCF) interaction. Methylation alterations can affect this interaction, leading to transcriptional dysregulation. In cancer, clustered PCDHs undergo a mechanism of long-range epigenetic silencing by hypermethylation.In this study, we detected frequent methylation alterations at CpG islands associated to these clustered PCDHs in all the solid tumours analysed (colorectal, gastric and biliary tract cancers, pilocytic astrocytoma), but not hematologic neoplasms such as chronic lymphocytic leukemia. Importantly, several altered CpG islands were associated with CTCF binding sites. Interestingly, our analysis revealed a hypomethylation event in pilocytic astrocytoma, suggesting that in neuronal tissue, where PCDHs are highly expressed, these genes become hypomethylated in this type of cancer. On the other hand, in tissues where PCDHs are lowly expressed, these CpG islands are targeted by DNA methylation. In fact, PCDH-associated CpG islands resulted hypermethylated in gastrointestinal tumours.Our study highlighted a strong alteration of the clustered PCDHs methylation pattern in the analysed solid cancers and suggested these methylation aberrations in the CpG islands associated with PCDH genes as powerful diagnostic biomarkers.
|
[
{
"section_content": "Protocadherins (PCDHs) are type I transmembrane proteins containing 6 or 7 extracellular cadherin repeats, structurally similar to cadherins.They are characterized by a large molecular diversity, are broadly expressed and participate in cell-cell adhesion, predominantly in the nervous system establishing complex neural circuits [1]. PCDHs are classified as clustered and non-clustered protocadherins.The clustered PCDHs map in tandem at human chromosome 5q31 and comprise cluster α, cluster β and cluster γ genes (Human Genome Organization nomenclature PCDHA@, PCDHB@ and PCDHG@, respectively), whereas the non-clustered PCDHs are distributed across the genome.A great variety of activities have been reported for clustered PCDHs.These molecules mediate homophilic interactions like most members of the cadherin superfamily [2].The formation of these macromolecular complexes leads to the activation or inhibition of different signalling pathways through binding to the cytoplasmic domains of the PCDHs [3].These transmembrane proteins regulate Wnt/β-catenin [4], PYK2 and FAK tyrosine kinases (involved in cell adhesion) [5,6] and mTOR pathways [4], among others. Wu and Maniatis first described the structure of protocadherin gene clusters [7,8].PCDHA and PCDHG gene clusters consist of variable exons that encode for the extracellular domain, the transmembrane domain and a short part of the cytoplasmic domain, and constant exons that encode for a shared C-terminal domain.In contrast, PCDHB gene cluster presents exons without a constant region.As PCDHB exons, each variable exon of PCDHA and PCDHG has its own promoter that is controlled by methylation [7,8].It has been reported that promoter stochastic choice, due to methylation changes and DNAbinding factor, and transcript splicing generate Pcdh diversity in neurons [9,10].This promoter choice and thus the transcription of clustered protocadherins depend on a complex mechanism where the CCTC binding factor (CTCF) plays an essential role.This zinc finger protein binds to a conserved sequence element (CSE) and a specific sequence element (SSE) located in the promoter, and to the enhancer element, a regulatory region downstream of each cluster, favouring genome looping [11,12].CTCF recognizes its DNA-binding sites, recruits the cohesion complex, whose members are Rad21, Smc1, Smc3 and SA2 [13], and allows the interaction of active promoters and specific enhancers through the formation of a hub [12].Recent works also suggested that the binding of CTCF to the Pcdha cluster is regulated by transcription of a long non-coding RNA (lncRNA), initiated at a newly identified promoter within each Pcdha exon.Transcription of this antisense lncRNA mediates DNA demethylation of the CTCF binding sites, thus promoting CTCF binding [14].Guo et al. found that forward-reverse orientation of the CTCF binding sites is also important for looping formation and enhancer-promoter interactions leading to cell-specific gene expression [15]. As mentioned before, PCDH expression is controlled by DNA methylation and its dysregulation is common in different types of cancer.In cancer pathogenesis, clustered PCDHs undergo a mechanism of long-range epigenetic silencing (LRES) by hypermethylation.Clustered PCDH gene silencing was found not only in tumour cell lines but also in different types of cancer including cervix, liver, lung, colon, breast and brain [4,[16][17][18].Novak et al. detected hypermethylation and transcription downregulation in the three clustered PCDHs in breast cancer [17].Other breast cancer studies showed that the abnormal DNA methylation of these gene families could be the consequence of the reduction of CTCF interaction with DNA due to CTCF aberrant expression or mutations in its binding domain [19,20].On the other hand, Guo et al. revealed that promoter methylation prevents or reduces CTCF binding to CSE [12].Dallosso et al. also found hypermethylation of the majority of PCDHA, PCDHB and PCDHG in both adenomas and colorectal carcinomas, relative to normal tissue [4].Moreover, these authors demonstrated that selected γ-PCDH are able to suppress Wnt activity in vitro [21].In particular, PCDHGC3 negatively regulates Wnt and mTOR signalling.Interestingly, PCDHGC3 has been found highly methylated only in carcinomas and not in previous stages and has been proposed as a driver for the progression from adenoma to carcinoma [4].Thus, although the role of protocadherins in tumour development has not been fully established, it is suggestive that these proteins are involved in the regulation of key cellular pathways of cell death and proliferation.More recently, Liu et al. demonstrated that PCDHGA7 downregulation is correlated with poor prognosis and KRAS genotypic status in colorectal cancer [22].Waha et al. detected hypermethylation in PCDHGA11 in astrocytoma, glioblastoma and glioma cell lines.Moreover, these authors found a significant correlation between PCDHGA11 hypermethylation and downregulation in astrocytomas and glioma cell lines [16].On the other hand, Kawaguchi et al. reported mosaic methylation and hypomethylation of the CpG islands (CGIs) associated with Pcdha cluster in mouse neuroblastoma cell lines [23].Other PCDHA@ genes, PCDHA4 and PCDHA13, have been found frequently hypermethylated in severe cervical neoplasia [18]. In the present study, we investigated the methylation status of clustered PCDHs in colorectal, gastric and biliary tract cancers (CRC, GC and BTC, respectively); pilocytic astrocytoma (PA); and chronic lymphocytic leukemia (CLL).Our results demonstrate that PCDHs frequently present alterations in their methylation status in solid cancers in contrast to blood cancer, suggesting the methylation alterations of these clustered genes as possible biomarkers for cancerogenesis. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Our experimental discovery dataset included DNA methylation data of four solid cancers (PA, CRC, GC and BTC) and one blood cancer (CLL) as summarized in Fig. 1.In particular, we analysed the following: - ",
"section_name": "Experimental discovery datasets",
"section_num": null
},
{
"section_content": "DNA was extracted from PA, CRC, CRA, GC and their respective normal fresh frozen tissues using DNeasy Blood & Tissue Kit (Qiagen). DNA from BTC and matched normal formalin-fixed and paraffin-embedded (FFPE) samples was carried out by QIAamp DNA FFPE Tissue kit (Qiagen). In the CLL study, DNA was isolated from peripheral whole blood lymphocytes using the DNA extraction 500 arrow® Kit (DiaSorin Ireland Ltd). DNA quantity of all samples was analysed by spectrophotometric reading (NanoDrop) and by fluorometric reading (Quant-iT™ PicoGreen® dsDNA Assay Kit) and its quality was evaluated by electrophoresis in a 0.8% agarose gel. All DNA samples were bisulfite converted using EZ DNA Methylation Gold Kit™ (Zymo Research). In the BTC study, DNA extracted from FFPE samples underwent an additional quality control step using the Infinium FFPE QC kit (Illumina) prior bisulfite conversion.Subsequently, they were subjected to a restoration step using the Infinium HD FFPE Restore Kit (Illumina). Genome-wide methylation analysis was performed by Illumina Infinium HumanMethylation27 BeadChips (27K) in PA study, Illumina Infinium HumanMethyla-tion450 BeadChips (450K) in CRC and CLL studies and Illumina Infinium methylation EPIC BeadChips in GC and BTC studies.The number of probes mapping in PCDHG@ cluster in the different BeadChips are reported in Additional file 1: [24,25]. ",
"section_name": "DNA extraction, bisulfite conversion and methylation assay",
"section_num": null
},
{
"section_content": "Illumina methylation 27K raw data were analysed as described in Antonelli et al. [24].Differential methylation levels (Δβ) between PAs and normal brain samples were calculated by Illumina Custom model, as implemented in the Illumina GenomeStudio software.We selected only differentially methylated probes (Δβ values ≥ 0.2 or ≤ -0.2, i.e. 20% differential methylation level) annotated in PCDH gene clusters with a p value threshold < 0.001.Hypermethylation was defined as Δβ values ≥ 0.2 and p value threshold < 0.001, while hypomethylation was defined as Δβ values ≤ 0.2 and p value threshold < 0.001 Illumina 450K and EPIC raw data were analysed using RnBeads as previously described [26,27].In brief, a differential methylation analysis between tumour and normal control samples was performed for each cancer type studied (CRC, CLL, GC and BTC).The normalization for the microarray signals was perfomed by Subsetquantile Within Array Normalization (SWAN) [28].Corrected p values (Benjamini & Hochberg) were computed as previously described [26,27].In particular, combined p values were adjusted for the entire CpG sites on the arrays using false discovery rate (FDR).CpG loci were annotated according to Illumina Manifest to obtain a gene list based on HUGO Gene Nomenclature Committee (HGNC) database.We selected only PCDHassociated differentially methylated CGIs with Δβ values ≥ 0.2 or ≤ -0.2 and an adjusted p value < 0.05.Hypermethylation was defined as Δβ values ≥ 0.2 and adjusted p value < 0.05, while hypomethylation was defined as Δβ values ≤ 0.2 and adjusted p value < 0.05.Since the results of this analysis were less robust in adenomas [25], we used the nominal threshold (p values < 0.05) in CRAs. Finally, for CRC, CRA, GC and BTC, the mean methylation value of each altered CGI for each sample has been used in an analysis of UHC and visualized by Bioconductor package \"ComplexHeatmap\" [29]. Contingency table 2 × 1 was used to evaluate the statistical significance between methylation levels and microsatellite instability (MSI) status. CGI annotations in tables and figures correspond to UCSC CGI names, indicating the number of CpG sites included in the CGI. ",
"section_name": "Methylation analyses",
"section_num": null
},
{
"section_content": "We explored whether the altered CGIs were associated with the CTCF binding sites.As mentioned before, both regions are included in the promoter [11].CTCF binding sites' genomic coordinates were obtained from ENCODE database [30].CTCF binding sites and CGIs were considered as associated if their distance was lower than 1000 bp. ",
"section_name": "CTCF binding site analysis",
"section_num": null
},
{
"section_content": "The power of the methylation analyses was estimated based on the calculation of mean delta betas and standard deviations using data retrieved from the NCBI Gene Expression Omnibus (GEO) portal [31] under accession number GSE48684.Based on this preliminary data, we performed a two-sample t test power calculation obtaining that a statistical power of 0.8 would be guaranteed by analysing 30 samples in order to detect a differential methylation level of at least 10%, using a type I error of 10e-8 (which takes into account the need to correct for multiple tests). ",
"section_name": "Power calculation",
"section_num": null
},
{
"section_content": "In silico methylation data from The Cancer Genome Atlas (TCGA), the NCBI GEO Portal and the International Cancer Genome Consortium (IGCG) Data Portal were used to validate the methylation alterations detected in the different cancer types analysed (Fig. 1).Methylation β values of the identified altered CGI were visualized using the web tool TCGA Wanderer [32,33]. ",
"section_name": "In silico validation datasets",
"section_num": null
},
{
"section_content": "Additional in silico analyses were conducted using data from TCGA. The database DNA Methylation and gene expression in Human Cancer (MethHC) [34] was used to compare the methylation status of selected PCDHs (PCDHGC3, PCDHGC4, PCDHGC5) in different types of cancer. Xena Functional Genomics Explorer [35] allowed to perform a survival analysis and to study the correlation between methylation and expression of PCDHG C-type in LGG and between the altered N-shelf region or altered CGIs associated with gene promoters and the expression of these genes in TCGA-LGG, TCGA-COADREAD, TCGA-STAD and TCGA-CHOL. ",
"section_name": "In silico analyses",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Differential methylation analyses between cancer and their respective normal tissue samples were performed using experimental datasets and the results have been cross-validated in silico (Fig. 1).Differential methylation levels (Δβ) revealed that clustered PCDH were aberrantly methylated in all the solid cancers analysed.In fact, hypermethylation of CGIs associated with PCDH genes was among the most significant methylation alterations detected, even in BTC where methylation differences between tumour and normal samples were fewer and less pronounced than in the other cancers analysed.In CRC, the most altered CGI associated with PCDHG@ was the fouth most hypermethylated CGI and the fifth most significantly altered CGI among the 74 CGIs found aberrantly methylated in both CRC and CRA in our previous study [25].The most altered PCDHG-associated CGIs in GC and BTC among the statistically significant hypermethylated CGIs (adjusted p value < 0.05) were ranked as 122/522 and 40/48, considering the Δβ, and 28/522 and 13/48, considering the p value, respectively.Interestingly, we detected a hypomethylation event in PCDHG cluster although we did not find any hypermethylated CGIs associated with PCDH in pilocytic astrocytoma.This region was 12/208 most hypomethylated in our discovery set.In contrast, we did not find any relevant methylation alterations in PCDHs in CLL.Overall, these data suggest that clustered PCDH methylation alterations are frequent events during tumorigenesis. ",
"section_name": "PCDH cluster: an aberrantly methylated region in solid cancer",
"section_num": null
},
{
"section_content": "We evaluated the methylation status of PCDH cluster in 20 PA and four normal brain samples.We detected DNA hypomethylation (Δβ value = -0.285) of a flanking region of a CGI (chr5:140871064-140872335, CpG 122) associated with the PCDHG cluster and two CTCF binding sites (Fig. 2a,b, Table 1) in PAs.The flanking region of this CGI is associated with PCDHGC5 gene promoter (Fig. 2a, Table 1).This hypomethylation event was successfully cross-validated using in silico methylation data of pilocytic astrocytoma (GSE44684) (Fig. 2c).We could not investigate the methylation status of the CGI (chr5:140871064-140872335, CpG 122) since we did not have enough epigenome coverage using the Illumina Infinium HumanMethylation27 BeadChips, but in silico analysis revealed that also this CGI was hypomethylated in PA (Fig. 2c). ",
"section_name": "PCDH alterations in pilocytic astrocytoma",
"section_num": null
},
{
"section_content": "The differential methylation analysis conducted on 18 CRC and four normal samples revealed four significantly hypermethylated CGIs related to the PCDHG cluster (Fig. 3a, b, Table 2).All these altered CGIs, except one (chr5:140864527-140864748, CpG 22), were associated with CTCF binding sites (Fig. 3a, Table 2).To elucidate if these aberrations were early events in cancer process, we also performed a differential methylation analysis on 21 CRA and three control mucosae.This analysis revealed methylations alterations in the same CGIs altered in CRC (Fig. 3b, Table 2).Three altered CGIs mapped to promoter regions (Fig. 3a, Table 2).Of note, one of these CGIs (chr5: 140892913-140893189, CpG 20) was not associated with PCDH@ according to Illumina Manifest since it is located downstream the cluster and upstream DIAPH1 gene.Nevertheless, we considered this altered CGI because it was significantly hypermethylated in both CRA and CRC.In general, the Δβ values were higher in carcinomas than in adenomas.On the contrary, one CGI (chr5:140750050-140750264, CpG 16) presented DNA methylation differences only in CRA samples ( tumour stages, i.e.Δβ value = 0.435 and 0.277, in CRC and CRA, respectively.Finally, beta values of the altered CGIs were visualized in a heatmap (Fig. 4).Unsupervised hierarchical clustering (UHC) showed a clear distinction between CRC and normal samples, except for 279T.In contrast, while 12 adenoma samples branched along with CRC samples, the methylation pattern of the other nine resembled that of normal samples.No association was observed between methylation values and clinical data (Fig. 4). We successfully validated all the aforementioned CGI alterations in data from TCGA (TCGA-COAD, TCGA-READ) (Fig. 3c).We observed that one CGI (chr5:140855386-140856620, CpG 95) was hypermethylated in COAD but not READ samples, with the exception of the most telomeric part of the CGI (cg04453180, cg07445963) (Fig. 3c).We observed the same methylation pattern in our discovery set.In general, the average CGI beta value was lower (0.125) in rectal cancer samples than in colon cancer samples (0.277).Moreover, this CGI presented lower methylation values in CRC than those observed in the other altered CGIs (Fig. 3, Fig. 4). ",
"section_name": "PCDH alterations in colorectal cancer",
"section_num": null
},
{
"section_content": "The differential methylation analysis between 22 gastric tumour and their matched normal samples revealed four significantly hypermethylated CGIs that were associated with CTCF binding sites, with the exception of CpG 22, and mapped to promoter regions (Fig. 5a, b, Table 3).These alterations were successfully cross-validated in silico using the TCGA stomach adenocarcinoma dataset (TCGA-STAD) (Fig. 5c).Of note, two of these CGI (CpG 22 and CpG 95) were also altered in CRCs.CpG 95 showed a similar methylation pattern as that observed in CRC, with low β values compared to the other altered CGIs (Fig. 5).UHC analysis allowed to distinguish a group of tumours (N = 7) characterized by high methylation values in all the altered CGIs, a group of tumours (N = 5) that branched along with normal samples and a third group of tumours (N = 10) whose alterations were intermediate between these two groups (Fig. 6).To note, eight out of nine MSI (microsatellite instability) samples were in the clusters of sole tumours and the remaining one clustered with the normal samples (Fig. 6).Thus, MSI was significantly more frequent in the group of tumours with high methylation values (p value = 2.0E-02).To validate these results, we performed a UHC analysis using in silico TCGA-STAD methylation data for the four altered CGIs of samples with available molecular subtype categorization (N = 248).UHC revealed two clusters of tumours with different methylation levels (Fig. 7).In particular, 47 out of 49 MSI samples branched within the cluster displaying high methylation values, confirming that MSI-positive samples were strongly The in silico validation could not berelated to the location (because only two control samples were available). ",
"section_name": "PCDH alterations in gastric cancer",
"section_num": null
},
{
"section_content": "The study conducted in BTC did not detect any CGI differentially methylated between BTC and matched normal samples according to our selection criteria.Nevertheless, two CGIs (chr5:140787447-140788044, CpG 45 and chr5:140797162-140797701, CpG 41), showed significant Δβ values with adjusted p values and were associated with two CTCF binding sites and promoter regions (Fig. 8a, b, Table 4).As previously mentioned, CpG 45 was altered in gastric cancer as well (Table 4).BTC samples were heterogeneous and included 20 gallbladder carcinomas and 22 intrahepatic and eight extrahepatic cholangiocarcinomas.Hence, we analysed each group separately and found significant differences for these loci between intrahepatic cholangiocarcinomas and extrahepatic cholangiocarcinomas/gallbladder carcinomas (Fig. 8b, Table 4). UHC analysis showed a clear distinction between normal and the majority of tumoral samples (68%) and underlined the methylation differences among the three tumoral localizations (Fig. 9).In fact, the majority of the gallbladder (85%) and extrahepatic (87.5%) samples clustered together in the branch of sole tumours, while intrahepatic cholangiocarcinomas were distributed almost equally between the two main clusters. The differential methylation of these CGIs was confirmed by the in silico methylation data (Δβ values = 0.370 and 0.278 for CpG 45 and CpG 41 respectively) (Fig. 8c) although these cases included 33 intrahepatic cholangiocarcinomas, two extrahepatic cholangiocarcinomas and one gallbladder cancer (TCGA-CHOL).To note, the normal samples of our discovery dataset included nine gallbladder and one extrahepatic tissues with average β values of 0.185 (CpG 45) and 0.227 PCDH methylation pattern is not altered in chronic lymphocytic leukemia Interestingly, these clustered genes behaved differently in a type of blood cancer, chronic lymphocytic leukemia, analysed by our group.Analysis of our experimental and in silico data (ICGC: CLLE-ES) did not reveal any significant methylation aberrations in PCDH clusters (Additional file 2: Table S1). ",
"section_name": "PCDH alterations in biliary tract cancer",
"section_num": null
},
{
"section_content": "To increase the robustness of our experimental results, we explored the methylation status of the altered CGIs associated with C-type PCDHG in different cancers, using the database MethHC (Fig. 10).As observed in Fig. 10a, PCDHGC3 was significantly hypermethylated (Δβ value = 0.224) only in COAD.Differently, PCDHGC4 and PCDHGC5 were commonly hypermethylated in a large variety of tumours (Fig. 10b,c). We tested whether the methylation alteration status of N-shelf region or CGIs annotated in promoter regions (Tables 1, 2, 3 and 4) could be associated with change in the expression pattern of the respective gene using TCGA-LGG, TCGA-COADREAD, TCGA-STAD and TCGA-CHOL data.We found a statistically significant negative correlation between methylation and gene expression (Additional file 3: Figure S2, Additional file 4: Figure S3, Additional file 5: Figure S4 and Additional file 6: Figure S5) except for CpG16 methylation and PCDHGB3 gene expression in TCGA-COAD (Additional file 4: Figure S3). We also investigated the correlation between the methylation status of the altered CGIs in the cancer types analysed in the current work and the overall survival using the web-tool UCSC Xena.Therefore, the survival curves were focused on the chromosome region, chr5:140750050-140893189 altered in CRC; chr5:140762401-140864748 in gastric cancer; chr5:140787447-140788044 in BTC; and chr5:140865433-140870165 in low grade glioma (LGG) (Fig. 11).Tumour samples were divided into high and low methylation β values groups.The Kaplan Meier plots showed a possible correlation only in LGG while the analyses did not reveal any significant differences between the two groups of patients with high and low methylation values in the other tumour types (Fig. 11).In fact, in LGG, we observed an abrupt decrease of the survival probability in the first period of the survival time (x-axis) in patients with low β values (blue line) (Fig. 11d).Therefore, we focused the analysis in the C-types PCDHGs (Fig. 12).Firstly, the survival curves of each C-type isoform indicated that the low methylation values of PCDHGC5 significantly correlate with a decrease of survival probability in the first period of this cancer type (Fig. 12c).Secondly, the methylation level of each of the three isoforms, PCDHGC3, PCDHGC4 and PCDHGC5, tended to negatively correlate with their expression levels, suggesting that aberrant methylation may be essential for their transcript regulation in LGG (Fig. 11d). ",
"section_name": "Further in silico analyses",
"section_num": null
},
{
"section_content": "The epigenetic dysregulation of clustered PCDHs has been associated with brain disorders and with cancer as well [2].In particular, the involvement of protocadherins in different types of tumours has been studied by several research groups [4,[16][17][18].In the current experimental study, the CGIs related to PCDH clusters were markedly and significantly altered in the solid tumours analysed (Table 5).We detected, with a high statistical power, significant methylation alterations in CGIs associated with clustered PCDHs that were successfully cross-validated using a larger sample size from in silico TCGA datasets (Fig. 2, Fig. 3 and Fig. 5).Interestingly, all the detected altered regions, except CpG 19 and CpG 20, were associated with promoter regions.Since promoter choice is regulated by DNA methylation [36] and the variable region of each gene cluster contains several CpG sites [8], we analysed the correlation between methylation and expression of the altered CGIs mapping in promoter regions.Interestingly, a negative correlation between methylation and expression was detected (Additional files 3: Figure S2, Additional file 4: Figure S3, Additional BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, COAD colon adenocarcinoma, HNSC head and neck squamous cell carcinoma, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, PAAD pancreatic adenocarcinoma, PRAD prostate adenocarcinoma, READ rectal adenocarcinoma, SARC sarcoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, THCA thyroid carcinoma, UCEC uterine corpus endometrial carcinoma file 5: Figure S4 and Additional file 6: Figure S5) except for CpG16 and PCDHGB3 (Additional file 4: Figure S3).Specific members of the PCDHG cluster were also observed to be downregulated in CRC [4,22].Of note, despite the different methylation and expression aberrations within the clustered PCDHs, it should be taken into consideration the concept remarked by Han et al. and Chen and Maniatis that functional compensation is likely to occur among these gene clusters [37,38]. In addition, differential methylation of C-type members of the PCDHG cluster was reported in silico in a great variety of cancers (Fig. 10).To explore whether the detected methylation alterations may also have an impact on tumour prognosis, we examined a possible association between high or low differential methylation values and the overall survival in silico (Fig. 11).The plots in Fig. 11 exhibited a trend in the correlation between patients with different levels of β values and the survival rate of LGG patients, while no association was found in the gastrointestinal tumour types.Furthermore, among the C-types PCDHG genes, PCHDGC5 showed association with LGG survival probability (Fig. 12c) and could be a predictive biomarker.However, in this cancer, all C-type PCDHGs presented a significant negative correlation between methylation values and expression levels (Fig. 12d).In fact, as mentioned before, clustered PCDHs are mainly expressed in the nervous system while their expression is lower in other tissues [2,39].Thus, our results confirmed that hypermethylated genes in cancer are already lowly expressed in the respective normal tissues [25,40,41], while a tumour in a tissue with high expression, as in this case of gliomas, can undergo hypomethylation in this gene cluster.Finally, our experimental discovery data and the in silico analyses indicated that PCDH cluster genes undergo methylation pattern changes during gastrointestinal tumorigenesis.The absence of significant methylation differences in clustered PCDHs genes in CLL suggested that they are not targeted by methylation during tumorigenesis in haematological neoplasms in contrast to solid tumours.An explanation for this result could be related to the cell adhesion function of PCDHs [1,42] that is not essential in blood cancer for cell contact and tumour mass formation.Besides, our CLL analysis was also supported by the crossvalidation in silico that revealed similar Δβ values (Additional file 2: Table S1).Further studies focused on other blood cancers are needed to support this finding. The analysis of our gliomas data revealed a hypomethylation event (Δβ value = -0.285) in the flanking region of a CGI associated with the PCDHG cluster, including the PCDHG C-type (Table 1).Although this hypomethylation did not involve the CGI itself but a flanking region, a previous work highlights the importance of alterations in this region in gene expression [43].Supportive evidence was provided by the survival analysis considering only the flanking region of the CGI in LGG (Fig. 11d).This survival curve indicated that in the low-β values group of patients (blue line), the survival probability had an early reduction compared to the high-β values group (Fig. 11d).Thus, this hypomethylation event may have a prognostic implication in PA samples.Moreover, at the expression level, PCDHGs are essential during neuronal development and their knockdown or deficiency leads to loss of different neuronal cell types, synapse decrease or dendritic arborisation decline [37,44,45].Therefore, the hypomethylation event could lead to the upregulation of this group of PCDHGs, suggesting that tumour cells need to behave as progenitor cells, i.e. returning to the conditions required during development.However, it should also be considered the possibility that the methylation status found in the tumour actually mirrors the cell of origin pattern clonally expanded [24,40,[46][47][48].In this case, it may not represent a cause or an effect of tumorigenesis, but still a cancer-specific clustered PCDH methylation pattern would remain a valuable biomarker.In addition, PCDHGs overexpression could be implicated in cell survival due to regulation of apoptotic signalling pathways [4] and interaction with cell-adhesion kinases [5,49].Our experimental data showed that the CGIs of clustered PCDHs in CRC are the most highly hypermethylated among the gastrointestinal tumours analysed (Tables 2, 3 and4).UHC analysis revealed that all CRCs clustered together separated from normal samples, with the sole exception of sample 279T (Fig. 4), suggesting a strong methylation alteration of clustered PCDHs in CRC.Moreover, the hypermethylation of these CGIs could be early events during carcinogenesis because they are frequently found in our adenoma samples although some of them did not present methylation alterations.Of note, the Δβ value was always higher in CRCs than in adenomas, except for the CGI located at chr5: 140750050-140750264 (CpG 16) (Table 2).As the values we are referring to, were average values, the differences observed between the two data sets could be due either to increased degree of methylation of each involved island in carcinomas compared to adenomas or to hypermethylation presence in more CRC samples than in adenomas.In this regard, we specifically looked at the β value for each sample for the selected islands.In fact, when we analysed adenomas, we found that while few of them branched nearby normal mucosa samples (CTE1279, CTE1434 and CTE1620), the remaining ones grouped on separated branches and some of them more closely resembled the methylation pattern of carcinomas (Fig. 4).To complement the analysis, we did not find any correlation between PCDHs methylation alterations and the grade of carcinogenesis in adenoma.In fact, as we observed in Fig. 4, the adenomas clustered randomly according to the disease grade.Interestingly, our experimental methylation studies showed that some CGI alterations were common in different cancers (gastric, biliary tract and colorectal cancer) and others were specific for each cancer-type but they were all associated with the PCDHG cluster (Table 5).Interestingly, CpG 22, the most hypermethylated CGI in CRC was also the most hypermethylated in GC.Since this CGI was also hypermethylated in CRA indicating that it is an early event in CRC tumorigenic, it is likely that this event can occur early also during GC tumorigenesis.Other studies have previously found methylation alterations of PCDHG cluster in gastrointestinal tumours, including colon cancer [4].We did not detect significant hypermethylation in the other two PCDH clusters in contrast to Dallosso et al.These events could be related to the wide expression of PCDHG cluster in embryonic and adult tissues, while PCDHA cluster is specifically expressed in the nervous system [4]. Furthermore, the detected methylation aberrations seem to be frequent events in gastrointestinal tumours, some involved in tissue-specific mechanisms and others in common mechanisms.In particular, we found a difference in the methylation pattern of CpG 95 between rectal and colon cancer samples suggesting that the identified alteration may be specific of colon localization.From a clinical point of view, this may be important because it could provide broad-spectrum and tissuespecific tumour biomarkers.Similar differences among localizations have been detected in GC and BTC samples.In fact, GC methylation analysis revealed that Δβ values of all the four altered CGIs did not reach our differential methylation threshold in paired samples localized in body/fundus.BTC results showed differences in Δβ values of two CGIs between localizations suggesting that the detected methylation alterations might reach higher β values in gallbladder/extrahepatic.It is important to mention that normal samples used in the current study were localized in gallbladder/extrahepatic ducts.Therefore, we cannot exclude that we did not observe any alterations in intrahepatic tumours because of the lack of their matched normal tissue samples.In fact, in silico data, where most of tumoral and normal samples had an intrahepatic localization, revealed marked methylation alterations of both CGIs.Thus, future analyses should be performed comparing tumoral samples to their coupled normal localization. We investigated whether other clinical characteristics were associated with methylation alterations in the different cancers analysed, finding an association between hypermethylation and MSI status only in GC as reported by other authors [50,51].In fact, tumour samples with MSI branched together except for 164PRH sample that clustered along normal samples (Fig. 6).We successfully validated these results using in silico TCGA-STAD methylation data (Fig. 7).Moreover, in silico EBVpositive samples displayed high methylation levels for the altered CGIs.This result agrees with previous evidence reported in TCGA-STAD cohort where EBVpositive samples presented extreme CpG island methylator phenotype (CIMP) [52]. The association between molecular subtypes and methylation values could be also observed in the other gastrointestinal tumours.Due to the lack of these molecular data for both our experimental and in silico cohort, we could not evaluate a possible association in BTC.Given that our CRC samples clustered together in a group with high values for all the altered CGI and included two MSI samples, no association between methylation and MSI status can be detected.Furthermore, our previous analysis of TCGA-COAD and READ methylation data for 74 CGIs, including two PCDH-associated CGIs (CpG 19 and CpG 22), revealed that most CRC samples clustered in the group of tumours displayed high β values [25], confirming that high methylation levels of the analysed CGI alterations are not related to different molecular status. Importantly, we included in our methylation analyses the position of the CTCF binding sites, possibly associated with the CGIs.As mentioned before, these sites are most likely related to the clustered PCDHs transcription through the formation of DNA loops mediated by CTCF interactions [11,15].Since methylation regulates CTCF binding [12], the methylation abnormalities detected in our experimental results could avoid or modify the hub formation by blocking the interaction between the CTCF protein and the neighbouring binding sites, consequently regulating PCDHG cluster transcription.Previous functional studies [12,14,20] have already shown that DNA methylation aberrations are associated to alteration of CTCF binding to DNA. This study, although suggesting the evaluation of the clustered PCDH-associated CGIs methylation levels as a tumour biomarker different types of cancer, has some limitations that can be overcome by more detailed future studies.A technical limitation is certainly due to the use of different types of arrays in the different cases, in particular, the 27K array for PAs, which therefore does not allow us to draw more definitive conclusions in the opposite methylation patterns observed in pilocytic astrocytomas compared to gastrointestinal tumours.In fact, further studies analysing DNA methylation alterations associated to PCDH cluster genes in additional brain tumours are needed to confirm the correlation between hypomethylation in cancer and normal tissue expression.Furthermore, an aspect that is certainly worth investigating is the lack of an experimental expression analysis and further functional analyses aimed to understand if and how the identified methylation alterations play a role in the tumorigenesis of the different tumours analysed. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Although several authors have conducted analyses in clustered PCDHs, this work highlighted that methylation alterations of PCDHG@ are among the most statistically significant aberrations in solid cancers.Moreover, our results suggest that in neuronal tissue, where PCDHs are highly expressed, this gene cluster becomes hypomethylated in pilocytic astrocytomas, while in tissues where PCDHs are lowly expressed, this cluster is targeted by DNA methylation.These epigenetic aberrations in the CGIs associated to PCDHG@ genes could be useful to consider specific members of this cluster as possible biomarkers.Nevertheless, further research is necessary to elucidate their function and their expression regulation in each tumour type. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "We gratefully acknowledge the CeSAR (Centro Servizi d'Ateneo per la Ricerca) of the University of Cagliari, Italy, for the use of the iScan platform (Illumina) for methylation assays. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Funding This work was supported by grants from Fondazione Banco di Sardegna (2012), Fondo per la Ricerca Locale (ex 60%), Università di Cagliari and POR FESR Sardegna (1C-177) to PZ and Regione Autonoma della Sardegna (CRP-79303) to PZ and LZ.Partly supported also by grants from Fondazione di Sardegna (2016) to AC, MS and PZ; Fondazione Neuroblastoma to FG; Italian Ministry for Education, University and Research (PRIN 2007 prot.2007WEJLZB and PRIN 2009 prot.20092ZELR2), and the Italian Association for Cancer Research (IG 2011/11855) to PC. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The datasets analysed during this study are available from the corresponding author on reasonable request. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Additional file 1: Authors' contributions AFVB helped in the statistical and bioinformatic analysis, interpretation of data, drafting of the manuscript and figure and table preparation.EL helped in the methylation data acquisition, management and analysis, supervision on statistical and bioinformatic analysis, interpretation of data, drafting of the manuscript and figure preparation.LM helped in the DNA extraction from fresh frozen tissues and from FFPE samples, DNA restoration, bisulfite treatment and DNA whole genome methylation assays.SB helped in the interpretation of preliminary data and study of clustered PCDHs and participated in drafting a first version of the manuscript.AF helped in the methylation data acquisition, management and analysis, particularly restricted to the CTCF binding sites.MA, AA, MB, FG and IM helped in the PA tissue collection and clinical data collection.AC, AR and LZ helped in the CRC/normal mucosa tissue collection and clinical data collection.VG and LV helped in the colon adenomas/normal mucosa DNA sample and clinical data collection.SEB and SG helped in the GC DNA sample and clinical data collection.MC, ACG, LF, MP, MS and PZ helped in the BTC FFPE and DNA sample collection and clinical data collection.GC, PC, MGE, GS and MZ helped in the CLL DNA samples and clinical data collection.DC adviced and supervised methylation analysis in clustered PCDHs, in relation to CTCF binding sites and C-type PCDHG gene expression in the brain.PZ helped in the study concept and design, supervision on methylation, statistical and bioinformatic analyses, interpretation of data, supervision and drafting of the manuscript.All authors discussed the results and commented on the manuscript.All authors read and approved the final manuscript Ethics approval and consent to participate All the biological samples analysed were obtained with written informed consent signed from patients and ethical approval granted by the relative Ethics Committee. Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Authors' contributions AFVB helped in the statistical and bioinformatic analysis, interpretation of data, drafting of the manuscript and figure and table preparation.EL helped in the methylation data acquisition, management and analysis, supervision on statistical and bioinformatic analysis, interpretation of data, drafting of the manuscript and figure preparation.LM helped in the DNA extraction from fresh frozen tissues and from FFPE samples, DNA restoration, bisulfite treatment and DNA whole genome methylation assays.SB helped in the interpretation of preliminary data and study of clustered PCDHs and participated in drafting a first version of the manuscript.AF helped in the methylation data acquisition, management and analysis, particularly restricted to the CTCF binding sites.MA, AA, MB, FG and IM helped in the PA tissue collection and clinical data collection.AC, AR and LZ helped in the CRC/normal mucosa tissue collection and clinical data collection.VG and LV helped in the colon adenomas/normal mucosa DNA sample and clinical data collection.SEB and SG helped in the GC DNA sample and clinical data collection.MC, ACG, LF, MP, MS and PZ helped in the BTC FFPE and DNA sample collection and clinical data collection.GC, PC, MGE, GS and MZ helped in the CLL DNA samples and clinical data collection.DC adviced and supervised methylation analysis in clustered PCDHs, in relation to CTCF binding sites and C-type PCDHG gene expression in the brain.PZ helped in the study concept and design, supervision on methylation, statistical and bioinformatic analyses, interpretation of data, supervision and drafting of the manuscript.All authors discussed the results and commented on the manuscript.All authors read and approved the final manuscript Ethics approval and consent to participate All the biological samples analysed were obtained with written informed consent signed from patients and ethical approval granted by the relative Ethics Committee. ",
"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
}
] |
10.3390/cancers13061336
|
Novel Agents in Chronic Lymphocytic Leukemia: New Combination Therapies and Strategies to Overcome Resistance
|
<jats:p>The approval of Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib and acalabrutinib and the Bcl-2 inhibitor venetoclax have revolutionized the treatment of chronic lymphocytic leukemia (CLL). While these novel agents alone or in combination induce long lasting and deep remissions in most patients with CLL, their use may be associated with the development of clinical resistance. In this review, we elucidate the genetic basis of acquired resistance to BTK and Bcl-2 inhibition and present evidence on resistance mechanisms that are not linked to single genomic alterations affecting these target proteins. Strategies to prevent resistance to novel agents are discussed in this review with a special focus on new combination therapies.</jats:p>
|
[
{
"section_content": "Over the last decade, the increasing knowledge on the pathogenesis and diseasedriving mechanisms of chronic lymphocytic leukemia (CLL) has finally translated into a multitude of new treatment options for patients with CLL [1,2].The approvals of the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and more recently acalabrutinib as well as the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax have transformed the treatment paradigm in both treatment-naïve and relapsed/refractory CLL [3][4][5][6][7][8][9][10][11][12].While venetoclax is mostly used in combination with anti-CD20 antibodies for a fixed duration of one (firstline) to two years (relapse setting), BTK inhibitors (BTKi) and PI3K inhibitors are exclusively approved for continuous treatment and mainly prescribed as monotherapies. Despite these new therapeutic options, CLL remains a chronic, incurable disease, with allogeneic hematopoietic stem cell transplantation being the only treatment with confirmed curative potential in a minority of patients [13][14][15][16][17]. Patients treated successfully with kinase inhibitors or venetoclax eventually relapse/progress or have to stop treatment due to intolerance.The mechanisms behind these clinical manifestations of resistance have been studied, and are in part explained by genetic resistance mechanisms [18][19][20].Besides mutations in the drug-targeting proteins, several non-genetic mechanisms rendering CLL cells resistant to treatment have been described [21][22][23][24].In this article, we will review the current state of research on resistance to novel agents, propose strategies to avoid resistance and provide guidance on treating patients who relapse or progress on novel agents. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "After proving exceptional activity in all analyzed patient groups, ibrutinib was approved for the treatment of CLL by the U.S. Food and Drug Administration (FDA) as well as the European Medicines Agency (EMA) in the relapsed/refractory setting in 2014 and for frontline use in 2016 [3][4][5]9,12].The second-generation BTKi acalabrutinib was recently approved based on marked survival benefits in two randomized controlled trials in treatment-naïve and relapsed/refractory CLL [10,25].In the US a third BTKi, zanubrutinib, has been approved for the treatment of mantle cell lymphoma and is expected to be approved for CLL soon [26]. While more recently, ibrutinib and acalabrutinib have been tested within fixedduration combinations, these combinations of novel agents are not yet approved.Hence, their main use remains as monotherapy that is given until disease progression or unacceptable toxicity [27][28][29][30].Adding to the financial burden and side effects associated with an indefinite therapy, the continued exposure to BTKi seems to promote the acquisition of resistance mutations. ",
"section_name": "Mechanisms of Resistance to BTK Inhibition",
"section_num": "2."
},
{
"section_content": "Ibrutinib, acalabrutinib and other covalent binding BTK inhibitors exhibit their main inhibitory effect on the B-cell receptor (BCR) pathway by irreversibly binding to the C481 position of BTK and thereby inactivating the enzyme.Not surprisingly, the first discovered mutations that conferred resistance to ibrutinib were mutations in the BTK gene at the ibrutinib binding site [11].Woyach and colleagues performed whole exome sequencing on six patients progressing on ibrutinib and discovered that five of these patients had acquired cysteine-to-serine mutations in BTK at position 481 (C481S) that were not present before treatment with ibrutinib.Through the replacement of cysteine with serine, ibrutinib can no longer bind to BTK irreversibly, leading to restored BCR signaling and clinical resistance to ibrutinib [11].Sequencing data from early trials have shown BTK mutations in approximately 80% of patients progressing on ibrutinib [11,[31][32][33].By sequencing earlier samples of those progressing patients, BTK-mutated clones were already detected at a median of eight and nine months before clinical progression [31,33].In a large cohort comprising 373 patients treated with ibrutinib at the Ohio State University (OSU), BTK mutations occurred in 23.3% at a median of 34 months following ibrutinib initiation [32].More recently, an analysis of a French registry cohort has shown an incidence of BTK mutations of 57% in patients who were still on ibrutinib, suggesting that a substantial proportion of patients with CLL on ibrutinib monotherapy already harbors resistanceconferring mutations when clinically still responding to treatment [34].Importantly, the acquisition of BTK mutations was clearly associated with the risk of subsequent clinical progression in this study, confirming findings of Woyach et al. who had also studied this phenomenon prospectively [33,34]. On rare occasions, other putatively resistance-conferring mutations have been reported in BTK.Some also affect the ibrutinib binding site (e.g., C481F, C481Y) [32], while some others affect BTK binding to B-cell linker protein (BLNK), enabling Phospholipase C gamma 2 (PLCy2) activation despite the presence of ibrutinib [35]. BTK mutations were often found to be accompanied by mutations in PLCG2, the gene coding for PLCy2, the substrate downstream of BTK [11,[31][32][33][34].Most PLCG2 mutations are gain-of-function mutations at the SH2/SH3 domain of the PLCG2 gene, leading to autonomous BCR signaling despite BTK inhibition [32].In two prospective studies, the cumulative incidence of PLCG2 mutations was 13% after at least 3 years of ibrutinib treatment and 10% after a median of 3 years after start of ibrutinib, respectively [32,34].The majority of patients with PLCG2 mutations harbored concurrent BTK mutations that occurred at similar time points in treatment (median 35 and 34 months after start of ibrutinib, respectively).In the context of extensive sequencing efforts, other rare genetic aberrations possibly conferring resistance to ibrutinib have been described, including del(8p) and 2p gain with subsequent XPO1 overexpression [36,37]. A recent study assessed the occurrence of BTK and PLCG2 mutations in a pooled cohort of 388 patients without clinical progression from various ibrutinib trials by nextgeneration sequencing [38].With a median follow-up on treatment of 35 months (previously untreated patients) and 36 months (relapsed/refractory patients), the analysis revealed that the incidence of these resistance-conferring mutations strongly differed between previously untreated patients and patients receiving ibrutinib in a relapsed/refractory setting.Among patients on first-line ibrutinib, only 3% harbored BTK mutations while 30% of the relapsed/refractory patients showed BTK mutations, consistent with differences in clinical progression rates between these populations. The pattern of genetically mediated resistance to acalabrutinib or other covalent binding BTKis such as zanubrutinib is thought to be similar to ibrutinib due to their formation of a covalent bond at the same binding site.The only study on resistance mutations in CLL patients treated with acalabrutinib in the frontline as well as relapse setting detected BTK C481 mutations in 69% of patients progressing on acalabrutinib, while 14% of these patients harbored concurrent subclonal PLCG2 mutations [39]. In patients with progressing CLL, BTK and PLCG2 mutations are often found in clones/subclones of variable size with reported ranges between 0.2% and nearly 100% [11,[31][32][33][34].However, even small subclones might lead to clinical resistance, as studies in Waldenström's macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL) suggest.In vitro and in vivo assays in those entities showed that BTK-mutant cells protect wildtype cells from ibrutinib-induced killing by releasing interleukin 6 (IL-6) and IL-10 and thereby triggering strong prosurvival signaling including the JAK/STAT pathway, providing a possible explanation for clinical resistance in patients with a BTK-mutated subclone [40].Furthermore, the variant allele frequency (VAF) of BTK mutations in circulating CLL cells appeared to be lower in patients with primarily nodal relapses indicating that genetic aberrations detected in peripheral blood CLL cells are not necessarily representative for clonal composition in other compartments [33]. ",
"section_name": "Genetic Mechanisms of Resistance to BTK Inhibition",
"section_num": "2.1."
},
{
"section_content": "Despite comprehensive genetic analyses, a substantial part of clinical progressions on ibrutinib are not explained by genetic alterations.Different non-genetic mechanisms of adaptation to ibrutinib treatment have been described in CLL cells.The main mechanisms are the maintenance of BCR signaling through alternative pathways and interactions of CLL cells and the tumor microenvironment (TME). Under the influence of BTK inhibition by targeted drugs, CLL cells and malignant B-cells in other lymphoid neoplasms may adapt and compensate for the blocked BTK axis by activating the PI3K/Akt/Erk pathway [21,22,41].Functional analyses by Spina et al. in cells from ibrutinib-treated patients revealed that the BCR pathway through Akt and Erk was still inducible upon stimulation of the B-cell receptor in spite of effective inhibition of the BTK/PLCy2 pathway [22].The group also showed that in CLL cells persisting under ibrutinib, genes involved in the MAPK/Erk pathway were upregulated [21].Similarly, CD40L stimulation of the non-canonical NF-kB pathway still led to nuclear translocation of NF-kB while the canonical NF-kB pathway was inhibited by ibrutinib [22]. Ibrutinib treatment was shown to effectively reduce chemokines involved in homing, retention and adhesion of CLL cells in their growth-and survival-supporting microenvironment [42,43].During BTK inhibition CLL cells may adapt their phenotype by upregulation of homing/adhesion proteins and increased surface IgM [22,44,45].Protective nurse-like cells (NLC) in the TME also seem to play a role in rescuing CLL cells from ibrutinib-induced killing, as ibrutinib does neither seem to fully antagonize the CLL cell-supporting function of NLCs nor to effectively mobilize them from their lymph node or bone marrow niches [46][47][48].It has also been hypothesized that extracellular vesicles of bone marrow stromal cells play a role in the development of drug resistance by rescuing CLL cells from apoptosis and thereby increasing chemoresistance to different drugs, including ibrutinib, idelalisib, venetoclax and fludarabine [49]. ",
"section_name": "Non-Genetic Mechanisms of Resistance to Ibrutinib",
"section_num": "2.2."
},
{
"section_content": "Two inhibitors of PI3K, idelalisib and duvelisib, have been approved for the treatment of relapsed/refractory CLL.Both drugs are used as continuous therapies and mostly applied in heavily pretreated patients, hence progression on these agents was observed comparably early with a median PFS of 16.4 months for idelalisib plus rituximab and 13.3 months for duvelisib in the trials leading to approval [50,51].In spite of various sequencing efforts and in contrast to BTKi and venetoclax, no resistance-conferring mutations were so far identified in the gene coding the target protein PI3K [52,53].Several mechanisms like activating mutations in MAPK pathway genes, an upregulation of Igf1r and an amplification or activating mutations of PI3KCA have been associated with resistance to idelalisib [53][54][55][56].However, in the absence of comprehensive analyses, it remains largely unclear whether these mechanisms are responsible for clinical resistance in a substantial proportion of patients. ",
"section_name": "Mechanisms of Resistance to PI3K Inhibitors",
"section_num": "3."
},
{
"section_content": "B-cell lymphoma-2 (Bcl-2) is an anti-apoptotic protein and part of a family of proteins, regulating B cells disposition to undergo apoptosis.In CLL, overexpression of Bcl-2 results in an inhibition of pro-apoptotic BH3-only proteins ensuring survival of the CLL cell [57][58][59].Venetoclax, a BH3 mimetic, has been developed to bind Bcl-2 at the same site as BH3-only proteins to effectively inhibit Bcl-2 [60].After clinical studies in CLL have consistently shown impressive activity of venetoclax in all therapeutic settings as monotherapy as well as in combinations, it has been approved for the treatment of patients with previously untreated and relapsed/refractory CLL [7,8,[27][28][29][30][61][62][63][64][65][66]. ",
"section_name": "Mechanisms of Venetoclax Resistance",
"section_num": "4."
},
{
"section_content": "The first discovered resistance-conferring mutation in the context of venetoclax treatment affects the binding site of the target protein, Bcl-2.In a study of 15 patients progressing on venetoclax, Blombery and colleagues could identify the BCL2 single-nucleotide variant G101V in seven of 15 patients by next-generation sequencing (NGS) and digital-droplet polymerase chain reaction (ddPCR) [20].The variant was not detected in these patients before initiation of venetoclax treatment and in a large group of venetoclax-unexposed patients with CLL.Using earlier samples of the seven patients, the mutation could already be found up to 25 months before clinical relapse [20].In a functional analysis, Blombery et al. could demonstrate that the ability of venetoclax to compete with BH3-only proteins for binding of Bcl-2 was strongly impaired in the presence of the G101V mutation [20].Structural analyses revealed the molecular basis of this reduction in affinity by reporting the crystal structure of Bcl-2 in complex with venetoclax [67]. Recently, a more sensitive sequencing approach revealed the presence of numerous other BCL2 mutations in patients with G101V variants who clinically progressed on venetoclax monotherapy administered as relapse treatment [68].Ten out of 11 (91%) of the analyzed patients harbored additional acquired BCL2 mutations in different CLL cells with strongly varying cancer cell fractions [68].Another report identified a putatively resistanceconferring D103Y mutation in BCL2, also affecting the binding site of venetoclax [18]. Given the often subclonal nature of the identified mutations in clinically progressing patients and the lack of BCL2 mutations in a large proportion of patients who relapse on venetoclax, it is questionable if BCL2 mutations are the sole cause of clinical resistance to venetoclax [69,70]. Another study of eight high-risk CLL patients carrying del(17p) and/or TP53 mutations progressing on venetoclax revealed other recurrent aberrations with resistanceconferring potential and described a rather heterogeneous clonal evolution in venetoclaxtreated CLL [19].Whole exome sequencing in this cohort identified acquired homozygous CDKN2A/B deletion in 3/8 (38%) and BTG1 missense mutations in 2/8 (25%) patients.Single patients harbored mutations in BRAF, SF3B1, RB1, MLL3, BIRC3 and a high-level focal amplification of CD274 (PD-L1) [19].While further functional analyses showed no increased resistance in CDKN2A/B-mutated cells, the BRAF mutation was associated with elevated Mcl-1 expression and showed resistance in transduced cell lines. ",
"section_name": "Mutations in BCL2 and Alterations in Cancer-Related Genes",
"section_num": "4.1."
},
{
"section_content": "Resistance to venetoclax treatment has also been detected on a non-mutational basis through upregulation of other anti-apoptotic Bcl-2 family members.As the sensitivity to BH3 mimetics depends largely on the ratio of the expression of proapoptotic and antiapoptotic proteins, it was hypothesized early on that an increase in anti-apoptotic proteins following venetoclax exposure would confer resistance to Bcl-2 inhibition [71,72].Various studies have demonstrated that venetoclax-associated overexpression of Mcl-1 and Bcl-X L can confer resistance to the drug in vitro and in vivo [23, 24,[72][73][74][75][76].Recently it has been postulated that among the Bcl-2 family members, Bcl-X L is more relevant for the development of venetoclax resistance, as functional analyses have shown that proapoptotic proteins preferably interact with Bcl-X L when both anti-apoptotic proteins are present [24].Guièze and colleagues have performed an extensive analysis including genome-scale screens in a Bcl-2-driven lymphoma cell line and integrated expression profiling and identified Mcl-1 overexpression and BIM sequestration as a resistance-conferring mechanism to venetoclax treatment [23].The group has also shown that reprogramming of the biology of the mitochondrial outer membrane can result in altered expression of Bcl-2 family members and an increase in oxidative phosphorylation (OXPHOS) activity, with both leading to resistance to Bcl-2 inhibition [23]. The above outlined mechanisms of resistance and potential approaches to overcome them are summarized in Figure 1. Cancers 2021, 13, x 5 of 19 treated CLL [19].Whole exome sequencing in this cohort identified acquired homozygous CDKN2A/B deletion in 3/8 (38%) and BTG1 missense mutations in 2/8 (25%) patients.Single patients harbored mutations in BRAF, SF3B1, RB1, MLL3, BIRC3 and a high-level focal amplification of CD274 (PD-L1) [19].While further functional analyses showed no increased resistance in CDKN2A/B-mutated cells, the BRAF mutation was associated with elevated Mcl-1 expression and showed resistance in transduced cell lines. ",
"section_name": "Changes in Cellular Metabolism/Bcl-2 Family Members",
"section_num": "4.2."
},
{
"section_content": "Resistance to venetoclax treatment has also been detected on a non-mutational basis through upregulation of other anti-apoptotic Bcl-2 family members.As the sensitivity to BH3 mimetics depends largely on the ratio of the expression of proapoptotic and antiapoptotic proteins, it was hypothesized early on that an increase in anti-apoptotic proteins following venetoclax exposure would confer resistance to Bcl- The above outlined mechanisms of resistance and potential approaches to overcome them are summarized in Figure 1. ",
"section_name": "Changes in Cellular Metabolism/Bcl-2 Family Members",
"section_num": "4.2."
},
{
"section_content": "The above cited studies have demonstrated that resistance to novel agents in CLL, especially when caused by acquired BTK and BCL2 mutations, appears to occur rather late in the course of treatment, though some reports show the detection of these mutations as early events [31].A recent analysis by Wiestner and colleagues showed that resistance mutations in BTK or PLCG2 seem to appear later in patients receiving ibrutinib as first-line therapy in comparison to patients in a relapsed/refractory situation [38].There are various promising strategies to possibly circumvent resistance to BTKi and Bcl-2 inhibitors. ",
"section_name": "Strategies to Prevent Resistance to Novel Agents",
"section_num": "5."
},
{
"section_content": "In the pivotal studies, acquired BTK and BCL2 mutations could be detected months and even years [11,21] before the patients fulfilled the iwCLL criteria [77] of clinical disease progression.Following these observations, Woyach and colleagues initiated a prospective study on 112 patients receiving ibrutinib monotherapy and performed serial screening for known resistance mutations [33].They demonstrated that mutations in BTK and PLCG2 occurred early and clearly correlated with consequent clinical disease progression and could thus be used as a biomarker for relapse and an opportunity to adapt treatment [33].Hence, a future strategy could be to monitor resistance similarly to strategies in antiviral treatments as for example HIV. ",
"section_name": "1.",
"section_num": null
},
{
"section_content": "Constant selection pressure by administering continuous monotherapies with BTKis or Bcl-2 inhibitors might contribute to the acquisition of resistance mutations in a significant number of patients.Hence, avoiding constant drug exposure and selection of BTKi-and venetoclax-resistant clones by using time-limited treatment approaches could be another option to circumvent the development of resistance. ",
"section_name": "2.",
"section_num": null
},
{
"section_content": "Another promising strategy to circumvent the acquisition of resistance to novel agents is the development of next generation inhibitors which bind non-covalently to the target kinase and are therefore still active in CLL cells harboring the most common resistance mutations. The strategies of time-limited combination as well as the current status of the development of non-covalent BTK inhibitors is discussed in the following segment. ",
"section_name": "3.",
"section_num": null
},
{
"section_content": "Time-limited approaches include combinations of different drugs, as monotherapies usually do not achieve sufficiently deep responses that would allow drug discontinuation [78,79].Combination therapies would possibly also reduce the selection of BTKor BCL2-mutated clones, as e.g., BTK C481S-mutated cells could still be eliminated by concomitant venetoclax or an anti-CD20 antibody while they would likely outgrow under ibrutinib monotherapy.On the other hand, it has been shown that the combination of venetoclax with a BTKi was able to reprogram apoptotic dependencies and venetoclax resistance could be overcome in malignant B cells [24,80,81].Also, resistance-mediating upregulation of Bcl-2 under ibrutinib monotherapy can increase the sensitivity towards venetoclax [82,83].Another probable advantage of combination therapies would be the lower minimal residual disease (MRD) these combination treatments could achieve.A CLL cell count of <10 -6 which was shown to be achieved by a substantial fraction of patients receiving a time-limited combination of venetoclax and obinutuzumab correlates to 10,000 times less measurable CLL burden compared to a MRD-positive (≥10 -2 ) patient under ibrutinib monotherapy [84].It is conceivable that in a substantially smaller CLL cell pool, resistance-conferring genetic alterations are less likely to develop.In addition, avoiding resistance by limiting duration and combining different drugs would possibly allow for re-exposure to the same drug combination.Data from studies evaluating the most promising time-limited combination treatments is listed in Table 1, while currently ongoing trials are shown in Table 2. TN: treatment-naïve, R/R: relapsed/refractory, uMRD: undetectable minimal residual disease (<10 -4 ), PFS: progression-free survival, OS: overall survival.Two time-limited combinations have recently been approved for the treatment of CLL.After the MURANO study demonstrated significantly superior survival in relapsed/refractory patients treated with 24 months of venetoclax and rituximab compared to bendamustine and rituximab, the scheme was approved and widely adopted in the relapsed/refractory setting [8].Follow-up publications showed durable responses in patients treated with venetoclax and rituximab with 57% of the patients still being progression-free 4 years after the start of treatment [90].No information on the occurrence of BCL2 mutations has been published so far, but first data of patients being retreated with the same combination at progression suggest that a large part of patients can respond to a re-exposure after time-limited venetoclax and rituximab [91]. More recently, the one-year combination treatment of venetoclax and obinutuzumab was approved after yielding impressive PFS advantages and high rates of undetectable MRD (uMRD) when compared to chlorambucil and obinutuzumab [7,61].The latest update showed durable and deep responses and it could even be demonstrated that the rate with which MRD increases after the end of treatment is lower in the venetoclax and obinutuzumab arm, suggesting that not only the depth but also the quality of the response is improved [84].As only a few patients have progressed after this combination, retreatment data is scarce, but it is conceivable that patients might respond again to venetoclax and obinutuzumab, as they have only been exposed for one year.Clinical trials evaluating retreatment after time-limited combinations are currently being planned. There are currently numerous studies ongoing testing combination approaches in all stages of clinical development [92].The most promising combination therapies consist of a BTKi in combination with venetoclax with or without the anti-CD20 antibody obinutuzumab and are currently studied in phase 3 trials. Jain and colleagues published an early interim analysis of their phase 2 trial of a fixed-duration treatment with ibrutinib and venetoclax in treatment-naïve patients with CLL [28].Ibrutinib was given alone for 3 cycles (28 days each) followed by 24 cycles of combined treatment.After one year of combination treatment, 88% of the evaluable patients had a complete remission or complete remission with incomplete count recovery and 61% of the evaluable patients showed uMRD (<10 -4 ).However, due to the early read-out, longer observation is required.An overview on selected other studies is shown in Table 1.Results from the Phase 3 FLAIR (2013-001944-76) and GLOW (NCT03462719) trials, testing this combination in a randomized setting, are eagerly awaited and expected soon.The phase 3 CLL17 trial of the German CLL Study Group (GCLLSG) (NCT04608318) is currently evaluating the approved options ibrutinib monotherapy and venetoclax plus obinutuzumab against ibrutinib and venetoclax. The shorter triple combination of ibrutinib, venetoclax and obinutuzumab was studied extensively as well.A phase 2 study of the combination in treatment-naïve and relapsed/refractory patients with CLL was recently published and yielded high response rates, high rates of uMRD and durable remissions [30].The same combination was studied in the phase 2 CLL2-GIVe trial, in which patients with detectable post-treatment MRD could continue ibrutinib treatment until cycle 36.After 15 months of combined treatment, 33 of 41 patients (81%) achieved uMRD in the peripheral blood and 22 patients stopped treatment at this time point due to uMRD and CR/CRi [86].The same concept was tested in the randomized phase 3 GAIA/CLL13 trial (NCT02950051) by the GCLLSG against standard chemoimmunotherapy and other fixed-duration venetoclax combinations.The study will also be the first to show results on the efficacy of the time-limited combinations of venetoclax plus obinutuzumab in fit patients as well as venetoclax plus rituximab in the first-line setting. Similar combination treatments using acalabrutinib instead of ibrutinib have yielded similarly promising results in phase 2 trials and are currently being evaluated against standard chemoimmunotherapy in a large phase 3 trial (NCT03836261) [87,88]. The GCLLSG currently runs two trials evaluating time-limited, MRD-driven combination treatments of venetoclax, obinutuzumab and acalabrutinib/zanubrutinib after an optional debulking with bendamustine (NCT03787264, NCT04515238).The trials are accompanied by an extensive real-time screening program for the occurrence of known resistance mutations to BTKi and venetoclax as well as other potentially druggable targets using ddPCR on circulating tumor DNA (ctDNA).In the induction phase, the screening is performed monthly followed by every three months and in the case of occurring resistance mutations, the individual results are discussed by the sponsor and the treating physician.The concept is currently too labor-intensive and costly to apply it in clinical practice, but it will certainly provide valuable information on the relevance of known resistance mutations and their early detection in time-limited combination approaches. ",
"section_name": "Time-Limited Combination Treatments",
"section_num": "5.1."
},
{
"section_content": "Second-generation BTK inhibitors like acalabrutinib and zanubrutinib have demonstrated their efficacy and safety in different B-cell malignancies and might be considered as an alternative to ibrutinib due to fewer off-target effects and different safety profiles [6,10,25,93,94].However, they also bind irreversibly to BTK at the same binding site as ibrutinib and are also not able to inhibit BTK in patients with the most common mutation associated with ibrutinib resistance (BTK C481S). Novel, third-generation, non-covalent BTK inhibitors have been designed to overcome this mechanism of resistance by reversibly binding to BTK without interacting with C481.Two of the new third-generation BTKi, vecabrutinib and fenebrutinib, have shown initial proof of concept but do not seem to be further evaluated in CLL [95,96].Two other thirdgeneration BTKi, ARQ 531 and LOXO-305, have yielded promising results in their early clinical trials and were advanced to the next stage of clinical testing [97,98].ARQ 531 is a non-covalent BTKi that binds to the ATP-binding region of BTK and has shown its activity in a phase 1 study in relapsed/refractory CLL patients [98].In a heavily pretreated population with 22/26 (85%) patients harboring BTK C481S mutations, seven patients achieved a partial response (PR).The phase 2 part of the study is currently ongoing.LOXO-305 is a non-covalent BTKi that has demonstrated its efficacy in a phase 1/2 trial of patients with relapsed/refractory CLL [97].In the BRUIN study, of 65 patients with CLL/SLL (58 BTKi-pretreated, 7 previously BTKi-naïve) who had a response assessment, 37 (57%) either had a PR or PR with lymphocytosis and only one patient progressed so far.The median follow-up of the study is still short and responses are thought to improve over time, as seen in other BTK inhibitors. Proteolysis targeting chimera (PROTAC) have been developed to target proteins that are otherwise difficult to inhibit.They consist of a ligand directed against the target of interest coupled with a ligand for binding of an E3 ubiquitin ligase and act by facilitating degradation of the target by the proteasome [99].Multiple BTK-targeting PROTACs were developed and the first analysis specifically examining their activity in BTK C481S-mutated CLL demonstrated activity of the construct MT-802, whereas ibrutinib expectedly showed no relevant activity in mutant cells [100].Another BTK-targeted PROTAC, L18I, was able to induce degradation of BTK C481S-mutated cells as well as other BTK variants with Thr, Gly, Trp or Ala substitutions at Cys [101].Compared with ibrutinib, PROTACs also seem to more selectively target BTK, whether this translates to less clinical toxicity will however only be clear when PROTACs are tested in clinical trials.PROTACs against Bcl-X L have also been reported and could be promising drugs for patients resistant to BTKi and venetoclax [102].Due to its Von Hippel-Lindau E3 ligase it is thought to not relevantly degrade Bcl-X L in platelets, which might lead to less thrombocytopenia when compared to previous Bcl-X L inhibitors like ABT263 and might therefore be more tolerable [102]. ",
"section_name": "Non-Covalent BTK Inhibitors and BTK Degraders",
"section_num": "5.2."
},
{
"section_content": "Time-limited combination treatments might effectively prevent the acquisition of certain types of resistance, but currently indefinite monotherapies are widely used in the frontline setting and it is important to know how to best treat patients with acquired resistance to those drugs.The next section will focus on experimental treatment options for patients relapsing or progressing on approved novel agents but begin with a short overview of the current evidence on optimal sequencing of approved treatment options.Approved and experimental cellular treatment options like allogeneic hematopoietic stem cell transplant (allo HCT), Chimeric Antigen Receptor (CAR) T-cell and CAR NK-cell therapies will not be discussed in this review. ",
"section_name": "Treatment Options in Case of Clinical Resistance to Novel Agents",
"section_num": "6."
},
{
"section_content": "Some patients may be resistant to either BTKi or venetoclax but have not yet been exposed to the other and studies have recently demonstrated that both, venetoclax after ibrutinib and BTKi after venetoclax are effective treatment options for patients who have discontinued treatment with either of the drugs [103,104].In earlier retrospective analyses by Mato and colleagues, idelalisib appeared inferior to both, ibrutinib after venetoclax and venetoclax after ibrutinib [105].Given the overall superior efficacy data of venetoclax and BTKi compared to PI3K inhibitors in CLL, a sequencing approach using venetoclax-based regimens after progression on ibrutinib and BTKi after progression on venetoclax is the current standard of care [92,106].While patients discontinuing ibrutinib due to toxicities can be treated with acalabrutinib as the efficacy and safety of this approach has recently been demonstrated, this is not the case for patients who progress on ibrutinib and likely harbor BTK mutations, as the binding site of ibrutinib and acalabrutinib is identical [107]. Despite the durable responses that can be achieved by optimally sequencing novel agents, combinations might be even more promising.Recently, concurrent BTK, PLCG2 and BCL2 mutations were found in patients who were treated with continuous ibrutinib monotherapy, had relapsed and then received continuous single-agent venetoclax [108].Upon progression on venetoclax, four of eight evaluable patients with CLL-type progression harbored both BTKi-specific (BTK/PLCG2) and venetoclax-specific (BCL2) resistance mutations, suggesting that sequencing of single agents might lead to a situation in a substantial fraction of patients in which re-exposure to either of the drugs would probably be unsuccessful. ",
"section_name": "Optimal Sequencing of Approved Agents",
"section_num": "6.1."
},
{
"section_content": "PI3K inhibitors have been investigated as monotherapy (duvelisib) or in combination (idelalisib plus rituximab, umbralisib plus ublituximab) [50,51,109,110].However, no specific information on their efficacy in patients progressing on ibrutinib and/or venetoclax has been published so far, as patients who were pretreated with BTKi or venetoclax were excluded from most of these studies. In the setting of ibrutinib-resistant CLL mediated by PLCG2 mutations, in vitro analyses have shown that inhibition of SYK and LYN, both upstream of BTK, can overcome persistent survival signaling [111].In a recent phase 2 study, the SYK inhibitor entospletinib has produced responses in patients previously treated with B-cell receptor pathway inhibitors, even in patients harboring BTK and PLCG2 mutations [112].The overall response rate was however low (33%) and the progression-free survival was short.In the CLLRUmbrella2 study, a combination treatment of entospletinib and the BTK inhibitor tirabrutinib with or without the addition of obinutuzumab was evaluated in patients with relapsed/refractory CLL [113].The overall response rates at week 25 were 100% for the double combination and 90% for the triple combination, undetectable MRD was only achieved by 10% of patients in the triple combination group [113].Cerdulatinib, a SYK/JAK-STAT inhibitor has shown activity specifically in ibrutinib-resistant CLL samples [114].In the first in human study, the drug showed promising activity against CLL, although not in patients that had progressed on ibrutinib before starting cerdulatinib treatment [115]. The inhibition of other proteins within the B-cell receptor pathway has also produced encouraging results.PKCβ inhibitors as well as MALT1 inhibitors have been shown to effectively kill ibrutinib-resistant CLL cells in vitro, though clinical data on these drugs have not been published yet [116,117]. ",
"section_name": "Other B-Cell Receptor Pathway-Targeting Approaches",
"section_num": "6.2."
},
{
"section_content": "Based on the observations that venetoclax resistance appears to be mediated by an upregulation of the anti-apoptotic proteins Mcl-1 and to a lesser extent Bcl-X L these proteins are considered as promising targets to overcome resistance to Bcl-2 inhibition [23, 24,73,75].AMG-176, a direct Mcl-1 antagonist has shown activity in a preclinical setting by effectively killing CLL cells while sparing normal blood cells and showing synergy with venetoclax; however, a phase 1 study of the drug had to be suspended due to safety concerns [118].Another direct Mcl-1 inhibitor, AZD5991, is currently studied in a phase 1/2 trial after demonstrating potent antitumor activity in vitro and in preclinical models of acute myeloid leukemia (AML) and multiple myeloma [119].CDK9 is the transcriptional regulator of Mcl-1 expression and the CDK9 inhibitor voruciclib has indeed shown to effectively reduce Mcl-1 expression in preclinical studies, a phase 1 study in patients with B-cell malignancies or AML is currently recruiting [120,121].The above mentioned Bcl-X L degrader might also be a promising approach to target upregulation of anti-apoptotic proteins often observed under venetoclax treatment [102]. Another therapeutic approach is targeted by cirmtuzumab, which is a humanized monoclonal antibody targeting Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).ROR1 is highly expressed on CLL cells but not on normal tissue and it acts as a receptor for Wnt5a which was found to enhance CLL cell proliferation [122,123].The antibody has been evaluated in patients with relapsed/refractory CLL alone and in combination with ibrutinib [122,124,125].While single-agent cirmtuzumab has not produced any objective responses, it led to decreasing lymphocyte counts in the majority of patients [122].In combination with ibrutinib it showed an overall response rate of 67%.Whether this was solely attributable to the BTK inhibitor will be shown in a randomized phase 2 study of ibrutinib vs. ibrutinib plus cirmtuzumab that is currently ongoing. Another promising target on the B-cell surface is the receptor of the B-cell activating factor (BAFF-R), that appears to be constantly expressed throughout treatment with ibrutinib in contrast to CD20 [126].The anti-BAFF-R antibody VAY-736 was found to enhance antibody-dependent cellular toxicity and block BAFF-mediated survival signaling in preclinical models of CLL [126].In a phase 1 trial, VAY-736 was added to ibrutinib in patients with BTKi-specific resistance mutations (mainly BTK C481S) or insufficient responses to ibrutinib [127].Six of 15 (40%) patients, including patients with resistance mutations, achieved complete remissions and three patients achieved undetectable MRD, allowing for ibrutinib discontinuation [127]. Other drug candidates have yielded encouraging activity in preclinical studies but have not yet been tested in clinical trials.For instance, a bromodomain and extra-terminal (BET) protein inhibitor, GS-5829, has demonstrated preclinical activity in CLL [128].It effectively induced apoptosis and reduced proliferation in primary CLL cells while also inhibiting growth of NLCs, suggesting activity against the CLL-supportive microenvironment.Another BET inhibitor, JQ1, was shown to increase venetoclax-induced apoptotic effects in vitro and exhibit anti-tumor activity in venetoclax-resistant CLL cell lines [129].Preclinical activity was also shown for Histone Deacetylase 6 (HDAC6) inhibition in CLL cell lines and euTCL1 transgenic mouse models, leading to the development and clinical testing of the selective HDAC6 inhibitor ACY-1215 in relapsed/refractory lymphoid malignancies [130,131]. Bispecific antibodies that are able to simultaneously bind antigens on effector and malignant cells, have also shown antitumor activity in CLL.A novel bispecific antibody targeting CD3 and CD19 induced potent T-cell mediated killing of CLL cells in vitro and in a patient-derived xenograft mouse model, whereas the established anti-CD3/CD19 antibody blinatumomab failed to induce a response in the same mouse model [132].The bispecific anti-CD3 and anti-CD20 antibody epcoritamab has been shown to be highly active in different lymphoma entities ex vivo and is currently being evaluated within a clinical phase I trial for CLL patients with multiple relapses, as well as other lymphoma entities [133]. ",
"section_name": "Other Currently Investigated Non-Cellular Experimental Treatments",
"section_num": "6.3."
},
{
"section_content": "A steadily increasing number of patients with CLL is treated with novel agents, particularly BTK inhibitors and venetoclax with or without anti-CD20 antibodies.These patients will eventually relapse/progress and many of them will harbor resistance-conferring mutations, mostly in the genes encoding the target proteins of these novel agents.New treatment options are emerging for patients with resistance mutations, most prominently and currently most promising third-generation BTK inhibitors that appear to be effective after progression on ibrutinib and in patients with the most common acquired BTK mutations.However, most of these new options have not been tested in phase 3 trials yet and patients who sequentially progress on BTKi and venetoclax still pose a major challenge and are currently mostly offered cellular therapies. As BTK/PLCG2 and BCL2 mutations are frequently acquired rather late in the course of mostly single-agent treatment, time-limited combination approaches aiming at undetectable MRD might be an excellent way to avoid the development of resistance mutations.In the absence of resistance-conferring mutations, patients could be successfully retreated with the same regimen.Systematic genetic analyses of patients relapsing after time-limited combinations and results of retreatment studies will provide answers to the question if timelimited combination treatments are indeed superior or equal to continuous monotherapy.The development of new treatment approaches in order to overcome resistance to targeted agents will be one of the major challenges in CLL research in the chemotherapy-free era, which has already begun. ",
"section_name": "Conclusions",
"section_num": "7."
}
] |
[
{
"section_content": "Funding: This research received no external funding. Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Funding: This research received no external funding. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Author Contributions: M.F. and B.E. prepared the first draft and wrote, reviewed and edited the final manuscript.All authors have read and agreed to the published version of the manuscript. Conflicts of Interest: M.F. has nothing to disclose.B.E. reports grants and personal fees from Janssen-Cilag, Roche, Abbvie and Gilead, personal fees from Novartis, Celgene, ArQule, AstraZeneca and Oxford Biomedica (UK) and grants from BeiGene, all outside of the submitted work. ",
"section_name": "",
"section_num": ""
}
] |
10.1186/s13073-015-0252-1
|
Landscape of gene fusions in epithelial cancers: seq and ye shall find
|
Enabled by high-throughput sequencing approaches, epithelial cancers across a range of tissue types are seen to harbor gene fusions as integral to their landscape of somatic aberrations. Although many gene fusions are found at high frequency in several rare solid cancers, apart from fusions involving the ETS family of transcription factors which have been seen in approximately 50% of prostate cancers, several other common solid cancers have been shown to harbor recurrent gene fusions at low frequencies. On the other hand, many gene fusions involving oncogenes, such as those encoding ALK, RAF or FGFR kinase families, have been detected across multiple different epithelial carcinomas. Tumor-specific gene fusions can serve as diagnostic biomarkers or help define molecular subtypes of tumors; for example, gene fusions involving oncogenes such as ERG, ETV1, TFE3, NUT, POU5F1, NFIB, PLAG1, and PAX8 are diagnostically useful. Tumors with fusions involving therapeutically targetable genes such as ALK, RET, BRAF, RAF1, FGFR1-4, and NOTCH1-3 have immediate implications for precision medicine across tissue types. Thus, ongoing cancer genomic and transcriptomic analyses for clinical sequencing need to delineate the landscape of gene fusions. Prioritization of potential oncogenic "drivers" from "passenger" fusions, and functional characterization of potentially actionable gene fusions across diverse tissue types, will help translate these findings into clinical applications. Here, we review recent advances in gene fusion discovery and the prospects for medicine.
|
[
{
"section_content": "Recurrent chromosomal rearrangements in cancers have been described for over half a century [1,2].The characterization of the oncogenic fusion BCR-ABL1 at t (9,22) translocation loci in chronic myeloid leukemia, which culminated in the development of a molecularly targeted therapy, provides a compelling \"bench to bedside\" paradigm for cancers [3,4].Numerous gene fusions have since been defined at cytogenetically distinct loci of recurrent chromosomal aberrations in hematological malignancies and sarcomas, as well as in solid cancers, albeit much less frequently, arguably owing to technical limitations in resolving karyotypically complex, heterogeneous sub-clones in solid tumor tissues [5,6].The serendipitous discovery of ETS family gene fusions in common prostate carcinoma [7,8], and of ALK and ROS kinase fusions in lung cancer [9,10] through transcriptomic and proteomic approaches, bypassing chromosomal analyses, provided a strong fillip to the search for gene fusions in common solid cancers and pointed to alternative approaches to gene fusion discovery.Developments in high-throughput sequencing techniques over the past decade [11] have made possible a direct, systematic discovery of gene fusions in solid cancers [12][13][14], rapidly revealing a diverse genomic landscape.Gene fusions have now been identified in several common carcinomas, including those of the prostate, lung, breast, head and neck, brain, skin, gastrointestinal tract, and kidney, which alongside the widely documented gene fusions in thyroid and salivary gland tumors support the notion that gene fusions are integral to the genomic landscape of most cancers. Here, we review the emerging landscape of gene fusions across solid cancers, focusing on the recent spurt of discoveries made through sequencing.We review common features of \"driver\" fusions (those that contribute to tumor progression), the major functional classes of fusions that have been described, and their clinical, diagnostic and/or therapeutic implications. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "The first gene fusions to be defined in solid cancers, RET/PTC [15] and NTRK1 [16] rearrangements in papillary thyroid carcinoma were identified through a \"transformation assay\" using cancer genomic DNA transfected into murine NIH3T3 cells, followed by retrieval and analysis of human genomic DNA from transformed cells [17].More typically, karyotyping and cytogenetic analysis of recurrent translocations helped define early gene fusions in solid cancers, such as CTNNB1-PLAG1 [18] and HMGA2 fusions [19] in salivary gland pleomorphic adenomas, PRCC-TFE3 in renal cell carcinomas [20], and ETV6-NTRK3 fusion in secretory breast carcinoma [21].Incorporating more molecular approaches, a recurrent 2q13 breakpoint locus, t(2;3)(q13;p25), in follicular thyroid carcinoma was fine mapped using yeast artificial chromosomes, and cloned through 3′ rapid amplification of cDNA ends (RACE) of the candidate PAX8 cDNA, leading to characterization of the PAX8-PPARγ gene fusion [22].Anticipating high-throughput genomics approaches, an expressed sequence tag (EST) mapping to the recurrent chromosomal breakpoint at t(15;19)(q13;13.1) in midline carcinoma was identified from an EST database and cloned through RACE to identify the pathognomonic gene fusion BRD4-NUT [23].The gene fusions defined in solid cancers thus far were localized at cytogenetically distinct, recurrent chromosomal aberrations, and were largely confined to relatively rare subtypes of solid cancers [5]. However, between 2005 and 2007, independent of a priori evidence of genomic rearrangements, recurrent gene fusions involving ETS family genes were discovered in prostate cancer, based on analysis of genes displaying outlier expression [7,8,24].Around the same time, a transformation assay with a cDNA expression library (not genomic libraries [17]) from a lung adenocarcinoma sample led to the discovery of EML4-ALK fusions [10], and a high-throughput phosphotyrosine signaling screen of lung cancer cell lines and tumors identified SLC34A2-ROS1 fusions in non-small-cell lung carcinoma (NSCLC) [9].Thus, analyses of cancer RNA and proteins provided a critical breakthrough in the identification of oncogenic gene fusions in common carcinoma.In Fig. 1, we summarize the timeline of gene fusion discoveries, 100 years since Boveri's prescient hypothesis that malignant tumor growth is a consequence of chromosomal abnormalities, including \"combinations of chromosomes\" [25]. ",
"section_name": "Detection of gene fusions in carcinoma",
"section_num": null
},
{
"section_content": "High-throughput sequencing of tumor samples provides a direct readout of chimeric sequences corresponding to putative gene fusions, and the available depth of coverage helps uncover even relatively minor, sub-clonal events.In a proof of principle study, high-throughput genomic sequencing was used to identify several gene fusions in a panel of breast cancer cell lines and tissues [14].However, considering that only a small subset of genomic breakpoints correspond to gene fusions encoding fusion transcripts or proteins, alternative approaches were explored.In a directed approach, focusing on chimeric transcripts as the readout of \"expressed\" gene fusions, Maher and colleagues used coupled short-and long-read transcriptome sequencing [12] and paired-end transcriptome sequencing [13] to detect chimeric RNAs that could be analyzed to characterize gene fusions.RNA sequencing has since been widely used in the discovery of numerous gene fusions in diverse epithelial cancers.Additionally, paired-end tag [26] and chromatin interaction analysis by paired-end-tag sequencing have been employed for gene fusion discovery [27], as well as phosphoproteome analysis, as in the discovery of a SND1-BRAF fusion in a gastric carcinoma sample [28]. ",
"section_name": "Next-generation sequencing",
"section_num": null
},
{
"section_content": "1. Gene fusions are an integral component of the landscape of somatic aberrations in all cancers. 2. Recurrent 5′ fusion genes are generally lineage-and/or cell-type specific. 3. Recurrent 3′ fusion genes in epithelial cancers are usually kinases or transcription factors, similar to the situation in hematological and soft tissue cancers. 4. High-throughput sequencing enables systematic discovery of gene fusions with high sensitivity and precision. 5. High-throughput sequencing often identifies multiple gene fusions in individual samples, presenting a challenge to distinguish oncogenic \"driver\" from unimportant \"passenger\" aberrations. 6. Chimeric RNAs expressed independent of chromosomal rearrangements are frequently observed in cancer (and benign) tissues. 7. Functionally recurrent gene fusions provide clinically relevant molecular subclassifications of existing morphological categories of tumors.8. Functionally recurrent gene fusions that are seen across tissue types define functionally distinct molecular subtypes of cancers.9. Gene fusions represent personalized therapeutic targets and prognostic and diagnostic markers. The DNA-or protein-based methods, however, are not as commonly used as RNA sequencing, likely owing to several additional, specialized steps that are involved. Interestingly, RNA sequencing has also identified a class of chimeric RNAs that do not involve chromosomal aberrations.For example, \"read-through\" chimeric SLC45A3-ELK4 transcripts, such as those detected in prostate cancer, result from runaway transcription of the androgen-inducible, prostate-specific gene SLC45A3 into ELK4, the adjacent ETS family gene in the same orientation [12,[29][30][31].Similarly, the VTI1A-TCF7L2 fusion, originally identified through genomic sequencing of colorectal carcinoma (CRC) samples [32], was found in a follow-up study using RNA analyses to be quite prevalent in other cancers, as well as in benign samples [33].Chimeric transcripts not associated with genomic translocation have also been observed between non-contiguous genes.Guerra and colleagues identified CCND1-TACSTD2 (TROP2) chimeric mRNA that involves genes located on different chromosomes in subsets of ovarian, breast, gastrointestinal, and endometrial cancers [34].The functional significance of these RNA chimeras is not clear at present, as their expression is typically seen to be relatively non-specific. Fig. 1 Timeline of gene fusion discoveries.A timeline representation of salient gene fusion discoveries starting with 1914, the year that marked the publication of Boveri's monograph \"Zur Frage der Entstehung maligner Tumoren\", in which he proposed that aberrant \"combinations of chromosomes\" underlie malignant transformation [25].The top bar shows recurrent chromosomal rearrangements or gene fusions in hematological (purple) and soft tissue (green) malignancies, and the bottom bar shows gene fusions in relatively rare (blue) and those in common (red) epithelial cancers.ACC adenoid cystic carcinoma, AML acute myeloid leukemia, ALL acute lymphoblastic leukemia, APL acute promyelocytic leukemia, cholangio cholangiocarcinoma, CML chronic myeloid leukemia, CRC colorectal carcinoma, MLL mixed lineage leukemia, PLGA pediatric low grade astrocytoma, Ph Philadelphia chromosome ",
"section_name": "Box 1. Summary points",
"section_num": null
},
{
"section_content": "High-throughput sequencing of cancer samples frequently identifies multiple gene fusions in individual samples, often presenting a challenge for identifying potentially oncogenic driver fusions among irrelevant passenger aberrations.Some useful generalizations have emerged from multiple analyses: first, driver fusions are typically marked by a continuous open reading frame (ORF) that retains functional domains, such as the kinase domain in gene fusions involving oncogenic kinases, or DNA-binding domains in the case of transcription factors; second, some fusions display loss of auto-inhibitory domains (for example, loss of the N-terminal inhibitory domain in the product of BRAF fusions, or loss of 3′ UTR sequences in FGFR or HMGA2 fusions that serve as binding sites for inhibitory microRNAs).Yet other types of fusions juxtapose the promoter of certain tissue-specific, inducible or highly expressed genes; for example, the prostate-specific, androgen-inducible genes TMPRSS2 or SLC45A3 fused in frame with the proto-oncogenes ERG or BRAF, respectively, generate the TMPRSS2-ERG and SLC45A3-BRAF gene fusions in prostate cancer. In the case of novel gene fusions involving less characterized genes, distinguishing candidate driver fusions from random events is complicated by the many false positive candidates resulting from alignment artifacts, such as multi-mapping of reads owing to homologous (pseudogenes) and/or repetitive sequences, and sequencing artifacts due to errors in library generation (particularly ligation and PCR artifacts) and sequencing.Incorporating these considerations, and additional bioinformatics filters, various bioinformatics pipelines have been developed to help prioritize fusion candidates from next-generation sequencing (NGS) data, including Chimerascan [35], FusionSeq [36], DeFuse [37], TopHat-Fusion [38], PRADA [39], and JAFFA [40].While useful to help reduce the number of false candidates, the output from bioinformatics pipelines needs to be further validated, preferably followed by functional assays, before designating candidate gene fusions as novel driver aberrations.Recurrence of fusions, fusion partners or partner gene families in gene fusion databases also helps to prioritize candidate fusions.Once validated, screening for novel gene fusions in larger cohorts of samples employs quantitative RT-PCR or more recent techniques such as nano-string-based detection [41][42][43]. ",
"section_name": "Driver and passenger gene fusions",
"section_num": null
},
{
"section_content": "From the first reported chromosomal rearrangements in the 1960s up to the year 2000 (roughly marking the advent of high-throughput molecular techniques), the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer catalogued more than 600 \"recurrent balanced neoplasia-associated aberrations\", in which solid cancers accounted for less than 20 % [44]; in its latest update (7 May 2015), this database lists 10,004 \"gene fusions\" [45], with solid cancers accounting for a much greater proportion, and with a large number of these fusions identified by recent high-throughput gene expression or sequencing analyses.Over the last decade, numerous gene fusions have been characterized in diverse solid cancers, including ETS family gene fusions in prostate cancer [7,8,12,30,[46][47][48][49][50][51][52][53][54][55][56]; ALK, ROS1 and RET kinase fusions in lung cancer [9,10,[57][58][59][60][61][62][63][64][65][66][67][68][69]; RAF kinase fusions in brain tumors [70][71][72][73][74][75][76][77][78][79][80], melanoma [81,82], gastric cancer [28,82], and prostate cancer [82,83]; R-spondin fusions in colorectal and prostate cancer [83,84]; CD44-SLC1A2 gene fusions in gastric cancer [85]; MAST-and NOTCH-family gene fusions in breast cancer [86]; MITF gene fusions in renal cancer [87]; and a number of FGFR family fusions in diverse cancer types [88] (Table 1).More than 8000 gene fusions across 16 different tumor types are tabulated in The Cancer Genome Atlas (TCGA) Fusion gene Data Portal (http://www.tumorfusions.org)[89].The key points regarding gene fusions in epithelial cancers are summarized in Box 1. These gene fusions in solid cancers encompass the diversity of fusion architectures, as shown in Fig. 2 and Box 2, and represent a spectrum of functional categories, including those described earlier such as kinases and transcription factors, as well as those involving newer pathways and loss-of-function fusions (discussed later).Notably, even as numerous novel gene fusions are being discovered fairly rapidly, most of these are either non-recurrent singletons, or are seen to recur at exceedingly low frequency in tumor subtypes or to recur across tumor types (Table 1).Incidentally, gene fusions displaying molecular recurrence involving both 5′ and 3′ partner genes, as in TMPRSS2-ERG, EML4-ALK, and BRD4-NUT, are relatively few.A large number of fusions display recurrence of a fusion gene in combination with multiple different partners; for example, BRAF/RAF1 [76,79,82,83] and FGFR1/2/3 [88][89][90][91][92][93][94] are fused to several different 5′ partners across different tissue types (Additional file 1).This heterogeneity is likely reflective of the diverse tissue-physiological milieu in which these oncogenes impart selective advantage to the cancer cells.Conversely, some lineage-specific genes are seen to serve as 5′ partners across multiple different 3′ genes; for example, TMPRSS2 and SLC45A3 in prostate cancer have been observed as 5′ partners of ERG, ETV1, ETV4, ETV5, BRAF, and ELK4 (Table 1 and Additional file 1).Another type of observed \"recurrence\" involves isoforms of a gene familyfor example, ETV1/2/3/4/5, FGFR1/2/3, BRAF/RAF1, BRD3/4, CRTC1/CRTC3, and NTRK1/3as fusion partners.Considering that individual fusions may be observed relatively rarely (even uniquely), the potential functional consequences of gene fusions assumes priority over considerations of recurrence. ",
"section_name": "Overview of the landscape of gene fusions in epithelial cancers",
"section_num": null
},
{
"section_content": "Functionally distinct molecular classes of gene fusions that are shared across tumor types can be identified in solid cancers. ",
"section_name": "Functional consequences of gene fusions",
"section_num": null
},
{
"section_content": "Given their therapeutic importance, identification of gene fusions involving kinases can often signify a clinically actionable observation.Kinase fusion genes detected across multiple cancer types include RET, NTRK1, NTRK3, ALK, ROS1, FGFR1/2/3, and serine threonine kinases including the RAF family genes BRAF, RAF1, CRAF, and MAST1/2 (Table 1 and Additional file 1).In most gene fusions involving kinases, the kinase domain is retained [95], and this provides a strong filtering criterion in high-throughput sequencing data analysis.Analysis of mRNA sequencing data from the TCGA compendium, comprising 4366 primary tumor samples from 13 tissue types, revealed kinase fusions involving ALK, ROS, RET, NTRK, and FGFR gene families, which were detected in several types of cancer: bladder carcinoma (3.3 %), glioblastoma (4.4 %), head and neck cancer (1.0 %), low-grade glioma (1.5 %), lung adenocarcinoma (1.6 %), lung squamous cell carcinoma (2.3 %), and thyroid carcinoma (8.7 %) [89]. ",
"section_name": "Kinases",
"section_num": null
},
{
"section_content": "Gene fusions involving dysregulated expression of transcription factors include ETS family gene fusions, seen in approximately 50 % of all prostate cancers and probably one of the most prevalent transcription factor gene fusions in common epithelial cancers.Among these, ERG represents the most common fusion partner and ETV1 the most promiscuous, with a dozen or more different fusion partners described to date (Additional file 1) [24,96]. Other gene fusions involving transcription factors include NUT (or NUTM1), POU5F1, MAML2, NFIB, PLAG1, TFE3, NOTCH, and PAX8 fusions, imparting spatially and/or stochastically dysregulated expression in multiple different cancer types.NOTCH1 and NOTCH2 fusions result in dysregulated transcriptional outcomes, because after ligand activation, the NOTCH intracellular domain (NICD) forms a transcriptional activator complex, activating genes involved in differentiation, proliferation and apoptosis, and those associated with carcinogenesis.MAML2 acts as a transcriptional coactivator for NOTCH proteins by amplifying NOTCHinduced transcription of HES1.TFE3, which belongs to the MITF/TFE family of basic helix-loop-helix leucine zipper transcription factors, is involved in TGF-βinduced transcription, and has important roles in cell ",
"section_name": "Transcription factors",
"section_num": null
},
{
"section_content": "An overview of the genomic architecture of gene fusions reveals that fusions may result from insertion, deletion, inversion, or tandem duplication or amplification, and may involve the same chromosome (intra-chromosomal) or different chromosomes (inter-chromosomal) (Fig. 2).A majority of chromosomal rearrangements have been associated with intra-chromosomal tandem duplications and amplifications in multiple whole-genome sequencing studies [14,26,80,150].Micro-homologies and repeat elements have been associated with loci of recurrent break points [151].In an analysis of RAF family gene fusion breakpoints in low-grade astrocytomas, tandem duplications generated by microhomology-mediated break-induced replication were identified as the mechanism of generation of fusions [74]. Spatial proximity between distant chromosomal loci has been associated with chromosomal rearrangements, as observed between RET and the H4 genes located 30 megabases (Mb) apart on chromosome 10, involved in RET gene fusions in papillary thyroid carcinoma [152].This proximity may be induced by genotoxic stress; for example, androgen stimulation coupled with the genotoxic stress of radiation was shown to generate gene fusions through \"induced proximity\" between TMPRSS2 and ERG (located on chromosome 21q22.2,approximately 3 Mb apart) as well as between TPMRSS2 and ETV1 (located on chromosome 7) [153,154] (Fig. 3a). Another phenomenon, called chromothripsis, describes the frequent occurrence of massive chromosomal aberrations localized to only one or two chromosomes, with fragments of chromosome joined randomly [155,156].Chromothripsis may be responsible for the generation of numerous, apparently random passenger gene fusions that are retained in the multiclonal cells of epithelial cancers, as well as loss-offunction fusions involving tumor suppressors, likely involving the non-homologous end-joining DNA repair system (Fig. 3b). Several cancer-causing viruses, such as Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human papilloma virus (HPV), hepatitis B and C viruses (HBV and HCV), and Merkel cell polyomavirus (MCV), integrate into human genomic DNA at defined hotspots as well as seemingly randomly [157].Viral integration events have been associated with chromosomal aberrations, such as MYC amplification in HPVpositive genital carcinoma [158], and not uncommonly, loss of gene function [159,160] or gene fusions involving viral-human sequences have been reported [161,162].The recent report of a recurrent gene fusion of UBR5 on 8q22.3 and ZNF423 on 16q12.1 (UBR5-ZNF423) in 8 % of EBVassociated primary nasopharyngeal carcinomas suggests a driver function of this gene fusion in a subset of nasopharyngeal cancers [163].The fusion retains all the functional domains of HMGA2, and removes the 3′ UTR sequence that contains several inhibitory let7 microRNA binding sites.Absence of the Let-7-regulated 3′ UTR in the fusion transcript results in overexpression of HMGA2 that is sufficient for neoplastic transformation [19] FGFR-PLAG1 FGFR is the 5′ partner, which, without its kinase domain, provides the promoter to drive the expression of the 3′ partner, PLAG1 This FGFR fusion product does not include the FGFR kinase domain, and therefore is not a target for FGFR inhibitors [91] Adenoid cystic carcinomas (salivary glands, lacrimal glands, ceruminal glands; also breast) FDA Food and Drug Administration, FTC follicular thyroid carcinoma, GBM glioblastoma multiforme, MASC mammary analog secretory carcinoma of salivary glands, MEC mucoepidermoid carcinoma, nccRCC non-clear-cell renal cell carcinoma, NMC NUT midline carcinoma, NSCLC non-small-cell lung carcinoma, PTC papillary thyroid cancer, RCC renal cell carcinoma, RMC renal medullary carcinoma, TF transcription factor growth and proliferation.TFE3 is involved chromosomal translocations that result in various gene fusions (such as PRCC-TFE3, RCC17-TFE3, PSF-TFE3, NON-O(p54nrb)-TFE3 and ASPL-TFE3) in papillary renal cell carcinomas.PLAG1 is an oncogenic transcription factor associated with the neoplastic transformation of pleomorphic adenomas of the salivary gland and lipoblastomas through upregulation of IGF2, CRLF1, CRABP2, CRIP2, and PIGF.NFIB binds viral and cellular promoters activating transcription and replication.POU5F1 and PAX8 are homeobox-containing transcription factors, a family of genes that play a role in cell fate and differentiation programs, and whose role in cancer is well recognized, particularly PAX8 in thyroid cancer [22]. ",
"section_name": "Box 2. Mechanisms of generation of gene fusions",
"section_num": null
},
{
"section_content": "",
"section_name": "MYB-NFIB",
"section_num": null
},
{
"section_content": "CD44-SLC1A2/EAAT2 gene fusions are detected in 1-2 % of gastric cancers involving the glutamate transporter SLC1A2 [85], and cause intracellular accumulation of glutamate, a growth-promoting amino acid associated with oncogenic functions [97,98].Thus, this gene fusion may be establishing a pro-oncogenic metabolic milieu, akin to the increased levels of sarcosine reported in prostate cancer [99]. Wnt/β-catenin signaling pathway RNA sequencing of 68 \"microsatellite stable\" subtype colorectal cancer samples revealed two recurrent fusions The likely mechanisms of chimera generation are indicated.Chr chromosome involving R-spondin family genes, EIF3E-RSPO2 in two cases and PTPRK-RSPO3 in five cases [84].these gene fusions retained the functional domain of the Rspondins that are known to be agonists of the canonical Wnt/β-catenin signaling pathway.Additionally, the LACTB2-NCOA2 chimeric transcript detected in 6 of 99 (6.1 %) colorectal cancer cases led to disruption of NCOA2 expression, thus activating the Wnt/β-catenin pathway [100].Recently, R-spondin fusions such as GRHL2-RSPO2 were described in prostate cancer as well [83]. ",
"section_name": "Other functional classes Metabolic enzymes",
"section_num": null
},
{
"section_content": "Recently, fusions involving SKIL (which encodes a SMAD inhibitor) 3′ to androgen-regulated promoters such as TMPRSS2, SLC45A3, and ACPP, were found in 6 of 540 (1.1 %) prostate cancers and one cell line xenograft, LuCaP-77 [101].SKIL overexpression in these tumors was associated with upregulation of the TGF-β pathway, likely providing the oncogenic mechanism in these tumors. ",
"section_name": "TGF-β pathway",
"section_num": null
},
{
"section_content": "In an analysis of fusion transcripts observed in TCGA data across multiple tumor types, fusions involving chromatin modifier genes, including histone methyltransferase and histone demethylase genes, were identified in 111 samples (2.5 %) [89].Chromatin modifier genes are potential therapeutic targets and these gene fusions thus represent a novel class of potentially actionable aberrations. ",
"section_name": "Chromatin modifier genes",
"section_num": null
},
{
"section_content": "Additional classes of genes represented among recurrent fusions in solid cancers include those encoding growth factor receptors (GABBR2, TACSTD2, ITPR2), adaptors and co-factors (WIF1, GAB2), Ras-Gap proteins (DOCK5, ARHGAP15), and cytoskeletal proteins (SNF8, SEC22B, HIP1R, STXBP4, MYO19, TPR).Although some of these fusions are scored as recurrent, they may represent passenger mutations associated with loci of recurrent chromosomal aberrations, while others may define tissuespecific or cooperative roles. ",
"section_name": "Further functional classes",
"section_num": null
},
{
"section_content": "While most reported gene fusions pertain to gain-offunction aberrations imparting neoplastic phenotypes, with high-throughput sequencing, fusions resulting in loss of function of tumor suppressors such as TP53 and PTEN have been identified as well [102].The LACTB2-NCOA2 fusion in colorectal cancer leads to disruption of NCOA2, which encodes an inhibitor of the Wnt/β-catenin pathway [100], thus acting to promote carcinogenesis. ",
"section_name": "Loss-of-function gene fusions",
"section_num": null
},
{
"section_content": "Some gene fusions are associated with distinct subtypes of carcinoma, while others have been detected across different tissues or lineages, defining molecular subsets of cancers transcending morphological distinctions. ",
"section_name": "Gene fusion signatures in personalized medicine of epithelial cancers",
"section_num": null
},
{
"section_content": "Some of the salient gene fusions that define molecular subtypes of epithelial within specific organs or tissue types are summarized in Table 1.The ETV6-NTRK3 fusion is a diagnostic biomarker of secretory breast carcinoma, as well as the acinic cell carcinoma or cystadenocarcinoma recently designated as \"mammary analog secretory carcinoma of salivary glands\" (MASC) [21,103].BRD-NUT fusions define NUT midline carcinoma [104,105].CRTC-MAML2 fusions are the defining molecular aberration of mucoepidermoid carcinoma (MEC) [106,107]; translocation-negative MECs are proposed to be designated as a distinct subgroup of adenosquamous carcinoma [108].CRTC-MAML fusions are also found in MEC of the lung [109][110][111][112], cervix [113], thyroid glands and oral cavity [114], as well as in clear cell hidradenoma of the skin [115,116].In all cases, MAML2 fusions characterize benign or low-grade tumors, and for reasons not described so far have been associated with a favorable prognosis [117].Interestingly, pulmonary MECs have shown clinical response to gefitinib in the absence of sensitizing EGFR mutations, suggesting a potential connection with CRTC-MAML2 and the possibility of therapeutic application in other MECs harboring this fusion [110,118].The diagnostic subclass of adenoid cystic carcinomas, including salivary gland and breast cancer, is characterized by MYB-NFIB gene fusions [119,120].Fusions defining subtypes within a cancer include RET and NTRK gene fusions in subsets of papillary thyroid carcinoma [121], while PAX8-PPARγ fusions characterize subsets of follicular thyroid carcinoma [22,122].ETS family gene fusions, primarily including ERG (and less frequently, ETV1, ETV4, ETV5 or FLI1), are found in approximately 50 % of prostate cancers, the most common fusion being TMPRSS2-ERG.The EWSR1-ATF1 fusion found in hyalinizing clear cell carcinoma of the salivary glands, a rare and indolent tumor, can potentially be used as a molecular marker of this subtype that is histologically similar to the more aggressive MEC [123].Gene fusions or fusion partners found across tissue types are common in solid cancers.The EML4-ALK fusion, initially identified in lung cancer [9,10] has since been reported in breast cancer [124], colorectal carcinomas [66,124], and in pediatric renal medullary carcinoma that affects young African-Americans with the sickle cell trait [125,126].Similarly, RET fusions, first characterized in thyroid cancer, are widely observed in lung cancers, and the EWSR1-POU5F1 fusion was detected in two rare epithelial tumors, hidradenoma of the skin and MEC of the salivary glands [127]. Gene fusions involving RAF kinase genes (BRAF, RAF1, CRAF) have been identified in low-grade tumors of the central nervous system (pilocytic astrocytomas and other low-grade gliomas), gastric cancer, melanoma and prostate cancer.RAF family fusions involve truncation of the N-terminal auto-inhibitory domain, thus generating constitutively active RAF protein.Curiously, BRAF gene fusions in low-grade astrocytomas have been associated with a tendency to growth arrest, conferring a less aggressive clinical phenotype and a better clinical outcome [75,128].Additionally, RAF family fusions have been defined across diverse solid cancers, including prostate, gastric, and skin cancers [82,83].A screen for BRAF gene fusions in 20,573 solid tumors, using the Foun-dationOne™ targeted gene panel, identified BRAF fusions involving 29 unique 5′ fusion partners in 55 (0.3 %) cases across 12 different tumor types, including 3 % (14/531) of melanomas, 2 % (15/701) of gliomas, 1.0 % (3/294) of thyroid cancers, 0.3 % (3/ 1,062) of pancreatic carcinomas, 0.2 % (8/4,013) of non-small cell lung cancers and 0.2 % (4/2,154) of colorectal cancers, as well as single cases of head and neck cancer, prostate cancer, rectal adenocarcinoma, ovarian, uterine endometrial, and mesothelioma [70]. Fusions involving FGFR tyrosine kinase family genes have also been observed across diverse cancers [88].The first FGFR fusion observed in epithelial cancers, FGFR1-PLAG1, was found in a subset of pleomorphic salivary gland adenomas, and involves FGFR1 as the 5′ partner upstream of PLAG1, the known driver of salivary gland tumors [91].Curiously, this fusion excludes the tyrosine kinase domain of FGFR.Fusions that retain the tyrosine kinase domain of FGFR include FGFR3-TACC3 in glioblastoma [92,129].Subsequently, diverse FGFR fusions, all retaining the tyrosine kinase domain, have been observed in bladder, lung, breast, thyroid, oral, and prostate cancers, involving FGFR1, 2, or 3 either as the 5′ or 3′ partners [88,94]. ",
"section_name": "Recurrent gene fusions as biomarkers of subtypes of solid cancers",
"section_num": null
},
{
"section_content": "In Additional file 2 we summarize recent clinical trials involving gene fusions in epithelial cancers.The RET inhibitor vandetanib shows antiproliferative activity in RET-mutant medullary thyroid cancer (MTC) [130], and was recently approved by the US Food and Drug Administration for treatment of metastatic MTC.Sensitivity to vandetanib was also observed in RET-fusionpositive papillary thyroid carcinoma [131] and lung cancer cells [68,132].Treatment with Pfizer's kinase inhibitor crizotinib (PF02341066) led to a dramatic clinical response in EML4-ALK-positive NSCLC patients [133,134], as well as in one patient with an SLC34A2-ROS1fusion-positive tumor [58].Unfortunately, resistance is inevitably observed, owing mutations in the kinase domain [134,135], or ALK gene fusion amplification, KIT amplification or increased auto-phosphorylation of EGFR [136].This is representative of the challenge of treating solid cancers and argues for the development of combinatorial therapeutic approaches from the start rather than sequentially, as is the practice currently.RAF or MEK inhibitors represent potential precision therapeutic options for several solid cancers with the diverse RAF family gene fusions described earlier.Several FGFR inhibitors currently in clinical trials represent potential therapeutics for cancers harboring FGFR fusions across multiple cancer types, including bladder cancer, prostate cancer, and others [88,90,94,137].The rare PIK3C family gene fusions in prostate cancer (for example, TBXLR1-PIK3CA and ACPP-PIK3CB) show overexpression of the PI3KC genes and may be sensitive to PIK3CA inhibitors [83]. For treatment of secretory breast carcinoma expressing the ETV6-NTRK3 fusion, therapeutic targeting of the downstream signaling axis of IGF1R, using the IGIFR/INSR kinase inhibitors BMS-536924 and BMS-754807 that are currently in clinical trials, was found to be effective [138].Breast cancer cells expressing NOTCH fusion products that retain the γ-secretase cleavage site were sensitive to γ-secretase inhibitor (GSI) in culture, and treatment with GSI reduced tumor growth in vivo [86].On the other hand, breast cancer cells harboring NOTCH fusions that encode NICD independent of the γ-secretase cleavage site were insensitive to GSI. In a recent clinical sequencing study of 102 pediatric cancers, among 37 non-sarcoma solid cancers, several functional gene fusions were identified, including TFE3 fusions in a colorectal cancer (SFPQ-TFE3) and renal cell cancer (ASPSCR1-TFE3)both cases were treated with pazopanib, the latter displaying stable disease for 10 months [139]. Efforts to target several other gene fusions are underway.The newly developed bromodomain inhibitors that have shown dramatic efficacy in hematological malignancies [140,141] are now being tested in multiple clinical trials for NUT midline carcinoma characterized by BRD3/4-NUT gene fusions, which represent a rare but highly aggressive class of tumors with no effective treatment currently available [104].Also, the R-spondin fusions observed in colorectal and prostate cancer may be sensitive to Wnt pathway antagonist porcupine inhibitors [142]. Gene fusions involving ETS transcription factors have been utilized in diagnostic applications.A non-invasive assay system has been developed based on the detection of TMPRSS2-ERG fusion transcripts in urine samples from patients, which in combination with the detection of urine PCA3 improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator in predicting cancer on biopsy [143].Detection of TMPRSS2-ERG in circulating tumor cells in therapynaive patients and in castration-resistant prostate cancer patients following treatment suggests potential applications in non-invasive monitoring of the therapeutic response [144].While therapeutic targeting of transcription factor oncogenes is intrinsically challenging, on the basis of the interaction of ERG with the DNA repair enzyme PARP1 and DNA protein kinase DNA-PKc, use of PARP inhibitors was shown to inhibit growth of TMPRSS2-ERG-positive prostate cancer xenografts [145].Additionally, PARP inhibition was associated with radiosensitization of TMPRSS2-ERG-positive prostate cancer cells [146,147].These experimental leads point to possible therapeutic avenues targeting a prevalent gene fusion in a common carcinoma. ",
"section_name": "Some gene fusions provide personalized therapeutic targets",
"section_num": null
},
{
"section_content": "Genomic or transcriptomic sequencing has virtually supplanted molecular and cytogenetic techniques as the primary modality for discovery of gene fusions, and detection of gene fusions is increasingly incorporated into the standard workflow for genomic characterization of tumors in both research and clinical settings.Transcriptome sequencing has been useful in helping to identify expressed gene fusions based on evidence of the fusion of exon boundaries, but putative promoter fusions that do not generate chimeric transcripts are likely to go undetected.Furthermore, typically recurrent gene fusions characterized in cancers represent gain-of-function events arising from the juxtaposition of cell-type-or lineage-specific regulatory elements and proto-oncogenes, or novel combinations of functional domains derived from two proteins that provide combinatorial or additive functionalities to normal genes.However, NGS data also reveal less frequently described loss-of-function chimeras involving tumor suppressor genes such as TP53, PTEN, and others.A systematic analysis of loss-of-function gene fusions could identify additional cancer samples with loss of tumor suppressors that might be currently going unreported, and could help broaden our understanding of the role of gene fusions in cancer. The rapid increase in detection of gene fusions across cancers has spawned multiple discovery and prioritization pipelines to help distinguish bona fide functional gene fusions from random chimeras (and experimental artifacts).However, the development of diverse pipelines following different analysis parameters underscores a need for standardization of the vocabulary and information content in recording and reporting gene fusions, along the lines of the Minimum Information About a Microarray Experiment [148,149].Furthermore, even as bioinformatics analyses help prioritize fusion candidates, the \"recurrence\" of fusion genes and/or retention of functional domains provide the most compelling rationale for functional characterization. The detection of distinct gene fusions across subtypes of common carcinoma also provides a basis for molecular subclassification of these cancers.Recurrent gene fusions that characterize distinct subtypes of cancers include BRD4-NUT in NUT midline carcinoma, ETV6-NTRK3 in secretory breast carcinoma, CRTC-MAML2 fusions in mucoepidermoid carcinoma, and RAF family fusions in pilocytic astrocytomas.It is expected that as more and more carcinomas are analyzed by sequencing, additional subclasses may be recognized on the basis of whether the detected molecular aberrations are driver fusions.Importantly, the emerging landscape of gene fusions in solid cancers also reveals many gene fusions involving oncogene families or isoforms that are seen across multiple tumor types or subtypes, for example, fusions involving RAF and FGFR family genes.This supports the notion that a molecular classification of tumors in terms of driver fusions (or SNVs) may complement histopathological descriptions. Many oncogenes involved in gene fusions (for example, RET, BRAF, ALK, NOTCH or PIK3CA/B) are also known to harbor activating mutations.However, fusions and mutations tend to be mutually exclusive.This indicates that either fusions or activating mutations can independently provide oncogenic function, and that either of these aberrations may render the tumors sensitive to therapeutic targeting.Thus, for example, MEK inhibitors that have been found to be useful for tumors with a BRAF activating mutation may also benefit tumors with the BRAF fusion. The development of technologies that enable the systematic detection of molecular aberrations in cancer has profound clinical implications, as high-throughput sequencing of individual tumor samples is expected to become available as a routine diagnostic modality (as for whole-body PET scans or MRI) in the not-too-distant future.Considering the important diagnostic and therapeutic implications, the integration of approaches for the detection of driver gene fusions into cancer genomics pipelines is crucial for precision cancer medicine. ",
"section_name": "Perspectives and discussion",
"section_num": null
}
] |
[
{
"section_content": "We thank Robin Kunkel for help with the artwork for the figures.AMC is supported by the Doris Duke Charitable Foundation Clinical Scientist Award and the Prostate Cancer Foundation.AMC is an American Cancer Society Research Professor and A. Alfred Taubman Scholar.This work was supported in part by the US National Institutes of Health (R01CA132874), Early Detection Research Network grant UO1 CA111275, Prostate SPORE grant P50CA69568, the Department of Defense Era of Hope grant BC075023 (AMC). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Additional file 1: Recurrent gene fusions in epithelial cancers.Summary of recurrent gene fusions in epithelial carcinoma across different tissues.a Gene fusions with common 5′ and 3′ genes. The University of Michigan has filed for a patent on recurrent gene fusions in prostate cancer and AMC is named as a co-inventor.The technology has been licensed to Hologic Inc. to develop a molecular diagnostic. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Recurrent gene fusions in epithelial cancers.Summary of recurrent gene fusions in epithelial carcinoma across different tissues.a Gene fusions with common 5′ and 3′ genes. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "The University of Michigan has filed for a patent on recurrent gene fusions in prostate cancer and AMC is named as a co-inventor.The technology has been licensed to Hologic Inc. to develop a molecular diagnostic. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.5167/uzh-13911
|
EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer
|
The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.
|
[
{
"section_content": "The Notch1 gene plays an important role in mammalian cell fate decision and tumorigenesis.Upstream control mechanisms for transcription of this gene are still poorly understood.In a chemical genetics screen for small molecule activators of Notch signaling, we identified Epidermal Growth Factor Receptor (EGFR) as a key negative regulator of Notch1 gene expression in normal human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs).The underlying mechanism for negative control of the Notch1 gene in the human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun.Suppression of Notch signaling in cancer cells counteracts the differentiation inducing effects of EGFR inhibitors, while, at the same time, synergizing with these compounds in induction of apoptosis.Thus, our data reveal a novel role of EGFR signaling in negative regulation of Notch1 gene transcription, of potential relevance for novel combinatory approaches of cancer therapy. Cell growth and differentiation are controlled by a complex interplay of signaling pathways functioning in an \"integrated\", rather than sequential or parallel fashion.Chemical Genetics is based on the principle of using small molecular weight compounds to abrogate or enhance specific regulatory pathways, providing a powerful approach to analyze complex regulatory systems.In the present study we utilized this approach to probe into the signaling network involved in control of Notch1 gene expression and function in human keratinocytes, skin and tumors.Notch signaling plays a key role in promoting keratinocyte differentiation and suppressing keratinocyte-derived tumors 1,2 .Notch receptors, with Notch1 and 2 being the main forms expressed in keratinocytes, are processed by a Ca 2+ -dependent protease in the Golgi prior to transport to the cell membrane.Upon interaction with transmembrane ligands (Jagged1 and 2 or Delta1-4) expressed on the surface of neighboring cells, Notch receptors are activated by consecutive cleavage by an ADAM metalloprotease and a presenilin/-secretase complex.The resulting Notch intracellular domain (ICN) translocates to the nucleus where it associates with the DNA binding protein CSL (CBF-1 or RBP-J in mammalian cells, converting it from a repressor into an activator of transcription 3 .Binding of a second ancillary protein, Mastermind-like 1 (MAML1) or related family members, is required for sustained levels of Notch/CSL-dependent transcriptional activation (through recruitment of further transcription co-activators such as p300) 4 . A 51 amino acid peptide (MAM51) corresponding to the amino terminal region (aa 13-74) of the MAML1 protein competes for MAML1 binding to the Notch/CSL complex, thereby preventing downstream transcription 5 .We recently showed that, in primary human keratinocytes, suppression of Notch signaling by this approach results in a lesser commitment to differentiation, expansion of stem cell populations and dramatically increased susceptibility to ras-induced oncogenic conversion 2 .Similar effects were observed after pharmacological suppression of endogenous Notch activity by a -secretase inhibitor 2 .These findings are likely to be of clinical significance, as Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under positive p53 control in these cells 2,6 . EGFR is among the most intensely studied and well understood determinants of epithelial cell proliferation, and EGFR inhibitors have surfaced as an outstanding example of rational-based drug design for tumors 7 .EGFR signaling is likely to function in the keratinocyte proliferative compartment of the epidermis as a \"built-in\" mechanism to maintain self renewal and, at the same time, suppress differentiation, in contrast to the upper layers where this pathway is down-modulated.In fact, abrogation of EGFR/ERK signaling in proliferating keratinocytes, by either chemical or genetic manipulations, induces differentiation, while sustained activation of this pathway, under conditions where it is normally down modulated, suppresses differentiation 8,9 .This has potentially important implications for keratinocyte-derived tumors, where EGFR signaling is persistently activated and promotes proliferation 10 . At the biochemical level, little is known on the link between EGFR/ERK pathways and control of differentiation.Here we report a novel role of this pathway in negative regulation of Notch1 gene transcription in both normally proliferating keratinocytes and cancer, which impinges on control of differentiation as well as apoptosis. ",
"section_name": "Abstract",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Little is known of pathways involved in upstream control of Notch1 gene expression and activity in keratinocytes, and mammalian cells in general.To address this issue we undertook a chemical genetics approach.Rather than screening a large collection of unknown chemicals, we chose a library of 489 compounds, approved by the Food and Drug Administration (FDA) and of established target selectivity, using a Luciferase Notch/CSL-responsive reporter as a read-out.The negative regulators of Notch signaling identified by this screen included inhibitors of metalloproteases (MMP) and -secretase, which are required for endogenous Notch activation 4 , confirming the validity of the assay (suppl.Table I).Statistical analysis of the results pointed to a number of other candidate pathways.In particular, the most significant compounds to induce Notch activity were kinase inhibitors that target components of signaling networks connected with EGFR signaling, which was of special interest, given the relevance of this pathway in keratinocytes and cancer 11,12 . To validate the findings of our screen, we compared the effects of EGFR inhibition and stimulation on endogenous Notch signaling in human primary keratinocytes.A dose-response of human primary keratinocytes to the EGFR inhibitor AG1478 was determined, on the basis of down-modulation of phospho-EGFR, -ERK1/2, -Jun and -Elk levels, as well as c-Fos expression (Supplemental Fig. 1).At the same doses, there was induction of the \"canonical\" Notch target genes Hes1, Hes5 and Herp1, while, conversely, EGF treatment suppressed expression of these genes (Fig. 1A).In parallel with this effect, Notch1 mRNA levels were increased by EGFR inhibition, while they were down-regulated by EGF treatment (Fig. 1B).Consistent with a transcriptional mechanism, no increase of Notch1 mRNA stability was observed in EGFR inhibitor-treated cells after Actinomycin D treatment (data not shown).The results were confirmed at the protein level, by immunoblotting of AG1478-and EGF-treated keratinocytes with antibodies against total and cytoplasmic activated forms of Notch1 as well as Hes1 (Fig. 1C).Effects similar to those of AG1478 were also elicited by Tarceva, an EGFR inhibitor approved for clinical use (Collins&Workman, Nat Chem Biol 2006) (Supplemental Fig. 2). Besides chemical inhibition, up-regulation of Notch1 activity and expression were also observed after knockdown of EGFR expression by transfection of keratinocytes with specific siRNAs (Fig. 1D).Unlike Notch1, Notch2 expression was modulated by EGFR signaling at the mRNA but not protein level (Suppl.Fig. 3A; Fig. 1C), while no consistent changes were found in expression of the Notch ligands Jagged 1 and Delta like 1 (Suppl.Fig. 3B,C).expression is only an indirect consequence of these events.However, treatment of keratinocytes with TNF- at pro-apoptotic concentrations had no effects on levels of Notch1 expression, which was also not affected by suppression of keratinocyte growth by TGF-ß treatment (Supplemental Fig. 2) or, as we recently reported, increased expression of cyclin/CDK inhibitors (ANNA's paper). The ERK1/2 kinases and the AP-1 transcription complex function as downstream effectors of EGFR activation 11 .Induction of Notch1 gene expression similar to that caused by EGFR suppression was observed after siRNA-mediated knockdown of the MEK1 and ERK1 genes while, consistent with their proposed distinct function in keratinocytes (Khavari Cancer Res.2004 where they show that Only Mek1, however, recapitulated Ras/Raf effects in increasing proliferation and integrin expression while suppressing differentiation, which are impacts characteristic of epidermal neoplasia.),knockdown of MEK2 or ERK2 had no such effect (Fig. 1E). In contrast to MEK1 and ERK1, no increase of Notch1 expression, or even suppression, was also observed after knock-down and/or pharmacological inhibition of the p38 and JNK kinases, Akt and PKA (Fig. 1E and Supplemental Fig. 2). Induction of Notch1 expression similar to that caused by EGFR and ERK suppression occurred also after knockdown of c-Jun and c-Fos, two key AP-1 family members (Fig. 1 F,G).Even in this case, the effects were specific, as they were not observed after knockdown of other AP-1 family members like JunB, Jun D and Fra1, nor of Elk-1, a transcription factor which is activated by EGFR activation though a separate mechanism from AP1 (Friday&Adjei, 2008 Clin Cancer Res.) (Fig. 1F). ",
"section_name": "Negative regulation of Notch1 gene expression by EGFR/ERK signaling.",
"section_num": null
},
{
"section_content": "We and others recently showed that the Notch1 gene is a direct transcriptional target of p53 in keratinocytes 2,6 ADD MANDINOVA.Consistent with these previous results, our chemical screen pointed to a p53 inhibitor, pifithrin, as a negative regulator of Notch signaling (suppl.Table I), a finding which we directly confirmed by treating keratinocytes with this compound (data not shown).We therefore surmised that a p53-dependent mechanism may underlie up-regulation of Notch1 expression by EGFR suppression.To test this possibility, p53 expression was suppressed in primary keratinocytes by siRNA knock-down.This resulted in reduced levels of Notch1 expression already under basal conditions and, much more substantially, in response to EGFR knock-down (Fig. 2A).Consistent with a p53-dependent transcriptional control mechanism, luciferase reporter activity of a 2.4 kbp Notch1 promoter region containing p53 binding sites (but not of a shorter region lacking these sites) was induced in HKCs after EGFR inhibition, with such induction being abrogated by p53 knock-down (Fig. 2B).Endogenous p53 activity, as assessed by expression of well-established target genes, p21 WAF1/Cip1 and Gadd45 13 , was induced as a consequence of EGFR inhibition (Fig. 2C).There was also a substantial increase of Mdm2, a negative regulator of p53 stability and itself a p53 target gene 13 (Fig. 2D).Consistent with the negative feedback loop between p53 and Mdm2 protein expression, induction of p53 protein expression by AG1478 became much more evident in cells concomitantly treated with Nutlin, an Mdm2 inhibitor 14 (Fig. 2E). Emerging evidence points to the importance of control of p53 activity by transcription of this gene ( 15-17 and refs. therein).Consistent with this possibility, real time RT-PCR analysis showed that p53 mRNA levels were significantly increased as a consequence of EGFR inhibition while, conversely, were reduced by EGF treatment (Fig. 2F).Previous work with mouse embryonic fibroblasts indicated that the p53 gene can be a direct target of c-Jun-mediated transcriptional suppression 18 .In agreement with this conclusion, chromatin immuno-precipitation experiments showed specific binding of the endogenous c-Jun protein to a predicted AP-1 binding region of the p53 promoter in control but not EGFR-inhibitor-treated cells (Fig. 2G). Functionally, p53 promoter activity (Fig. 2H) as well as endogenous p53 RNA expression (Fig. 2I) was induced by siRNA-mediated knockdown of c-Jun, which, as shown before, induces Notch1 expression.A link between the two was demonstrated by the fact that induction of Notch1 expression by c-Jun knock-down was blocked by the concomitant down-modulation of p53 expression (Fig. 2J). ",
"section_name": "Modulation of Notch1 gene transcription by EGFR signaling through p53.",
"section_num": null
},
{
"section_content": "EGFR signaling provides a break to differentiation, while increased Notch activity promotes this process 1 .Real time RT-PCR as well as immunoblot analysis showed that down-modulation of EGFR signaling, by either transfection with siRNAs against EGFR or AG1478 treatment, induced expression of several terminal differentiation markers in keratinocytes, including Keratin1 and 10 and Involucrin (supplemental Fig. 4 and data not shown).To assess whether induction of differentiation by EGFR suppression is due to up-regulation of Notch signaling, primary human keratinocytes were infected with a retroviral vector expressing a 51 amino acid peptide (MAM51) that competes for MAML1 binding to the Notch/CBF-1 complex, thereby preventing downstream transcription 5 .Treatment of MAM51expressing keratinocytes with AG1478 caused a similar induction of Notch1 expression as control cells; by contrast, induction of Hes1 and differentiation markers was suppressed (Fig. 3A).Chemical inhibitors of -secretase activity like DAPT suppress proteolysis-dependent activation of endogenous Notch receptors 19 .As with MAM51 expression, treatment of primary keratinocytes with DAPT counteracted induction of Hes1 and differentiation marker expression caused by EGFR downmodulation (Fig. 3B).Similar counteracting effects were also observed after siRNAmediated knock-down of Notch1 and p53 expression, indicating that up-regulation of these genes by EGFR inhibition is responsible for the observed induction of differentiation (Fig. 3C).A more indirect downstream mechanism, like that reported in Drosophila with EGFR signaling suppressing Groucho-dependent transcription 20 , is unlikely in our system.In fact, consistent with our previous findings, EGFR activation exerted no counteracting effects on induction of differentiation by activated Notch1 (Fig. 3D) and increased Hes1 expression, a mammalian Groucho effector 21 , suppressed rather then induced differentiation (Fig. 3E), confirming what previously observed for Hes1 effects in mouse primary keratinocytes 31 . Growth/differentiation control of keratinocytes in culture is likely to differ in significant aspects from control of these cells in intact skin.For further validation of our findings, we resorted to several complementary approaches.For the first, mice with a GFP reporter for Notch activity were injected with the EGFR inhibitor AG1478. Immunofluorescence analysis showed significantly elevated GFP expression in the epidermis of the AG1478 treated mice versus the control (Fig. 4A). For confirmation and quantification of the results, the epidermis of these mice was separated from the underlying dermis by a brief heat treatment, followed by total RNA preparation and real time RT-PCR analysis.This confirmed increased GFP expression, which paralleled increased expression of the endogenous Notch1, p53 and Keratin 1 genes (Fig. 4B,C).As a second approach, the epidermis of homozygous mice for a hypomorphic EGFR mutation was analyzed, in parallel with heterozygous littermates.Even in this case, decreased EGFR activity was found to result in increased p53 and Notch1 expression (Fig. 4D). In senescence driven skin aging and data not shown).Similar results were obtained with an organ culture system of freshly excised human skin, using an optimized method that allows maintenance of viable tissue with no sign of degeneration and/or altered differentiation for up to 7 days (our unpublished observations). Immunohistochemical as well as real time RT-PCR analysis indicated that, even in these conditions, EGFR inhibition caused a parallel induction of Notch1 activity and expression, and differentiation in keratinocytes, together with p53 and p21 WAF1/Cip1 (Fig. 4F, Supplemental Fig. 5).Using this approach, we tested whether the increase in keratinocyte differentiation is Notch dependent.For this, human skin cultures were treated with AG1478 plus/minus DAPT.As shown in Fig. 4G, induction of Keratin 1 and Involucrin expression by EGFR inhibition was counteracted by the concomitant treatment with the Notch / -secretase inhibitor, while, as expected, induction of the Notch1 gene itself, or of p53, was unaffected or even increased. ",
"section_name": "EGFR-p53-Notch control of differentiation in primary keratinocytes and intact skin.",
"section_num": null
},
{
"section_content": "To assess whether the p53/Notch regulatory loop discovered here applies to conditions where increased EGFR signaling has been causally linked with cancer development, we analyzed transgenic mice expressing a constitutive active form of the EGFR/Ras adaptor protein SOS under control of a keratin 5 promoter (K5-SOS-F).These mice develop spontaneous skin tumors strictly dependent on the presence of functional EGFR 22 .In K5-SOS-F transgenics with a concomitant keratinocytespecific deletion of the c-Jun gene, skin tumor development is impaired, correlating with reduced EGFR expression and increased differentiation 23 .The present results suggested that EGFR/c-Jun regulation of p53 and Notch1 expression may also be involved.In fact, real time RT-PCR analysis showed significantly higher levels of Notch1 and p53 expression in the small tumors formed by K5-SOS-F transgenic mice with epidermal deletion of the c-Jun gene (c-Jun ep SOS + ) relative to tumors formed in K5-SOS-F transgenics with the intact c-Jun gene (c-Jun f/f SOS + ) (Fig. 5A).These data were confirmed at the protein level by immunoblot analysis of a separate set of tumors, as well as by immunofluorescence for Notch1 expression (Fig. 5B,C). To assess whether similar EGFR regulation of Notch1 expression applies to human cancer, keratinocyte-derived SCC cells (SCCO28, SCC12 and SCC13) with wild type p53 (http://www.sanger.ac.uk/genetics/CGP/CellLines/) were treated with EGFR inhibitor.Besides mutations, p53 activity can also be reduced in tumors as a consequence of decreased p53 gene transcription 17,[24][25][26][27] .Consistent with this mode of regulation, EGFR inhibition of SCC cells induced expression of the p53 gene as well as of p21 WAF1/Cip1 , indicative of increased p53 activity (Fig. 6 A,B).This was paralleled by a substantial increase of Notch1 mRNA and protein levels and differentiation markers (Fig. 6C,D: Supplemental Fig. 6A).As with primary keratinocytes, p53 knockdown experiments showed that even in cancer cells, induction of Notch1 expression by EGFR-inhibition is p53-dependent (Fig. 6E). Cancer cell lines can differ substantially in their control mechanisms from cells in primary tumors.Therefore, as a further validation of our findings, the same organ culture system described above for intact skin was adapted to the analysis of clinically occurring SCCs freshly excised from patients.The dissected more homogeneous parts of tumors were cut into small pieces of the same size (2x2 mm), and placed into multi-well dishes as for skin organ cultures.In five independent tumors, EGFR inhibition resulted in reduction of c-Fos expression, indicative of EGFR signaling suppression, and concomitant induction of Notch1, p53 and Keratin 1 (Fig. 6F; Supplemental Fig. 6C).In four other tumors no such effects were observed, consistent, in two cases, with resistance of EGFR inhibition (as assessed by no decrease in c-Fos expression) and, in the other two, undetectable p53 expression or activity (data not shown). ",
"section_name": "Inhibition of EGFR signaling in cancer cells induces Notch1 gene expression through p53.",
"section_num": null
},
{
"section_content": "As with primary keratinocytes, even in SCC cells inhibition of EGFR signaling caused up-regulation of differentiation markers expression, through a Notch dependent mechanism (Supplemental Fig. 6A,B).We have recently found that Notch-dependent differentiation of keratinocytes render these cells more resistant to apoptosis (REF Mandinova et al.).Thus, an attractive possibility was that suppression of Notch signaling, while suppressing the pro-differentiation effects of EGFR inhibitors, may synergize with these compounds in triggering apoptosis.To assess this possibility, SCC cells were treated with DAPT plus-minus EGFR inhibitor.As shown in Fig. 7A, the concomitant treatment led to a substantial increase of apoptosis.These findings were paralleled by a synergistic induction of Bim1 expression (Fig. 7B), a proapoptotic Bcl2 family member that has been recently implicated in the response of cancer cells to EGFR inhibitors 28 . To further validate the relevance of these findings for the behavior of cancer in vivo, immune-compromised mice were injected with SCC cells expressing the Notch inhibitory MAM51 peptide, in parallel with control cells.After formation of sizable tumors (4 weeks after injections), mice were treated with AG1478 for a week.RT-PCR analysis of tumor RNAs showed substantially higher levels of Hes1 and differentiation marker expression in tumors formed by control than MAM51-expressing cells, while levels of Bim1 were oppositely regulated (Fig. 7C).This was paralleled by a higher apoptotic fraction in tumors with suppressed Notch signaling (Fig. 7D). ",
"section_name": "Inhibition of Notch signaling in cancer cells suppresses differentiation induced by EGFR suppression while it synergizes for apoptosis.",
"section_num": null
},
{
"section_content": "Loss-and gain-of function experiments in mice as well as work with human keratinocytes have established that EGFR signaling plays a key role in positive control of keratinocyte growth potential and carcinogenesis 10 .A similar role has been found for downstream effectors of this pathway at the level of transcription, like c-Jun 9 .Besides enhancing proliferation, we have shown here that EGFR signaling plays a significant role in suppressing differentiation through negative regulation of Notch1 gene expression and activity.This mechanism is likely to provide a break for the commitment to differentiation of keratinocytes in the basal proliferative compartment of the epidermis as well as in cancer, where EGFR signaling is characteristically elevated 10 . In the upper epidermal layers, EGFR signaling is normally down-modulated and therefore ceases to be relevant.In fact, differentiation of keratinocytes as they migrate to the upper epidermal layers is induced by multiple EGFR-unrelated events including, most notably, loss of integrin-mediated adhesion to the matrix and establishment of cadherin-dependent cell-cell adhesion 29 .This is consistent with control of the Notch1 gene by EGFR signaling -via p53 -being relevant for the behavior of proliferating keratinocytes in normal skin and cancer, while additional multiple mechanisms are responsible for the increase of Notch signaling in normally differentiating keratinocytes of the upper epidermal layers 1 . We have shown that suppression of the EGFR signaling cascade leads to upregulation of Notch1 gene expression through a mechanism involving transcriptional up-regulation of the p53 gene.Previous studies pointed to NF-B control of p53 gene expression 30 , which could be of relevance to the present situation, as NF-B activity is induced in keratinocytes with differentiation 31 , while its suppression promotes tumor development 32 .However, expression of NF-B responsive genes, as an indication of endogenous activity, is not induced in keratinocytes by EGFR suppression (data not shown).As an alternative mechanism, we have found that control by AP-1 family members is involved.AP-1 is a heterodimeric DNA-binding complex formed by proteins of the c-Jun and c-Fos families, which differ in their ability to activate target genes and can also function in an inhibitory fashion 33 . Previous work with mouse embryo fibroblasts has indicated that c-Jun can be a direct negative regulator of p53 gene expression 18 .Consistent with this conclusion, we have found that endogenous c-Jun binds to the p53 promoter in both normal and SCCderived keratinocytes, and that expression of the p53 gene in these cells is enhanced by c-Jun knock-down.Notch1 expression is also induced by c-Jun knockdown, in a p53-dependent fashion.This concomitant mode of regulation of p53/Notch1 expression by EGFR signaling was further validated by our findings with organ cultures of intact human skin and SCCs, as well as a mouse model of skin cancer formation dependent on EGFR and c-Jun function. EGFR has become an important target of cancer drug design, and several selective EGFR inhibitors have now been approved for clinical use.Recent data have highlighted the fact that inhibitors directed to critical receptors like EGFR utilize the cell death pathway for inducing tumor regression 28 .However, the exact molecular mechanisms underlying sensitivity and resistance of tumor cells to EGFR inhibitionincluding the contribution of other integrated pathways like the ones we have identified here -remain to be elucidated 34 .Importantly, suppression of Notch signaling in squamous carcninoma cells counteracts the differentiation inducing effects of EGFR inhibitors, while, at the same time, synergizing with these compounds in induction of apoptosis.Other ongoing studies in the laboratory indicate that the enhancing effects of Notch suppression on apoptosis may extend also to lung cancer cells.This suggests an attractive new avenue of combination approaches for cancer therapy that may enhance the potency of EGFR inhibitory agents on tumors, while at the same time ameliorating their well known toxic effects on the skin which have been attributed, at least in part, to aberrant differentiation 12 . ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Cell culture and viruses. Culturing of primary human keratinocytes and SCC 12, 13 and 028 cells, and infection with the MSCV-MAM51 5 and control retrovirus were as previously reported 2 .Adenoviruses for Notch1, Hes1 and GFP and adenoviral infections were previously described 31 .NCI-H1299 and NCI-H1299 were cultured in RPMI medium (Invitrogen) supplemented with 10% bovine serum.Chemical inhibitors, AG1478 (LC Labs), Nutlin (Calbiochem), DAPT (Calbiochem), ERK inhibitory peptide-cell permeable (Calbiochem) were dissolved in DMSO and applied at the indicated concentrations.For knock down experiments, cells were transfected as described 31 with validated stealth siRNAs for human EGFR, p53, and c-Jun in parallel with corresponding Stealth siRNA controls (Invitrogen), or ERK1, ERK2 and Notch1 (GeneGlobe, Qiagen) and analyzed 48-72 hours after transfection. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Conditions for real time PCR analysis, chromatin immunoprecipitation Chip), immunoblotting and immunofluorescence were as previously described 2 .The list of gene-specific primers is provided in Supplemental Table II.We used the following antibodies: Notch1 (Santa Cruz, C-20), activated Notch1 (Cell Signaling), Hes1 (Chemicon AB5702), Keratin1 (Babco AF87), Involucrin (Babco PRB140C), EGFR (Cell Signaling), p53 (Cell Signaling), MDM2 (Cell Signaling), Integrin 4 (Santa Cruz), -Tubulin (Sigma GTU87), for immunoblotting for mouse proteins: Notch1 (Pharmingen), p53 (Novacastra), c-Jun (Transduction Labs) and Actin (Sigma), c-Jun for Chip assays (Santa Cruz, H79). ",
"section_name": "Quantitative real time RT PCR, chromatin immunoprecipitation and immunodetection techniques.",
"section_num": null
},
{
"section_content": "Discarded human skin samples from abdominoplasty procedures were obtained from the Centre Hospitalier -Universitaire Vaudois (Lausanne, Switzerland) under patients' agreement and institutional approval.Skin samples, sterilized in 70% ethanol and cut, after removal of subcutaneous fat, into 1x1cm pieces, were placed in keratinocyte serum-free medium (KSF, GIBCO-BRL) supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), in 0.25% agar (Sigma).The epidermis was maintained at the air-medium interface.For RNA collection, skin samples were placed in preheated PBS at 60 °C for 45 seconds, then chilled (on ice) in 0.1M PBS for 1 minute, followed by mechanical separation of epidermis and dermis.The epidermis was homogenized in TRI Reagent (Sigma) for RNA preparation.Human SCC samples were obtained as discarded material from Mohs micrographic surgery at Massachusetts General Hospital (Boston, MA) with patients' and institutional approvals.Tumor samples were sterilized in 70% ethanol, cut into pieces of approximately 2x2 mm and placed in semi-solid medium similarly to skin organ cultures. ",
"section_name": "Organ cultures",
"section_num": null
},
{
"section_content": "For in vivo tumorigenicity assays, control and MAM51 expressing SCCO28 cells were brought into suspension, admixed with Matrigel (BD Biosciences), and injected (5x10 6 cells/injection) subcutaneously in 8 weeks old female athymic nude mice.Four weeks later animals were treated three times (every other day) with AG1478 (1 mg/animal, dissolved in 200 l 50% DMSO:DMEM) or DMSO vehicle control by i.p. injections.Mice were sacrificed 2 days after the last treatment and tumors processed for RNA preparation and analysis. ",
"section_name": "Tumorigenicity assays",
"section_num": null
},
{
"section_content": "Cells were trypsinized, recovered by centrifugation at 300 g and fixed in 2% paraformaldehyde in PBS for 16 h.Permeabilization and enzymatic labeling with TMR red-conjugated-dUTP were performed according to the manufacturer's protocol (Roche, IN).The percentage of cells that incorporated the fluorescence-conjugated dUTP was determined by flow cytometry.TUNEL assay on histological sections was analyzed with fluorescent microscopy and IPLab software. ",
"section_name": "TUNEL Assays",
"section_num": null
}
] |
[
{
"section_content": "Drs. Jim Follen and Caroline Shamu of the Harvard Institute for Chemistry and Cell Biology and Nichola Tolliday from the Broad Institute (Harvard/MIT, Cambridge, MA) for assistance with screening, Drs.W. Austen (MGH, Boston, MA) and W. Raffoul (U. of Lausanne) for human skin material, Drs.Jeff Settleman and Ultan McDermott (MGH, Boston, MA) for the lung cancer cell lines, Dr. Rainer Zenz for mouse skin tumor samples, Vikram Rajashakera for technical help, and Drs.C. Brisken and C. Missero for careful reading of the manuscript,.This work was supported by NIH Grants AR39190, CA16038 and CA73796, the Swiss National Foundation, a grant from the European Union (Epistem, Sixth Framework Program, LSHB-CT-2005-019067) and, in part, by the Cutaneous Biology Research Center through the Massachusetts General Hospital / Shiseido Co. Ltd.Agreement. ",
"section_name": "Acknowledgments.",
"section_num": null
},
{
"section_content": "",
"section_name": "Acknowledgments.",
"section_num": null
},
{
"section_content": "Drs. Jim Follen and Caroline Shamu of the Harvard Institute for Chemistry and Cell Biology and Nichola Tolliday from the Broad Institute (Harvard/MIT, Cambridge, MA) for assistance with screening, Drs.W. Austen (MGH, Boston, MA) and W. Raffoul (U. of Lausanne) for human skin material, Drs.Jeff Settleman and Ultan McDermott (MGH, Boston, MA) for the lung cancer cell lines, Dr. Rainer Zenz for mouse skin tumor samples, Vikram Rajashakera for technical help, and Drs.C. Brisken and C. Missero for careful reading of the manuscript,.This work was supported by NIH Grants AR39190, CA16038 and CA73796, the Swiss National Foundation, a grant from the European Union (Epistem, Sixth Framework Program, LSHB-CT-2005-019067) and, in part, by the Cutaneous Biology Research Center through the Massachusetts General Hospital / Shiseido Co. Ltd.Agreement. ",
"section_name": "We thank",
"section_num": null
}
] |
10.3324/haematol.2012.069369
|
Clinical implications of the molecular genetics of chronic lymphocytic leukemia
|
Genetics and molecular genetics have contributed to clarify the biological bases of the clinical heterogeneity of chronic lymphocytic leukemia. In recent years, our knowledge of the molecular genetics of chronic lymphocytic leukemia has significantly broadened, offering potential new clinical implications. Mutations of TP53 and ATM add prognostic information independently of fluorescence in situ hybridization cytogenetic stratification. In addition, next generation sequencing technologies have allowed previously unknown genomic alterations in chronic lymphocytic leukemia to be identified. Mutations of NOTCH1, SF3B1 and BIRC3 have been associated with short time to progression and survival. Each of these lesions recognizes a different distribution across different clinical phases and biological subgroups of the disease. The clinical implications of these molecular lesions are in some instances well established, such as in the case of patients with TP53 disruption, who should be considered for alternative therapies/allogeneic stem cell transplant upfront, or in patients with ATM disruption, who are candidates to rituximab-based immunochemotherapy. On the contrary, NOTCH1, SF3B1 and BIRC3 mutations appear to have a specific significance, the clinical value of which is currently being validated, i.e. association to Richter syndrome transformation for NOTCH1 mutations, and short progression-free survival after treatment for SF3B1 mutations. Certainly, these new lesions have helped clarify the molecular bases of chronic lymphocytic leukemia aggressiveness beside TP53 disruption. This review covers the recent advancements in our understanding of the molecular genetics of chronic lymphocytic leukemia and discusses how they are going to translate into clinical implications for patient management.
|
[
{
"section_content": "The clinical course of chronic lymphocytic leukemia (CLL) is extremely heterogeneous.[3][4][5][6][7][8][9][10][11][12][13][14][15] The Rai and Binet clinical staging systems still remain the cornerstone for identifying CLL patients with advanced disease stages for whom treatment-free survival (TFS) and overall survival (OS) are usually short. 2,5,6However, these staging systems do not provide risk stratification in early stage disease, that nowadays includes most cases of newly diagnosed CLL, and also fail to identify those patients who will develop chemorefractoriness. [11][12][13][14][15] Understanding CLL genetics may help clarify the molecular bases of the clinical heterogeneity of this leukemia.In the 1990s, Juliusson et al. 16 applied conventional karyotype banding analysis to systematically assess the prevalence and prognostic impact of chromosomal abnormalities associated with CLL.Despite its technological limitations, this approach revealed that more than half of CLL patients had clonal chromosomal changes.Importantly, this pivotal analysis indicated that chromosomal abnormalities affect CLL outcome and served as a proof of concept to document that genetic alterations in CLL may be prognostically relevant in a hierarchical order. 16n 2000, this notion was unequivocally documented by the seminal study by Döhner et al. 17 that established interphase fluorescence in situ hybridization (FISH) analysis as a standard technique to evaluate cytogenetic lesions in CLL, detecting chromosomal abnormalities in over 80% of patients, thus overcoming the limited applicability and resolution of conventional karyotyping.By correlating FISH lesions with the course of the disease, a hierarchical model based on five risk categories was established.CLL cases harboring the 17p13 deletion independent of concomitant abnormalities (prevalence 7%) had the worst prognosis (median survival 32 months), followed by cases carrying the 11q22-q23 deletion (prevalence 18%, median survival 79 months), trisomy 12 (prevalence 16%, median survival 114 months), normal karyotype (prevalence 18%, median survival 111 months) and 13q14 deletion (prevalence 55%, median survival 133 months). 17ytogenetic lesions, however, do not entirely explain the genetic basis of the clinical heterogeneity of CLL.Additional information has come from the detailed definition of the molecular correlates of CLL chromosomal aberrations.In fact, TP53, the tumor suppressor gene affected by 17p13 deletion, and ATM, the gene targeted by 11q22-q23 deletion, are not only deleted, but also recurrently mutated in CLL.][20][21][22][23][24][25] In recent times, the improvements in next generation sequencing technologies have provided a novel opportunity to examine the CLL genome, and have allowed previously unknown genomic alterations to be identified, such as mutations of NOTCH1 (neurogenic locus notch homolog protein 1), SF3B1 (splicing factor 3B subunit 1) and BIRC3 (baculoviral ",
"section_name": "Genetic heterogeneity of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Genetics and molecular genetics have contributed to clarify the biological bases of the clinical heterogeneity of chronic lymphocytic leukemia.In recent years, our knowledge of the molecular genetics of chronic lymphocytic leukemia has significantly broadened, offering potential new clinical implications.Mutations of TP53 and ATM add prognostic information independently of fluorescence in situ hybridization cytogenetic stratification.In addition, next generation sequencing technologies have allowed previously unknown genomic alterations in chronic lymphocytic leukemia to be identified.Mutations of NOTCH1, SF3B1 and BIRC3 have been associated with short time to progression and survival.Each of these lesions recognizes a different distribution across different clinical phases and biological subgroups of the disease.The clinical implications of these molecular lesions are in some instances well established, such as in the case of patients with TP53 disruption, who should be considered for alternative therapies/allogeneic stem cell transplant upfront, or in patients with ATM disruption, who are candidates to rituximab-based immunochemotherapy.On the contrary, NOTCH1, SF3B1 and BIRC3 mutations appear to have a specific significance, the clinical value of which is currently being validated, i.e. association to Richter syndrome transformation for NOTCH1 mutations, and short progression-free survival after treatment for SF3B1 mutations.Certainly, these new lesions have helped clarify the molecular bases of chronic lymphocytic leukemia aggressiveness beside TP53 disruption.This review covers the recent advancements in our understanding of the molecular genetics of chronic lymphocytic leukemia and discusses how they are going to translate into clinical implications for patient management. ",
"section_name": "Clinical implications of the molecular genetics of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "",
"section_name": "ABSTRACT",
"section_num": null
},
{
"section_content": "",
"section_name": "Pattern and distribution of genetic lesions affecting chronic lymphocytic leukemia outcome",
"section_num": null
},
{
"section_content": "Molecular defects of TP53 and ATM are well-established genetic lesions carrying clinical relevance in CLL.The tumor suppressor gene TP53 maps on the short arm of chromosome 17 (17p13) and codes for a central regulator of the DNA-damage-response pathway. 34Activation of TP53 leads to cell-cycle arrest, DNA repair, apoptosis, or senescence via both transcription-dependent and transcriptional-independent activities.Consistently, TP53 plays a central role in mediating the pro-apoptotic and antiproliferative action of several DNA-damaging chemotherapeutic agents, including alkylators and purine analogs. 348][19][20][21] Most cases with 17p13 deletion also carry TP53 mutations on the second allele (~70%), while the remaining cases have a monoallelic 17p13 deletion in the absence of TP53 mutations (~20%), or TP53 mutations in the absence of 17p13 deletion (~10%). 35In line with the genetic instability associated with defective DNA-damage checkpoints, TP53 abnormalities frequently couple with complex cytogenetic abnormalities, particularly with unbalanced translocations. 21t the molecular level, approximately 75% of all mutations are missense substitutions, while the remaining lesions (~25%) are represented by truncating events, including frameshift insertions or deletions, non-sense substitutions and splice site mutations. 35,36Most missense mutations are localized within exons 5-8, which encode the central DNA-binding domain of TP53, thus impairing DNA binding and target gene transactivation (Figure 1). 35he ATM gene is a member of the phosphatidylinositol-3 kinase (PT3K) gene family and encodes a nuclear serine/threonine kinase whose activity is induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and chemotherapeutic drugs. 37ATM protects the integrity of the genome by regulating the cellcycle arrest at G1/S and G2/M to prevent processing of damaged DNA, and by activating DNA-repair pathways and inducing apoptosis if the DNA damage cannot be repaired.Many of these effects are mediated by the activation of both TP53-dependent and TP53-independent cellular pathways. 37TM is a large gene of 62 coding exons that maps on haematologica | 2013; 98(5) ",
"section_name": "Molecular characteristics of clinically relevant genetic lesions of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "chromosome 11q22-q23, which is a minimally common deleted region in CLL.As for TP53, the ATM gene in CLL may be inactivated by both deletion and/or somatic mutations. 22,23At the molecular level, ATM mutations consist in a mixture of missense substitutions distributed across the ATM coding sequence, with no hotspots. 22,23Only a proportion of ATM mutated CLL (10%-20%) show a concomitant 11q22-q23 deletion.37][38][39] Insights into the pathogenicity of ATM mutations in CLL are provided by the observation that two recurrent mutations (p.R2691C and p.P2699S) localize within functionally relevant sites, including the ATP-binding pocket of ATM, and critically impair ATM kinase activity. 25ATM mutants with an impaired kinase activity sequester ATM wild-type proteins with a dominant negative effect and inhibit their activation in response to physical and chemical insults. 25ecent studies based on next generation sequencing have revealed new genes implicated in CLL and potentially carrying clinical relevance.NOTCH1 encodes a transmembrane protein that acts as a ligand-activated transcription factor and regulates multiple target genes, including MYC, TP53 and molecules of the NF-κB pathway. 40,41In 2009, it was shown that the constitutive NOTCH signaling activation was implicated in CLL cell survival and apoptosis resistance.In an attempt to identify the underlying mechanism, the first identification of the NOTCH1 PEST domain mutation in CLL patients was reported. 42,43t the molecular level, NOTCH1 mutations in CLL are mainly represented by frameshift or non-sense events clustering within exon 34, and including the highly recurrent c.7544_7545delCT deletion (approx.80%-95% of all mutations) (Figure 1). 26,27,31,32NOTCH1 mutations in CLL are selected to disrupt the C-terminal PEST domain of the protein, that is responsible for the proteosomal degradation of the activated form of NOTCH1.Indeed, truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated NOTCH1 signaling. 44onsistent with this notion, a number of cellular pathways, including those controlling cell metabolism and cell cycle progression, are deregulated in CLL harboring NOTCH1 mutations.NOTCH1 is preferentially targeted in specific biological groups of CLL. 27,45In fact, NOTCH1 mutations are significantly more common in CLL with unmutated immunoglobulin heavy variable (IGHV) genes and are enriched in CLL harboring +12, where they identify a distinct clinico-molecular subgroup characterized biologically by deregulated cell cycle and clinically by short survival. 26,27,31,32,46,479][50] SF3B1 mutations in CLL are almost always represented by missense substitutions affecting the HEAT domains of the SF3B1 protein and recurrently target five hotspots (codons 662, 666, 700, 704 and 742), with the K700E substitution accounting for approx.][30] Among CLL genetic events, SF3B1 lesions have shown a preferential, though not consistent, association with 11q22-q23 deletion and ATM mutations. 29,30The functional consequences of SF3B1 mutations in CLL are currently under scrutiny.5][56][57] BIRC3 is also involved in maintaining wildtype TP53 levels by preventing NF-κB-mediated transcriptional and post-translational modifications of MDM2 expression and function.Consistently, BIRC3 knockdown contributes to cancer promotion through downregulation of the TP53 protein via MDM2. 58IRC3 is recurrently disrupted in CLL by mutations, deletions or a combination of both. 33BIRC3 inactivating mutations are mainly represented by frameshift or nonsense substitutions causing the truncation of the C-terminal RING domain of the BIRC3 protein (Figure 1), whose E3 ubiquitin ligase activity is required to prime MAP3K14 towards proteosomal degradation.][29][30]64 ",
"section_name": "A B D C",
"section_num": null
},
{
"section_content": "CLL course may proceed through distinct clinical phases, ranging from a pre-malignant condition known as monoclonal B-cell lymphocytosis (MBL) to overt CLL, and even to transformation into an aggressive lymphoma known as Richter's syndrome (RS). 1,26][67][68][69][70] This observation is consistent with the notion that 13q14 deletion and +12 represent first step genetic abnormalities in CLL.All the other genetic events accumulate in the more advanced phases of the disease, suggesting that they represent second hit lesions that are progressively selected or acquired during the evolution of the clone. As in other pre-malignant conditions, also MBL frequently harbor some of the genetic lesions that can be observed in the overt phases of the disease.][67][68][69][70][71][72] Two categories of MBL exist. 73One is represented by clinical MBL, which are detected in the context of a lymphocytosis investigated with laboratory techniques.The second category is represented by low count MBL, which are discovered while screening normal individuals of the general population for research purposes, and in whom the absolute number of lymphocytes is not increased. 73While the impact of high-risk genetic lesions on clinical MBL survival is currently unknown, their occurrence associates with an increased rate of progression to overt CLL. 67,69igh-risk cytogenetic abnormalities have been occasionally described also in low count MBL, but the clinical implications of this observation are currently unknown. 66,68hree major clinical phases can be distingushed in overt CLL, including: i) newly diagnosed CLL; ii) progressive CLL; and iii) relapsed and fludarabine-refractory CLL. Deletion of 17p13 occurs below 5% in newly diagnosed CLL. 11,1421][74][75][76][77][78][79][80] Deletion of 11q22-q23 occurs in less than 10% newly diagnosed CLL, while its prevalence rises to ~20% at the time of first treatment and ~20% at the time of fludarabine-refractoriness. 11,14,74-779 These frequencies make ATM alterations the most common genetic lesion predicting poor outcome at CLL presentation and at the time of treatment requirement (Table 1). NOTCH1, SF3B1 and BIRC3 mutations follow the same distribution across CLL clinical phases as other high-risk abnormalities.NOTCH1 mutations characterize ~5%-10% newly diagnosed CLL, while their prevalence increases to 13%-20% in progressive CLL requiring first treatment and in relapsed cases. 26,27,31,32,6430]64 Though occurring at low rates in newly diagnosed CLL (~5% of cases), BIRC3 lesions are enriched among relapsed and fludarabine-refractory CLL (~25% of cases). 33Due to the lack of information from clinical trials, the precise rate of occurrence at BIRC3 lesions at the time of first treatment requirement still remains to be clarified (Table 1). RS transformation is a very aggressive and an almost always lethal complication of CLL that combines the effects of both chemoresistance and rapid disease kinetics. 1The genetics of RS strongly influences its clinical behavior.The high rate of TP53 abnormalities (~60% of cases) accounts for the chemoresistant phenotype that is commonly observed in RS. 70 NOTCH1 mutations and MYC network abnormalities are the second most frequent genetic lesions in RS, where they occur in ~30% of cases. 26In RS, NOTCH1 mutations are largely mutually exclusive with MYC oncogenic activation by translocation/amplification of the gene or by disruption of MGA, its negative regulator. 26,81This finding is consistent with the observation that NOTCH1 directly stimulates MYC transcription and suggests that activation of oncogenic MYC may be one common final pathway selected for tumorigenesis in ~60% RS. 26,81 Lesions affecting ATM, BIRC3 and SF3B1, that are otherwise frequent at the time of chemorefractory relapse, occur at low rates in RS, thus corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation. 29,33,70 ",
"section_name": "Molecular lesions at different clinical phases of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "",
"section_name": "Clonal evolution of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "17p13 deletion 11,14, 72,74,75 4-5 5-7 8-10 1.5-2 TP53 mutation [19][20][21]77,78 5-10 5-6 8-11 1.5-3 11q22-q23 deletion 11,14,72,74,75 8-9 8-9 21-23 3-5 ATM mutation 24,25,39 10-15 7-8 15 4 NOTCH1 mutation 26,27,29,30,32,62 5-11 4-8 10 4.5 SF3B1 mutation [28][29][30]62 6-9 4-9 17 4.5 BIRC3 disruption 33 4 3.5 na na ease, CLL may undergo clonal evolution in a substantial fraction of cases. Compason of the profile of cytogenetic lesions from primary and relapsed CLL samples has revealed differences in ~20-40% cases, illustrating the dynamic nature of clonal evolution in this leukemia.70,[82][83][84][85][86][87][88][89][90][91] The risk of developing new genetic lesions during the course of the disease is positively correlated with the duration of the follow up.82,83 Additional factors contributing to clonal evolution in CLL are the IGHV mutation status and the selective pressure of treatments given during the disease history. 82,83 From a clical standpoint, clonal evolution has been associated with poor outcome, treatment resistance and transformation. 70,82,83 Based on conventil and FISH cytogenetic analysis, clonal evolution mainly consists in the development of 17p13 or 11q22-q23 deletions. 82,83 Mution analysis of sequential samples has revealed that the development of new TP53 mutations also contributes to clonal evolution, especially in chemorefractory patients and in patients complicated by RS. 18,70 Consistent with this observation, current guidelines recommend to repeatedly test for TP53 lesions at treatment requirement also in cases that were previously wild-type.2,92 NOTCH1, SF3B1 and BIRC3 lesions may emerge during the course of CLL, thus expanding the spectrum of genetic events currently associated with clonal evolution. 93imilar to TP53 abnormalities and 11q22-q23 deletion, also the development of new NOTCH1, SF3B1 and BIRC3 lesions may occur at the time of shift to a more aggressive clinical phenotype.In this context, a fraction of NOTCH1 mutations may develop at the time of transformation to RS. 26 Consistently, the acquisition of high-risk genetic lesions over time, including NOTCH1, SF3B1 and BIRC3 mutations, affects survival in a manner that is independent of modifications of other time-varying factors, such as patient age and disease stage.93 Open issues in the field of clonal evolution of CLL are: i) whether mutations detected from a certain timepoint onward were already present at subclonal levels in earlier disease phases and are thus subsequently selected (as demonstrated for a few cases), or whether they are acquired de novo during the course of the disease; and ii) whether the presence of small subclones harboring highrisk genetic lesions from the early phases of the disease may affect CLL outcome.A conclusive demonstration of the precise timing of mutations in CLL awaits studies aimed at tracking these lesions with high sensitivity techniques in sequential disease phases. ",
"section_name": "Diagnosis First treatment Prevalence % Overall survival (years) Prevalence (%) Overall survival (years)",
"section_num": null
},
{
"section_content": "",
"section_name": "Clinical relevance of genetic lesions in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "TP53 abnormalities represent strong predictors of poor survival and refractoriness in CLL and, for these reasons, they have direct implications for the clinical management of this leukemia. Among newly diagnosed CLL, patients harboring 17p13 deletion show a median overall survival (OS) of only 5-7 years. 17Although the outcome of patients with 17p13 deletion is generally considered poor, it is important to underline that there is a small subgroup of cases, generally expressing mutated IGHV genes, who may exhibit a slowly progressive disease without treatment indications for years. 94t the time of treatment requirement, the outcome of patients with 17p13 deletion is almost always very poor.Patients with 17p13 deletion respond poorly (5% of complete response vs. ~50% in non-17p13 deleted CLL) to fludarabine-cyclophosphamide-rituximab (FCR), that is the most effective regimen available today for CLL.The poor response rate in 17p13 deleted patients translates into very short progression-free survival (PFS) (11.2 months vs. 51.8months) and OS (38.1% at 36 months). 77This is in line with the central role of the wild-type TP53 protein in priming CLL cells to apoptosis and in mediating the cytotoxic effect of DNA-damaging agents, including purine analogs. Recently, by assessing the impact of CLL genetics on disease outcome, a number of prospective studies have consistently documented that TP53 mutations, even in the absence of 17p13 deletion, represent a predictor of poor response to treatment and short survival in CLL.In the GCLLSG CLL4 trial (F vs. FC), 74 none of the TP53 mutated CLL achieved a complete response, and the median PFS (23.3 vs. 62.2 months) and OS (29.2 vs. 84.6 months) were significantly shorter in patients with TP53 mutations compared to TP53 wild-type cases. 79In the GCLLSG CLL8 trial (FC vs. FCR), the presence of TP53 mutations associated with the lowest complete and overall response rates (6.9% vs. 36.4% and 62.1% vs. 95.3%,respectively), the shortest PFS (12.4 months vs. 45 months) and the shortest OS (39.3 months vs. not reached in all other patients) compared to TP53 wild-type cases. 95In the UK LRF CLL4 trial (chlorambucil vs. F vs. FC), TP53 mutated patients showed a complete remission rate of only 5%, a 5-year PFS of 5%, and a 5-year OS of 20%. 80lthough clinical trials have consistently shown a clear association between TP53 mutations and poor outcome in CLL, some controversial issues still remain to be clarified in this field.Indeed, it is currently a matter of debate whether monoallelic TP53 abnormalities have the same poor prognostic effect as biallelic TP53 lesions, and whether the TP53 mutation type and position in the protein might impact on patient oucome.In fact, in contrast to the GCLLSG CLL4 trial, cases from the LRF CLL4 trial harboring isolated TP53 mutations or deletions showed a longer PFS and OS after treatment compared to cases harboring biallelic TP53 disruption. 79,80Also, in a retrospective study, patients harboring truncating TP53 mutations or missense substitutions mapping outside the DNA binding domain seem to have a longer OS from diagnosis than cases harboring mutations within the DNA-binding motifs. 96n these bases, at least three main clinical implications need to be considered: 2,92 i) patients with TP53 abnormalities should not be treated until disease progression, since they can occasionally experience a prolonged TFS; ii) alongside 17p13 deletion, TP53 mutation analysis should be performed in all CLL patients before treatment initiation, since cases with TP53 disruption should be considered for alternative therapies upfront (see below); iii) a thorough search for TP53 mutations/deletions should be performed repeatedly before each line of therapy. Chemorefractoriness is due to TP53 disruption in ~40% of CLL patients failing treatment, but the molecular basis of this aggressive clinical phenotype remains unclear in a sizeable fraction of patients (~60%). 78In order to optimize the early diagnosis of chemorefractory CLL, it is crucial to understand the molecular basis of chemorefractoriness beyond TP53 disruption.The new molecular lesions recently identified may shed some light on this (see below). ",
"section_name": "Clinical relevance of TP53 abnormalities in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Deletion of 11q22-q23 was historically associated to CLL progression, poor response to alkylator-and fludarabine-based chemotherapy and, ultimately, short survival. 17,74,76The introduction of chemo-immunotherapy based regimens has changed the prognosis of patients with this genetic abnormality.In fact, in the GCLLSG CLL8 trial, treatment with FCR significantly improved the complete response rate in CLL patients with 11q22-q23 deletion compared to FC alone (51% vs. 15%, P<0.0001), making these patients more similar to standard-risk patients in terms of response and PFS. 77he prognostic impact of ATM mutations in CLL has been investigated in few studies, due to the complexity of the DNA sequencing procedure of such a large gene in the absence of well-defined mutational hotspots, and also because of the difficulty in discriminating somatic mutations and damaging germline mutations from single nucleotide polymorphisms.][24][25] The impact of ATM mutations on response to treatment and chemorefractoriness is still an open issue.In vitro, CLL cells with mutations affecting either one or both ATM alleles show defective apoptosis in response to radiation and chemotherapy-induced DNA damage.On the contrary, 11q22-q23 deleted CLL with a normal residual ATM allele preserve the DNA damage response, suggesting that loss of a single ATM allele might not be sufficient to induce chemorefractoriness. 97 Consistent with these pre-clinical observations, in the UK LRF CLL4 trial, patients with both ATM mutation and 11q22-q23 deletion showed a significantly reduced PFS (median 7.4 months) compared to those with wild-type ATM (28.6 months), 11q22-q23 deletion alone (17.1 months), or ATM mutation alone (30.8 months).Consistently, OS for patients with biallelic ATM alterations was significantly reduced compared to those with wild-type ATM or ATM mutations alone (median 42.2 vs. 85.5 vs. 77.6 months, respectively). 39On these bases, at least in the context of CLL treated with alkylating agents or purine analogs, only the co-occurrence of 11q22-q23 deletion and ATM mutation associated with poor outcome.Importantly, in the UK LRF CLL4 trial, the PFS of CLL harboring biallelic ATM lesions was similar to that of patients with TP53 alterations. 39Therefore, when using chemotherapy alone, ATM mutation could be one mechanism that accounts for a fraction of chemorefractory CLL in which no aberrations of TP53 are detected. In conclusion, ATM disruption represents an independent marker of poor prognosis in CLL patients, particularly if they are treated with chemotherapy regimens not containing rituximab.Since the addition of rituximab to intensive combination chemotherapy (i.e.FCR) leads to an improved outcome in CLL harboring 11q22-q23 deletion, this regimen represents the treatment of choice in clinically fit patients belonging to this genetic subgroup.Nonetheless, even in the immunochemotherapy era, 11q22-q23 deleted patients have a short PFS and, therefore, may be particularly suited for investigational agents combined with immunochemotherapy or maintenance strategies aimed at prolonging disease remission. 98 ",
"section_name": "Clinical relevance of ATM abnormalities in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Beside their pathogenetic role, NOTCH1, SF3B1 and BIRC3 lesions may also represent new biomarkers for the identification of poor-risk CLL patients. Retrospective studies have consistently shown the impact of NOTCH1 and SF3B1 mutations on newly diagnosed CLL outcome.NOTCH1 mutated patients have a more rapidly progressive disease and a significantly shorter OS probability (21%-45% at 10 years) compared to NOTCH1 wild-type cases (56%-66% at 10 years). 26,27,329][30] In a retrospective analysis of newly diagnosed CLL, BIRC3 disruption identifies patients with a poor survival (median OS 3 years) similar to that associated with TP53 abnormalities. 33ata from prospective studies and clinical trials validate the clinical importance of NOTCH1 and SF3B1 mutations in CLL. 64,99In a well-characterized population-based cohort of newly diagnosed CLL patients who were included in the Scandinavian Lymphoma Etiology study, the presence of NOTCH1 or SF3B1 mutations was strongly associated with poor outcome, both in terms of shorter time to treatment and decreased survival. 99In fact, time to treatment was only 4.8 months in patients harboring NOTCH1 mutations and 2.4 months in patients harboring SF3B1 mutations.This higher propensity to progression translated into a short OS of 66 months in NOTCH1 mutated patients and 63 months in SF3B1 mutated patients, that was significantly reduced compared to that of wild-type cases. 99In addition, NOTCH1 and SF3B1 mutations in this study had a similarly poor impact on prognosis as TP53 aberrations .99In the UK LRF CLL4 trial, patients harboring NOTCH1 and SF3B1 mutations have an OS (55 and 54 months, respectively) that is significantly shorter compared to wild-type patients (83 months) and longer than that of patients carrying TP53 abnormalities (26 months). 64verall, these data document that, at the time of treatment requirement, patients with NOTCH1 and SF3B1 mutations display an outcome that is intermediate between the one marked by TP53 abnormalities and the one characterizing wild-type cases.While both NOTCH1 and SF3B1 mutations are independent predictors of OS by multivariate analysis in this trial, only SF3B1 mutations, but not NOTCH1 mutations, significantly correlate with a short PFS. 64These data point to chemoresistant progression as a potential reason for the poor outcome in SF3B1 mutated patients, while NOTCH1 mutations apparently have no impact on disease sensitivity to treatment.The short survival associated with NOTCH1 mutations can be explained, at least in part, by a substantial risk (~50% at 15 years) of developing RS in patients harboring this genetic lesion. 100he GCLLSG is currently exploring the role of new mutations in CLL patients treated with first-line FC or FCR (CLL8 trial), as well as the role of alemtuzumab in overcoming NOTCH1 and SF3B1 alterations in relapsed/refractory patients (CLL2H trial).Preliminary data from the GCLLSG CLL8 trial 77 indicate that both SF3B1 and NOTCH1 mutations represent independent predictors of short PFS after treatment with FCR. 101In particular, in this trial, NOTCH1 mutations appear to identify a subset of CLL patients that may not benefit from the addition of rituximab to FC. 101 Conversely, based on a preliminary analysis of the GCLLSG CLL2H trial, 75 patients harboring NOTCH1 mutations may have a superior PFS after alemtuzumab treatment compared to NOTCH1 wild-type cases, at least in the relapsed/refractory setting. 102hough information on the impact of BIRC3 lesions on response to treatment is currently lacking, their enrichment among fludarabine-refractory CLL might suggest an association of these molecular defects with chemorefractory progression. 33he integration of the most recurrent and clinically relevant new molecular lesions into the backbone of the FISH hierarchical model has allowed a better understanding of the genetic basis of CLL heterogeneity and a significant improvement in patient prognostication. 93According to a proposed model, four genetic groups of patients are hierarchically classified: 93 i) high-risk patients, harboring TP53 and/or BIRC3 abnormalities independent of co-occurring genetic lesions, that account for approximately 15%-20% newly diagnosed CLL and show a 10-year survival of 29%; ii) intermediate-risk patients, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 in the absence of BIRC3 and TP53 abnormalities, that account for ~15%-20% newly diagnosed CLL and show a 10-year survival of 37%; iii) low-risk patients, harboring +12 or a normal genetics, that account for approximately 40% of newly diagnosed CLL and showed a 10-year survival of 57%; and iv) very low-risk patients, harboring del13q14 only in the absence of any additional abnormality, that account for ~20%-25% newly diagnosed CLL and a nearly normal life expectancy with a 10-year survival (69%) that did not significantly differ from a matched general population (Figure 2). 93 ",
"section_name": "Clinical relevance of novel molecular lesions",
"section_num": null
},
{
"section_content": "TP53 disruption is at present the only molecular marker that changes the therapeutic approach to CLL patients.The median OS of TP53 disrupted CLL treated with intensive immunochemotherapy regimens (i.e.FCR) is within 2 to 3 years, 77,95 thus resembling the outcome of acute leukemias.As a consequence, the occurrence of TP53 abnormalities represents a strong indication for treating patients with drugs with a TP53-independent mechanism of action and for performing an allogeneic stem cell transplant (SCT) consolidation in eligible cases. The anti-CD52 monoclonal antibody alemtuzumab has so far been the single agent with the highest efficacy in CLL with 17p13 deletion.4][105][106][107][108][109][110][111][112] At first-line, patients with TP53 disruption and treated with alemtuzumab-based regimens can achieve a response rate of 60%-90%, a complete response rate of 20%-60% and a PFS of 10-17 months.][105][106][107][108][109][110][111][112] The efficacy of alemtuzumab is increased by its combi-nation with high-dose steroids.In the UK CLL206 trial, alemtuzumab combined to high-dose methylprednisolone was administered to 39 CLL with 17p13 deletion (17 untreated and 22 previously treated). 111This combination resulted in a response rate of 85%, a complete response rate of 36%, a median PFS of 11.8 months and a median OS of 23.5 months.Based on these results, alemtuzumab combined to high-dose methylprednisolone represents the most effective cytoreductive therapy to be considered for fit patients with TP53 disruption as a bridge to allogeneic SCT, which is still required because otherwise responses are of short duration.4][115][116] On these bases, according to the EBMT guidelines, young CLL patients requiring treatment and harboring TP53 abnormalities have an indication for allogeneic SCT. 1174][115][116] Consistently, the actual strategy for fit patients with TP53 abnormalities is to induce the disease at least in partial remission to perform a reduced intensity conditioning allogeneic SCT. Despite these advances, a number of unmet clinical needs remain in the setting of CLL patients requiring treatment and harboring TP53 abnormalities, including: i) the design of alternative and safer approaches for remission induction, as remission rates with the current regimens are reached only in approximately one-third of patients with relatively high costs in terms of toxicity and infections; and ii) the design of alternative strategies for those patients who are not eligible to transplant procedures because of age and comorbidities. Novel compounds that act through mechanisms completely different from chemotherapy may overcome the refractoriness induced by TP53 abnormalities.Among fludarabine-refractory CLL with 17p13 deletion, ofatumumab, a new anti-CD20 antibody, led to a response rate of 41%; however, responses were of short duration. 118reliminary results obtained from ongoing clinical trials employing novel drugs interfering with the B-cell receptor signaling suggest that these novel agents may circumvent the negative impact of 17p13 deletion. 119,120urrently, data on genetic alterations of NOTCH1, SF3B1 and BIRC3 are not sufficient to substantiate a role, if any, for these mutations in guiding therapeutic choices.This issue still awaits clarification by the analysis of these new mutations in the context of multiple clinical trials. ",
"section_name": "Molecular genetics as a guide for choosing therapy",
"section_num": null
},
{
"section_content": "In recent years, our understanding of the complexity of the molecular genetics of CLL has broadened profoundly and this has translated into important implications for an optimized management of patients.Indeed, a workup at diagnosis for TP53 and ATM disruption, and NOTCH1, SF3B1 and BIRC3 mutations enables a more refined prognostic stratification of patients.None of these markers, however, represents per se an indication for early treatment, but prompts a closer clinical follow up.For patients Molecular genetics of CLL haematologica | 2013; 98 (5) with disease progression, it is mandatory to perform TP53 mutation analysis alongside 17p13 deletion, since cases with TP53 disruption should be considered for alternative therapies upfront, including alemtuzumab plus steroids followed by an allogeneic SCT in young and fit patients.Although there are currently no treatment strategies for TP53 disrupted CLL in the elderly, the use of low doses of alemtuzumab has proven feasible. 109ATM disruption represents a strong indication for the use of the FCR scheme in clinically fit patients, whilst in elderly/frail patients with this lesion rituximab-based alternative approaches should be explored. NOTCH1, SF3B1 and BIRC3 mutations at present do not guide therapeutic choices.However, they represent markers of short time to progression and survival, with potentially different clinical implications that need to be conclusively validated.NOTCH1 mutations apparently do not impact on CLL chemosensitivity, but are associated to a substantial risk of RS transformation, particularly if associated to TP53 disruption; therefore, a close follow up and early node biopsy is recommended in patients with this/these lesion(s).SF3B1 mutations are associated to a chemoresistant progression of the disease after alkylating agents and/or fludarabine therapy.BIRC3 mutations are also associated to a chemorefractory disease, although their impact on response to treatment has not yet been proven in the context of clinical trials.Thus, the molecular bases of CLL aggressiveness have been extended beyond TP53 disruption.Due to the clonal evolution of the disease, a thorough search for TP53 mutations/deletions should be repeatedly performed before each line of therapy in view of the clinical relevance of these abnormalities; we know that 11q22-q23 deletions can be also acquired over time and there is evidence of the acquisition of NOTCH1 mutations.Finally, the efficacy of new drugs needs to be tested according to the presence of these molecular lesions for a future personalized medicine approach based on the genetic profile of CLL patients. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "The work by the authors described in this review was supported by AIRC Special Program Molecular Clinical Oncology, 5 x 1000, n. 10007, Milan, Italy (to RF and to GG); Compagnia di San Paolo, Torino, Italy (RF); Progetto FIRB-Programma \"Futuro in Ricerca\" 2008 (to DR); PRIN 2008 (to GG) and 2009 (to DR), MIUR, Rome, Italy; Progetto Giovani Ricercatori 2008 (to DR), Ministero della Salute, Rome, Italy; and Novara-AIL Onlus, Novara (to GG), Italy. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.",
"section_num": null
}
] |
10.1038/s41408-023-00870-2
|
Early-stage measurable residual disease dynamics and IGHV repertoire reconstitution during venetoclax and obinutuzumab treatment in chronic lymphocytic leukemia
|
<jats:title>Abstract</jats:title> <jats:p>In chronic lymphocytic leukemia, measurable residual disease (MRD) depth following fixed-duration treatment is associated with progression-free survival. MRD is usually reported at the end of treatment as a static parameter, but successive MRD measurements allow more accurately characterization of MRD dynamics, which may reveal additional prognostic information. To study the value of MRD dynamics, we used the IGHV leader-based NGS assay to longitudinally measured MRD in 60 patients treated in the HOVON-139/GIVE trial, in which previously untreated patients received two cycles of pre-induction with obinutuzumab, followed by one year induction treatment with obinutuzumab and venetoclax, followed by randomization to consolidation treatment with venetoclax or MRD-conditional consolidation. Induction treatment resulted in undetectable MRD (uMRD) (<10-5) in 93% of assessed patients, with 85% and 74% of patients retaining uMRD at six and twelve months after randomization. High levels of early-stage MRD, assessed during venetoclax ramp up, were associated with failure of achieving uMRD after induction treatment, whereas low levels of early-stage MRD predicted a low probability of loss of uMRD and disease progression after randomization. Venetoclax consolidation treatment significantly impaired polyclonal IGHV repertoire reconstitution after induction treatment with venetoclax and obinituzumab. In conclusion, very early-stage MRD levels were associated with treatment outcomes.</jats:p>
|
[
{
"section_content": "Measurable residual disease (MRD) is an important posttreatment biomarker that predicts progression-free survival (PFS) following fixed-duration treatment in chronic lymphocytic leukemia (CLL) [1,2].MRD is often reported as a static parameter, crosssectionally assessed at the end of treatment.However, as treatment is initiated and subsequently withheld, MRD levels follow an L-shaped trajectory, characterized by a rapid decrease followed by a delayed logistic regrowth phase.Successive MRD measurements, which more comprehensively capture MRD dynamics, may reveal additional prognostic information.Indeed, serial MRD measurements in the CLL14 and MURANO trials have demonstrated that compared to chemoimmunotherapy, treatment with venetoclax and anti-CD20 monoclonal antibodies reduced the rate of clonal regrowth [3,4].However, whether in the context of venetoclax and obinutuzumab, very early MRD measurements, performed shortly after treatment initiation, may already predict MRD levels and prognosis at later time points, has not been thoroughly investigated. In this study, we used the IGHV leader-based next-generation sequencing (NGS) assay to longitudinally measure MRD in samples from the HOVON-139/GIVE trial [5,6].The HOVON-139/GIVE trial is a first-line randomized phase-II trial, intended to evaluate the efficacy and safety of pre-induction with two cycles of obinutuzumab, followed by induction with six cycles of obinutuzumab and venetoclax and six cycles of venetoclax monotherapy, followed by randomization to either 12 cycles of venetoclax maintenance or MRD-conditional treatment cessation [5]. Of the patients treated in the HOVON-139/GIVE trial, 60/67 had ≥1 sample available for MRD measurement (Supplementary Table 1).In this cohort, 28 patients were randomized to receive an additional year of venetoclax consolidation, 27 were randomized to receive MRD-conditional consolidation treatment, four patients went off protocol at the time of randomization and one patient died before randomization (Supplementary Fig. 1).In the consolidation arm, 28/28 patients received the additional 12 cycles of venetoclax.In the MRD-conditional arm, 26/27 did not receive additional venetoclax consolidation, whereas one patient received 8/12 cycles of venetoclax consolidation.To allow a perprotocol comparison of a venetoclax consolidation arm to a no consolidation arm, we censored this patient at the time of randomization.The four patients who went off protocol at the time of randomization were included in the no consolidation arm, as they had not received any further anti-leukemic treatment.The median follow-up was 43 months (IQR 38-50). MRD was measured in 185 samples, representing four time points: during the fourth week of venetoclax ramp-up in induction cycle 1, at the end of the twelfth cycle of induction treatment (EOiT), and 6 and 12 months post-randomization (R+6 and R+12) (Supplementary Fig. 1).In 131/185 (71%) samples, MRD was undetectable (<10 -5 ).MRD dynamics for the total cohort are depicted in Fig. 1A.MRD was undetectable (<10 -5 ) in 7/36 (19.5%) patients in induction cycle 1 (C1), 40/43 patients (93.0%) at EOiT, 45/53 (84.9%) patients at R+6 and 39/53 (73.6%) at R+12.MRD levels were comparable between the randomization arms and are depicted in Supplementary Fig. 2A,B.An analysis of the interassay agreement between the IGHV leader-based NGS assay and a multicolor flow cytometry approach is available in the Supplementary Data. In the fourth week of induction cycle 1, following 11 weeks of treatment, MRD levels ranged from an upper bound of 3.5*10 -1 (35% of PBMCs) down to MRD < 10 -5 in 7/36 (19%) patients.In patients with MRD measurements available at both C1 and EOiT (n = 25), high levels of MRD (>10 -2 ) at C1 were associated with failure of reaching MRD < 10 -5 at EOiT (2/3 versus 0/22, P = 0.01) (Fig. 1B).In addition, in patients who achieved MRD < 10 -5 at EOiT and with MRD measurements available at C1 and R+12 (n = 27), loss of MRD < 10 -5 at R+12 exclusively occurred in patients with MRD ≥ 10 -4 at C1 (8/18 vs 0/9, P = 0.03, PPV 44%, NPV 100%) (Fig. 1C), suggestive of a shallow depth of response in this group.There was a positive rank correlation between the MRD level at C1 and at R+12 (Spearman's ρ = 0.48, P = 0.01).Most importantly, no patient with MRD < 10 -4 at C1 experienced disease progression during follow-up, resulting in superior PFS at 40 months, compared to patients with MRD ≥ 10 -4 at C1 (100% [95%CI 100-100] versus 83% [95%CI 67-99], log-rank P = 0.048) (Fig. 1D).Reaching MRD < 10 -4 at C1 was not associated with pre-treatment biomarkers of high-risk disease (Supplementary Table 3). We estimated the rate of disease eradication by calculating the log 10 fold change in disease burden between baseline and C1 (ΔMRD).Mean ΔMRD was 5.0 log 10 (i.e., >100.000-foldreduction), ranging from 2.6 to 7.3.Patients reaching MRD < 10 -5 at EOiT had higher ΔMRD, compared to patients that failed to reach MRD < 10 -5 (mean ΔMRD 5.1 versus 2.9, P = 0.02).The optimal ΔMRD cut-off to predict reaching MRD < 10 -5 at EOiT was estimated at 4.0 log 10 (AUC 0.93).ΔMRD was not associated with pre-treatment biomarkers of high-risk disease (Supplementary Table 4). Besides MRD detection, IGHV sequencing captures the entire IGHV repertoire, a proxy of the diversity of the healthy B cell pool..52],P = 0.002) (Fig. 2B). ",
"section_name": "Dear Editor,",
"section_num": null
},
{
"section_content": "In this study, we characterized the MRD dynamics and IGHV repertoire reconstitution of treatment-naive CLL patients treated with venetoclax and obinutuzumab in the HOVON-139/GIVE trial.Induction treatment induced deep and durable levels of MRD, resulting in uMRD (<10 -5 ) in 93% of assessed patients, with 85 and 74% of patients retaining uMRD (<10 -5 ) at 6 and 12 months after the end of induction therapy.Reaching low levels of MRD (<10 -4 ) in the fourth week of induction cycle 1, having received 11 weeks of treatment, was associated with a 100% PFS rate in this cohort.Conversely, high MRD (>10 -2 ) at C1 and a slow rate of early MRD eradication were associated with failure to achieve uMRD (<10 -5 ) at the end of induction treatment. Very early MRD measurements are a proxy for the rate of disease eradication.Our data indicate that a rapid reduction in disease burden can be used as a biomarker which, irrespective of established genomic features, signifies therapy-sensitive disease, resulting in deep MRD levels at the end of treatment and a long time to progression.Conversely, slow disease eradication is a prognostically poor sign.Whereas these patients still reach deep levels of MRD (<10 -4 in this cohort), their response is shallower, culminating in the earlier loss of uMRD and a shorter time to progression.This notion is in line with the recent results of the UK TAP Clarity trial, where, in the relapsed and refractory setting, achieving a 100-fold or greater reduction of disease burden after 2 months of treatment with venetoclax and ibrutinib was associated with sustained low MRD and clinical response after a follow-up of 3 years [7,8].Moreover, in treatment-naïve patients receiving zanubrutinib, obinutuzumab and venetoclax, achieving a 2.6 log-reduction (400-fold) in MRD between baseline and cycle 5 was predictive of reaching undetectable MRD at cycle 8 and retaining MRD < 10 -5 after 1 year of treatment cessation [9]. IGHV repertoire sequencing through the IGHV-leader NGS assay allows for simultaneous detection of MRD and characterization of the healthy polyclonal IGHV repertoire.High IGHV diversity is indicative of a large pool of non-malignant healthy B cells.As anticipated, following treatment with obinutuzumab and venetoclax, IGHV repertoire diversity was low.After cessation of obinutuzumab, IGHV repertoire diversity increased to near-normal levels in patients who did not receive venetoclax consolidation treatment.However, in patients who received venetoclax consolidation, reconstitution of the IGHV repertoire was impaired.Venetoclax is toxic to both CLL cells and healthy B cells [10].Here, we demonstrate that consolidation treatment with venetoclax hampers recovery from the state of B cell aplasia induced by obinutuzumab, possibly resulting in a longer period of impaired humoral immunity.Whether this may have contributed to the higher rate of grade 2 or higher infectious adverse events in patients randomized to the venetoclax consolidation arm compared to the MRD-conditional arm (16/ 32 [50%] versus 6/30 [20%], as previously reported [5]), warrants a more in-depth analysis, incorporating more detailed clinical and serological data. In conclusion, in treatment-naïve CLL patients treated with venetoclax and obinutuzumab, high levels of early MRD (>10 -2 ) and a slow rate of MRD eradication, measured after 11 weeks of treatment, predicted failure to reach uMRD (<10 -5 ) at the end of induction treatment.Conversely, patients with low levels of early MRD (<10 -4 ) had a low probability of uMRD loss and disease progression.In addition, venetoclax consolidation treatment impaired polyclonal IGHV repertoire reconstitution after induction treatment with venetoclax and obinutuzumab, potentially associated with a higher chance of adverse infectious events.IGHV diversity was quantified using Shannon's diversity index.Statistical significance was evaluated using Welch's t-test.C1 induction cycle 1, EOiT end of induction treatment, IGHV immunoglobulin heavy-chain variable, R+6 6 months after randomization, R+12 12 months after randomization. ",
"section_name": "MRD depth",
"section_num": null
}
] |
[
{
"section_content": "The authors would like to thank all patients, their families, study monitors and investigators that participated in the HOVON-139/GIVE trial.The HOVON-139/GIVE trial was funded by F Hoffmann-La Roche (ML29995), who had the opportunity to review and comment on this paper. ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "For the methods, please refer to the Supplementary Data.P. J. Hengeveld 1,2 , J. Schilperoord-Vermeulen 2 , M. Y. van der Klift 2 , J. M. N. Dubois 3 , P. M. Kolijn 2 , F. G. Kavelaars 4 , M. Rijken 4 , J. A. Dobber 3 , K. Nasserinejad 5 , S. Kersting 6 , P. E. Westerweel 1 , A. P. Kater 3 , A. W. Langerak 2,7 and M-D.Levin 1,7 ✉ 1 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, the Netherlands. 2Department of Immunology, Erasmus MC, Rotterdam, the Netherlands. 3Department of Hematology and Experimental Immunology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands. 4Department of Hematology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands. 5 JMND has received research funding from Roche/Genentech.SK has received personal fees from Janssen, AbbVie, Novartis, Gilead and Celgene; and research funding from AbbVie, Janssen, AstraZeneca and Roche/Genentech.APK has received personal fees from AbbVie, LAVA, Genmab, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb; and research funding from AbbVie, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb.AWL has received research funding via an unrestricted grant from Roche-Genentech and speaker fees from Janssen.M-DL has received personal fees from AbbVie, Janssen, and Roche; and research funding from AbbVie, Janssen, AstraZeneca, and Roche/Genentech.The remaining authors declare no competing financial interests. The online version contains supplementary material available at https://doi.org/10.1038/s41408-023-00870-2. Correspondence and requests for materials should be addressed to M-D.Levin. 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": "METHODS",
"section_num": null
},
{
"section_content": "For the methods, please refer to the Supplementary Data.P. J. Hengeveld 1,2 , J. Schilperoord-Vermeulen 2 , M. Y. van der Klift 2 , J. M. N. Dubois 3 , P. M. Kolijn 2 , F. G. Kavelaars 4 , M. Rijken 4 , J. A. Dobber 3 , K. Nasserinejad 5 , S. Kersting 6 , P. E. Westerweel 1 , A. P. Kater 3 , A. W. Langerak 2,7 and M-D.Levin 1,7 ✉ 1 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, the Netherlands. 2Department of Immunology, Erasmus MC, Rotterdam, the Netherlands. 3Department of Hematology and Experimental Immunology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands. 4Department of Hematology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands. 5 ",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "JMND has received research funding from Roche/Genentech.SK has received personal fees from Janssen, AbbVie, Novartis, Gilead and Celgene; and research funding from AbbVie, Janssen, AstraZeneca and Roche/Genentech.APK has received personal fees from AbbVie, LAVA, Genmab, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb; and research funding from AbbVie, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb.AWL has received research funding via an unrestricted grant from Roche-Genentech and speaker fees from Janssen.M-DL has received personal fees from AbbVie, Janssen, and Roche; and research funding from AbbVie, Janssen, AstraZeneca, and Roche/Genentech.The remaining authors declare no competing financial interests. ",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-023-00870-2. Correspondence and requests for materials should be addressed to M-D.Levin. 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
}
] |
10.3390/cancers14143376
|
Optical Genome Mapping: A Promising New Tool to Assess Genomic Complexity in Chronic Lymphocytic Leukemia (CLL)
|
<jats:p>Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical–biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations (<15–20%). OGM underscored additional abnormalities, providing novel structural information on known aberrations in 55% of patients. Regarding genomic complexity, the number of OGM abnormalities had better accuracy in predicting TTFT than current methods (C-index: 0.696, 0.602, 0.661 by OGM, CBA, and CMA, respectively). A cut-off of ≥10 alterations defined a complex OGM group (C-OGM, n = 12), which included 11/14 patients with ≥5 abnormalities by CBA/CMA and one patient with chromothripsis (Kappa index = 0.778; p < 0.001). Moreover, C-OGM displayed enrichment of TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median: 2 vs. 43 months, p = 0.014). OGM is a robust technology for implementation in the routine management of CLL patients, although further studies are required to define standard genomic complexity criteria.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is characterized by the clonal proliferation and accumulation of abnormal mature B cells, typically CD5 positive, in the blood and lymphoid tissues.This disease is the most common leukemia in Western countries and displays a highly variable clinical course, ranging from very indolent cases to patients with aggressive disease and rapid progression [1].This clinical heterogeneity has important consequences for clinical follow-up, therapeutic strategies, and patient survival.Because of this, several prognostic and predictive biomarkers have been identified over the years [2]; the most relevant being deletions and/or mutations in TP53, located at 17p13, and the mutational status of the variable region of the immunoglobulin heavy chain (IGHV) gene [1].FISH analysis of the four-probe set included in Dohner's hierarchical model [targeting deletions on 13q14, 11q22 (ATM), 17p13 (TP53), and trisomy of chromosome 12] is currently considered the gold standard for cytogenetic assessment in CLL [3].However, this strategy underestimates the true genetic complexity of the tumor cells, which also predicts a more aggressive clinical course [4,5].Although the negative impact of complex karyotypes (CK) has been extensively demonstrated in patients treated with standard chemoimmunotherapy regimes [6][7][8][9], its clinical relevance in patients receiving new treatment modalities has not been fully established.While initial data from clinical trials with novel agents suggested an adverse significance of CK [10][11][12][13], recent studies, including extended follow-ups of older trials, pooled analyses, or new drug combinations, reported controversial results regarding its adverse significance [14][15][16][17][18][19][20][21].Thus, additional analyses are required to clarify the prognostic/predictive impact of genomic complexity in the new treatment era. To date, most of the outcome analyses of genomic complexity published in CLL have been reported using chromosome banding analysis (CBA) data and defined CK as the presence of three or more chromosomal alterations detected in the same cell clone [22,23].An increasing number of chromosomal abnormalities in the karyotype has been correlated with a worse clinical evolution of CLL patients, and, notably, the types of aberrations detected also modulates its prognostic impact [8,[24][25][26].Moreover, chromosomal microarray analysis (CMA) has replaced CBA and FISH in some laboratories for the detection of copy number variants (CNVs).Two large retrospective studies from the European Research Initiative on CLL (ERIC) demonstrated that genomic complexity, detected by either CBA or CMA, is an independent adverse prognosticator in CLL, five or more being the number of aberrations that best predicted a worse evolution [9,27].In addition, our group compared the performance of CBA and CMA to detect genomic complexity in a cohort of 340 patients enriched in CK.Although the prognostic utility of both methods was validated in this study, we demonstrated that risk stratification by CBA and CMA was not fully equivalent due to the intrinsic limitations of each technique [28].The ERIC group recently published their recommendations, in which CBA still remains the gold standard methodology, capable of providing information on structural and numerical aberrations at a low resolution of the whole genome while also allowing the overview of the clonal landscape and intraclonal hierarchy [29].It is noteworthy that, while CBA results rely on the in vitro proliferative capacity of the altered clones, CNV profiles can be defined at high resolution by CMA without having to culture samples.In some cases, CMA could reveal multiple CNVs or even chromothripsis events associated with monosomies or unknown cytogenetic elements reported in the karyotype.On the contrary, balanced abnormalities are only detectable by CBA, and subclonal alterations present at low percentages (<20%) could be missed by CMA due to its lower sensitivity [28][29][30][31]. Optical genome mapping (OGM) has emerged as a promising novel technique that could potentially overcome the aforementioned limitations in a single test.OGM relies on high-throughput imaging of long DNA molecules (>250 Kb) fluorescently labeled at a specific 6 bp sequence motif, occurring approximately 15 times per 100 Kb in the human genome.The unique labeling pattern throughout the genome confers the ability to unambiguously identify the genomic location of every imaged molecule, generating a local consensus map that can be compared to a reference genome to detect structural variants (SVs).This analysis is performed via the so-called rare variant pipeline, which specifically addresses mosaic samples and automatically detects both balanced and unbalanced SVs from single molecules, genome-wide, starting at 5 Kb and down to a 1% allele fraction.In addition, genome coverage depth information is also used to address copy number variant (CNV) and whole-chromosome aneuploidy identification [32,33].Several recent publications have demonstrated OGM's good performance in the cytogenomic assessment of different hematological malignancies, especially focused on myeloid neoplasms (acute myeloid leukemia and myelodysplastic syndromes) and acute lymphoblastic leukemia cases.In these studies, OGM effectively detected most of the clinically relevant abnormalities reported by standard methods and, in some cases, also provided new cytogenomic information [34][35][36][37].Conversely, the assessment of genomic complexity by OGM or the global characterization of the cytogenetic abnormalities identified in each patient was beyond the scope of the published studies, which focused on the identification of SVs currently interrogated in diagnostic procedures or that overlapped with genes defined as potentially relevant in leukemogenesis. In the present study, we aimed to assess the performance of OGM for the cytogenomic characterization of CLL patients in comparison with previous CBA and CMA information, with a special focus on genomic complexity.Moreover, we analyzed the potential use of OGM for the prognostic stratification of CLL patients based on genomic complexity. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "A total of 42 patients diagnosed with CLL (n = 39; 92.9%) and monoclonal B-cell lymphocytosis (n = 3; 7.1%) with available CBA, FISH and CMA results were included.Due to the purpose of the study, 18 of them presented CK by CBA (42.9%).All analyses were performed on cryopreserved peripheral blood mononuclear cells (PBMCs) obtained simultaneously to CBA or within one year.The median time from diagnosis to sampling was 10 months (range: 0-174 months), and only two patients (4.8%) received treatment prior to the genetic analyses.The main characteristics of the entire cohort are summarized in Table 1.The study was carried out in accordance with national and international guidelines (Professional Code of Conduct, Declaration of Helsinki) and approved by the Hospital del Mar Ethics Committee (2017/7565/I).Of note, CBA, CMA, and FISH data from 17 patients were included in a previous publication [28]. ",
"section_name": "Patients and Samples",
"section_num": "2.1."
},
{
"section_content": "For each sample, 1.5 million cells were used to extract ultra-high molecular weight (UHMW) DNA following manufacturer's instructions (Bionano Prep Frozen Cells DNA Isolation Protocol, Bionano Genomics, San Diego, CA, USA).The Direct Label Enzyme (DLE-1) was used to enzymatically transfer DL-green fluorescent labeling to UHMW DNA following the Bionano Prep Direct Label and Stain (DLS) Protocol (Bionano Genomics).Labeled molecules were directly loaded onto a Saphyr chip and imaged via the Saphyr instrument (Bionano Genomics).The Saphyr Chip's nano-channels allow only a single linearized DNA molecule to travel through via electrophoretic migration.Multiple cycles were run to generate approximately 1300 Gb of data per sample and reach an average genome coverage of 300×. ",
"section_name": "DNA Extraction, Labelling and Data Collection for Optical Mapping",
"section_num": "2.2."
},
{
"section_content": "Genomic analysis was performed with the rare variant pipeline included in Bionano Solve software (v.3.5) and visualized with Bionano Access software (v1.6) (Bionano Genomics).Briefly, the imaged molecules were aligned against a reference assembly (hg19), and two algorithms were applied to identify SVs and large CNVs.The structural variant calling algorithm assessed any deviation in the labeling pattern to detect insertions, inversions, deletions, duplications, and intrachromosomal or interchromosomal translocations.Of note, interstitial deletions or inversions >5 Mb were called as intrachromosomal translocations by this pipeline.Copy number variant calls were inferred from the label coverage detected in each genomic interval, indicating the presence of gains or losses in those regions with a significant increase or decrease from baseline.All abnormalities called by the software were prefiltered according to default recommended confidence scores for Bionano Access version 1.6 (confidence scores: insertion: 0, deletion: 0, inversion: 0.7, duplication: -1, intra-translocation: 0.3, inter-translocation: 0.65, and CNV: 0.99) or whether they were described in the OGM control sample dataset provided by Bionano Genomics.In addition, as certain regions of the genome had an unusually high variance in their coverage that could be due to an unreliable molecule alignment (often concentrated around centromere and telomere regions), both pipelines masked SV and CNV calls occurring in these regions.Finally, we also applied size filters to further reduce the number of variants and obtain results comparable to those observed by CMA.The cut-off size for SVs (inversions, insertions, deletions, and duplications) was set at 100 Kb, while only those CNVs larger than 500 Kb were called by the CNV tool.Prefiltered SV and CNV data were downloaded and visually reviewed with the Bionano Access software in order to merge segmented CNVs, discard those SVs duplicated in the results, and variants found as benign polymorphisms in the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home;accessed on 4 April 2022).In addition, the curated outputs from both algorithms were also compared in order to identify duplicities.In this regard, CNV gains and losses which overlapped with duplications and deletions or intrachromosomal translocation breakpoints, respectively, were considered as the same abnormality, and coordinates from the SV pipeline were taken.The final aberrations found in each patient were recorded for comparison with the results obtained by standard techniques.Additionally, less restrictive filters for confidence scores were applied in four patients with undetected known abnormalities to ascertain whether these were missed due to filtering issues or due to real OGM technical limitations. ",
"section_name": "Data Assembly, Structural Variant Calling and Filtering",
"section_num": "2.3."
},
{
"section_content": "Peripheral blood (PB) samples were used to set two parallel cultures using phorbol-12myristate-13-acetate (TPA) and immunostimulatory cytosine guanine dinucleotide (CpG)oligonucleotide DSP30 plus interleukin 2 (IL-2) as mitogens.At least 20 metaphases from each culture were analyzed when possible, and a minimum of 10 were evaluated in three patients with abnormal karyotypes.Karyotypes from the most altered culture (more abnormal clones or altered metaphases detected) were collected and described according to the International System for Human Cytogenetic Nomenclature (ISCN 2020) [38].The number and type of abnormalities were recorded as previously described [28]. Data from the routine FISH panel for TP53 (17p13), ATM (11q22), D13S319 (13q14), and chromosome 12 centromeric region (Metasystems, Altlussheim, Germany) assessed in uncultured PB were also collected.In order to validate some of the OGM findings, additional FISH analyses were performed using commercial locus-specific, whole chromosome painting probes (Metasystems; Abbot Molecular, Abbott Park, IL, USA) or custom bacterial artificial chromosome (BAC) probes from the Children's Hospital Oakland Research Institute library (Oakland, CA, USA).Details regarding the additional FISH probes tested are shown in Table S1. ",
"section_name": "Chromosome Banding Analyses and Fluorescence In Situ Hybridization",
"section_num": "2.4."
},
{
"section_content": "Chromosomal microarrays were assessed in DNA from PBMCs (n = 12) or PB CD19+ purified cells (n = 28).DNA was amplified, labeled, and hybridized to CytoScan HD (n = 30) or CytoScan 750K (n = 10) platforms (ThermoFisher Scientific, Eugene, OR, USA) according to manufacturer's protocols.Copy number variants found as benign polymorphisms in the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home;accessed on 4 April 2022) were excluded.The remaining aberrations, irrespective of size, were collected and reported using annotations of genome version GRCh37/hg19.CNV counting for risk classification of patients was performed using previously described criteria [27,28].As the current analysis pipelines used for OGM results do not enable copy-number neutral loss of heterozygosity (CN-LOH) detection, these abnormalities were not considered in the present study. ",
"section_name": "Chromosomal Microarray Analyses",
"section_num": "2.5."
},
{
"section_content": "First, we assessed the capability of OGM to detect the abnormalities previously defined by CBA, FISH, and CMA.In this regard, results obtained from standard techniques were compiled to set the list of abnormalities to be identified by OGM.The following criteria were applied: (i) for CNVs, coordinates defined by CMA were prioritized while FISH abnormalities or well-defined gains or losses identified in the karyotype but missed by CMA for technical reasons were also included; (ii) for balanced rearrangements (inversions or translocations), breakpoints defined in the karyotype were collected; (iii) among complex abnormalities in the karyotype affecting material of unknown origin, only the chromosomal breakpoints where additional material was identified were recorded to assess the presence of translocations by OGM in these regions.OGM results were considered concordant if the same abnormality was identified, even if it showed some variability in size or breakpoints.The percentage of detection by OGM was recorded, and potential causes of discrepancy were evaluated.Regarding additional abnormalities called by OGM, images from the available karyotypes and CMA data were reviewed to ascertain the potential relevance of OGM findings.Those abnormalities that could be compatible with any element observed in the karyotype were considered 'real' if validated by additional FISH analyses that allowed karyotype reinterpretation.In addition, rearrangements associated with chromothripsis events or CNVs detected by CMA were considered 'real' if validated by FISH or 'potentially real' if they were not specifically studied by FISH.Results from the additional FISH validations performed were used to finally classify some uncertain variants as 'real' or 'false positive' calls and to define custom criteria to identify novel OGM translocations with poor quality.The latter were classified as 'potential false positive' and were not considered in the final OGM abnormalities count. In addition, we assessed the potential use of OGM for the identification of higher-risk patients based on their genomic complexity.To this end, the number of abnormalities reported by OGM was compared with that identified by standard methods in the whole cohort.Furthermore, patients were categorized based on the complexity detected by OGM and compared with the previously defined risk groups in the two ERIC studies [9,27]. ",
"section_name": "Comparison among Techniques",
"section_num": "2.6."
},
{
"section_content": "Descriptive statistics were used to provide frequency distributions of discrete variables, while statistical measures were used to provide median values and ranges for quantitative variables.Chi-square or Fisher exact tests were used to compare discrete variables and the Mann-Whitney U test to assess continuous variables.The number of abnormalities recorded by OGM and CBA or CMA were correlated by Spearman's Rho, and the concordance among techniques was established via Cohen's Kappa index.Time to first treatment (TTFT) was defined as the time interval between the sampling date and the date of first treatment.The Kaplan-Meier method was used to estimate the distribution of TTFT in the 40 treatment-naïve patients.Comparisons among patient subgroups were performed via the Log-rank test.The concordance statistic (C-index) for right-censored data was calculated to assess the accuracy of each technique in predicting TTFT.As only four patients died during follow-up, overall survival analyses were not assessed.Likewise, multivariate analysis could not be performed due to the limited size of the present cohort.Statistical analyses were performed using SPSS v.23 software (SPSS Inc, Chicago, IL, USA) and R v3.5.2 (R Foundation for Statistical Computing, Vienna, Arustria).p-values < 0.05 were considered statistically significant. ",
"section_name": "Statistical Analyses",
"section_num": "2.7."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "OGM results from all 42 samples run met the recommended quality metrics, showing an average molecule N50 (for molecules > 150 Kb) value of 305 Kb and an average 363-fold effective coverage (Table S2).Globally, a total of 2202 abnormalities were called with the recommended prefiltering criteria for the Bionano Access version 1.6.Nonetheless, when the output was further filtered based on size (≥100 Kb) and visually reviewed, the number of detected abnormalities reduced to 579.In all, 36 deletions, 3 inversions, 4 duplications, 142 intrachromosomal translocations, 185 interchromosomal translocations, 59 CNV gains, and 150 CNV losses were considered for comparison with standard techniques.Of note, 42 of these CNVs were also called as duplications (n = 6), deletions (n = 24), or intrachromosomal translocations (n = 12) by the SV calling pipeline and subsequently not counted in the genomic complexity estimation (Figure S1). ",
"section_name": "Global SV and CNV Detection by OGM",
"section_num": "3.1."
},
{
"section_content": "In the whole cohort, a total of 309 abnormalities previously detected by CBA, FISH, and/or CMA were considered to explore the concordance with OGM results.While CNVs represented the vast majority of these (193 losses and 67 gains), information from only 49 translocations was previously available.Of note, in 29 of the rearrangements, only one of the translocation breakpoints was identified by CBA, as they involved additional material of unknown origin in the karyotype (\"add\").Globally, 90.3% of the previously known alterations (279/309) could be identified by OGM.Of note, no significant differences in the detection rate were observed between patients with non-CK and those carrying a CK by CBA (detection of 83/89 (93.3%) and 196/229 (89.1%), respectively; p = 0.396).In order to reach this concordance, a lower confidence score threshold was applied to properly detect five known translocations, and more permissive filtering criteria were used to pick up 15 alterations initially missed by the CNV pipeline.The latter included nine small CNVs (range: 292-488 Kb) filtered by the 500 Kb cut-off size used for CNVs, and six larger CNVs (range: 1282-22,049 Kb), which had been detected in less than 20% of cells by FISH and/or CMA and could also be visually suspected in the whole chromosome CNV view of the Bionano Access software.It is noteworthy that, when comparing only those abnormalities included in the routine CLL FISH panel, the concordance rate increased to 95.6% (detection of 55/57 known deletions on 13q14, 11q22 (ATM), 17p13 (TP53) or trisomy 12).In this regard, OGM failed to detect a 13q14 deletion and a TP53 deletion found in 15% and 17% of nuclei by FISH, respectively, and did not pick up any additional one of these recurrent CLL alterations. As well as their detection, we could evaluate OGM's accuracy regarding the definition of genomic location and size for 238/247 CNVs previously defined by CMA.To this end, CNVs detected by both techniques showed a high overlap with a median difference of only 22 Kb in the defined coordinates (range: 0-8.1 Mb).Major discordances were due to the known OGM masking of the terminal 2-5 Mb from large CNVs or whole chromosome aneuploidies, which were located at repetitive regions (12/238, 5.0%).Once these discrepancies found to be masked were excluded, differences larger than 2 Mb in size (range: 2.1-5.7 Mb) were only observed in five CNVs (2.1%).It is noteworthy that OGM allowed the definition of the genomic coordinates from eight known CNVs that had been previously detected by FISH and/or CBA but missed by CMA.In addition, similar results were observed when the alterations called by the SV and CNV pipelines were compared. Among the 30 missed abnormalities, 14 were not expected to be detected by OGM as they were translocations with breaks in the (peri-)centromeric or telomeric regions (n = 11), which are not covered by OGM labels, two relatively small telomeric deletions (1.5 and 2.5 Mb) and one gain involving chromosome Y (4.1 Mb) that were masked by the CNV analysis pipeline to avoid the calling of false positive in these highly variable regions.On the other hand, sensitivity explained the discrepancy in 13 of the remaining 16 aberrations that were susceptible to detection by OGM (Table 2).In this regard, nine CNVs undetected by OGM were identified in a median of 17% of cells by FISH and/or CMA (range: 3-25%).Among these, three were not detected by CMA either.Similarly, sensitivity could explain four missed translocations that had been detected in minor clones by CBA related to the culture selection of abnormal clones intrinsic to this technique.Although the percentage of abnormal cells could not be set for these translocations, it is important to note that the SV pipeline correctly detected some other abnormalities found in only 5% of cells by FISH. Finally, OGM was not able to identify a small 138 Kb deletion encompassed in a complex chromothripsis event and two additional abnormalities that could not be associated with any feasible cause of discrepancy (Table 2).Percentage not available; although the \"add(9)(q34)\" could be a minor clone (detected as clonal evolution in only two out of 13 abnormal metaphases), other abnormalities from the same clone were properly detected.WCP revealed that the additional material was from chr. 17 and, as seen by CBA, the chr.17 telomeric region could be involved in the fusion (telomeric regions are masked by the SV pipeline) Abbreviations: NA = Not available; WCP: whole chromosome FISH painting. Detailed results obtained by each of the standard techniques, the 309 known abnormalities considered, and their detection by each of the OGM analysis pipelines are shown in Table S3. ",
"section_name": "Concordance Rate of OGM Results with Standard Techniques",
"section_num": "3.2."
},
{
"section_content": "As OGM provided data not only on CNV genomic location but also structural information, we next assessed whether OGM could provide a more comprehensive structural overview of the known aberrations detected compared to a CBA, FISH, and CMA combination.Of note, the number of interchromosomal translocations called by OGM (n = 194) was much higher than expected, taking into account previous CBA data, even in non-CK patients.In this regard, additional analyses were performed to ascertain which calls should be considered real or, on the contrary, filtered as potential false-positive calls.A total of 72 translocations were considered unsuitable for these focused comparisons as they were distributed among only three patients (with 16, 20, and 36 translocations, respectively) that showed highly complex karyotypes including several abnormalities involving material of unknown origin, difficult to compare with OGM or validated by FISH.Thus, these three cases were excluded from the comparison.Among the remaining 122 translocations with CBA and CMA data available, 12 (9.8%)corresponded to previously well-defined translocations (seven known translocations, five of them having two breakpoints identified in each derivative chromosome), and 63 (51.7%) could potentially be reflecting unknown rearrangements associated with previously described abnormalities.Conversely, 47/122 (38.5%) were not supported by CBA and were called in apparently normal regions by CMA (Figure 1).Visual inspection of OGM calls revealed that, contrarily to that observed for those concordant or potentially real calls, most apparently false-positive calls displayed few labels mapped in at least one of the translocation partners (≤10), which also correlated with low 'RawConfidenceLeft' or 'RawConfidenceRight' parameter values (<10) in the SMAP file generated by the rare variant pipeline (Figure 2).Three of these translocations with low labels [t(5;6)(q22.3;q27)called in patients 3 and 5, and t(11;19)(q23.3;p13.3) in patient 13] were assessed by interphase FISH using targeted custom BACs break-apart probes and could not be validated.Moreover, 51 additional calls with high label coverage in both translocation partners were indirectly validated by whole chromosome FISH painting (Table S4).These findings suggested that the final set of translocation calls to be considered could be manually filtered based on these criteria.On the other hand, high variability was observed in the number of self-molecules supporting the translocations called by OGM (median: 45; range: 5-175).A total of 27 of the apparently well-covered translocations were based on less than 10 molecules, which is the cut-off used by some authors in hematological studies [39,40].Thereby, we next evaluated if these low values could also be associated with a higher rate of false-positive calls, but no conclusive results were obtained.Fourteen translocations were consistent with CBA or whole chromosome FISH painting (WCP) information, whereas two were not validated by interphase FISH with custom BAC probes and were classified as false-positive calls.Notably, five of the latter were subclonal abnormalities by CBA detected in only 5-7% of nuclei by FISH or confirmed in clones initially missed by CBA (Table S4).These results suggested that the number of molecules supporting the translocation calls could be impaired by both potential errors in the mapping of the sample or by intrinsic characteristics of the abnormal clone.Thus, this parameter was not considered in the final filtering of the translocations found in the whole cohort.Likewise, contrarily to that described by other authors [37], as most of the validated translocations failed the assembly chimeric score provided by the software, this parameter was not considered either.Globally, 45/192 (23.4%) interchromosomal and 5/142 (3.5%) intrachromosomal translocation calls were manually discarded from the analysis, and none corresponded to any known abnormality by conventional techniques (Figure 1). Once data were further manually filtered, novel structural findings on previously known aberrations were found in 23/42 patients (54.8%).On the one hand, when compared with CBA, OGM identified 23 intra-and 37 interchromosomal translocations associated with 33 of the previously known structural rearrangements: 15 called as translocations, inversions, insertions, or deletion, and 18 reported as \"add\" in the karyotype.Interestingly, OGM showed rearrangements involving at least three chromosomes in 13/18 (72.2%) abnormalities in which only one of the breakpoints could be identified in the karyotype (\"add\").Moreover, it also revealed higher complexity in 7/15 (46.7%) apparently well-described abnormalities (Figure 3).On the other hand, 93 intra-and 30 interchromosomal translocations underscored by OGM were associated with CNVs identified by CMA.Apart from unbalanced rearrangements underlying large terminal CNV or rearrangements associated with complex chromothriptic events, unexpected rearrangements between apparently unrelated small interstitial CNV were also recorded.Interestingly, apart from the three translocations with breakpoints at 13q14 defined by CBA, OGM detected novel translocations associated with known 13q14 or 11q22 (including ATM) deletions in 5/29 (17.2%) and 2/11 (18.2%), respectively (Figure S2).Examples of cases in which OGM revealed that abnormalities apparently well-described by CBA were more complex than initially assumed.(A) Patient #24 showed an abnormal karyotype described as containing a t(5;18)(p15;q21) and a del(16)(q12).Whole-genome Circos plot and Circos plot from chromosomes 5, 16 and 18 obtained by OGM revealed that the initially reported abnormalities were a three-way translocation between these chromosomes.OGM results were confirmed by WCP, in which material from both chromosomes 16 and 18 was detected in the der(5) chromosome; (B) The karyotype defined in patient #CK11 contained two deletions [del(X)(q25) and del(14)(q22q32)] and additional material of unknown origin at chromosomes 5 and 8 [add(5)(q31) and add(8)(q24)].Circos plots show the four translocations identified by OGM that shared breakpoints on chromosomes X, 5 and 8, and two breakpoints on chromosome 14 were located at the start and end coordinates from a known CNV loss identified by CMA.FISH analysis allowed their validation and karyotype reinterpretation.The corrected ISCN formula for this abnormal clone was finally defined as 47,XY,der(X)t(X;5)(q22.1;q32),der(5)t(5;14)(q32;q32.2),der(8)t(X;8)(q22.1;q23.3),+12,der(14)t(8;14)(q23.3;q24.1). In addition to the aforementioned translocation calls, OGM also identified three inversions and six deletions ≥100 Kb previously undetected by conventional techniques.Among these deletions, three could be identified in the revision of raw CMA data (sizes: 0.8, 1.8, and 4.1 Mb), and one was validated by FISH (6% of the nuclei presented the deletion using a custom BAC probe within the 4.2 Mb region lost by OGM).The two remaining discordant deletions were of just 230 Kb and 239 Kb and were not further assessed.In addition, in the two patients with no CMA study available, OGM underscored the presence of two large CNVs associated with a cryptic translocation by CBA (validated by FISH) and a 1.2 Mb deletion associated with a known t(13;17)(q14;p13). ",
"section_name": "Novel Abnormalities Detected by OGM",
"section_num": "3.3."
},
{
"section_content": "Regarding the genomic complexity identified by OGM, after the filtering and manual revision of raw results, the median number of abnormalities among the whole cohort was five (range: 1-70).As expected, those patients showing a CK by CBA displayed a significantly higher median number of alterations compared with non-CK patients (20 [range: 4-70] vs. 3 [range: 1-48]; p < 0.001).These differences were related to higher detection of both translocations and CNVs in the CK group (Figure S3).A significant strong correlation with the number of anomalies recorded by CBA and CMA was observed (r s = 0.734 and r s = 0.845, respectively).Nonetheless, it is noteworthy that OGM detected a significantly higher complexity than conventional techniques, which detected a median of two and three abnormalities by CBA and CMA, respectively (range: 0-16 for CBA and 0-30 for CMA) (Figure S3).In terms of prognostic impact, considering the number of abnormalities as a continuous variable, OGM presented a higher accuracy in predicting TTFT than CBA and CMA (C-index: 0.696, 0.602, 0.661; by OGM, CBA, and CMA, respectively). Next, we evaluated if OGM could be a useful tool for risk stratification based on genomic complexity in CLL.Thus, considering the abnormalities found by OGM and comparing their distribution among the CBA and CMA risk categories defined in previous studies [28], an arbitrary cut-off of ≥10 alterations was used to define two risk groups by OGM.Based on this criterion, genomic complexity by OGM was assigned to 12 patients (28.6%) (Complex by OGM, C-OGM), while the remaining 30 patients were classified into a lower risk category (Non-complex by OGM, NC-OGM).Globally, the C-OGM group included 11/14 patients considered to be in the highest risk category by at least one standard technique (CBA and/or CMA) and one additional patient with a normal karyotype that, despite being considered to be in the CMA intermediate-risk group, displayed chromothripsis (Figure 4).Detailed OGM information from the three higher-risk patients, according to conventional methods, not included in the C-OGM group is shown in Figure 3B (patient CK11) and Figure S4 (patients 12 and CK16).Globally, for the identification of higher-risk patients, a moderate-strong agreement was observed between OGM and the combination of CBA and CMA methods (κ = 0.778; p < 0.001).As expected, the C-OGM group also showed an increased proportion of patients with TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median TTFT: 2 vs. 43 months, p = 0.014) compared with NC-OGM patients (Figure S5).Conversely, none of the other patient characteristics compared presented significant differences (Table 3).Interestingly, all patients with multiple trisomies by CBA (namely +12, +19, +/-other abnormalities; patients 21, CK1, CK15, CK18), previously described to exhibit very favorable outcomes [24], were categorized into the NC-OGM group with four to eight abnormalities detected by this technique. ",
"section_name": "Global Genomic Complexity Found by OGM",
"section_num": "3.4."
},
{
"section_content": "Over the last decade, several works have highlighted the prognostic and predictive value of genomic complexity in patients with CLL.Recent large ERIC cooperative studies have validated the performance of both CBA and CMA in the risk stratification of CLL and suggested the detection of five or more chromosomal abnormalities as an adverse biomarker to be implemented in routine CLL management [9,27].As both techniques had some intrinsic limitations that led to discordant risk stratification in up to 30% of patients [28], herein, we aimed to evaluate whether the recently developed OGM technology is a suitable alternative to overcome these discrepancies.To the best of our knowledge, this is the largest cytogenetic characterization via OGM conducted to date in CLL patients. Overall, we demonstrated the feasibility of OGM in detecting 90% of the alterations previously defined by the combination of standard techniques (CBA, FISH, and/or CMA) in a single assay.In addition, this technique afforded not only high accuracy in the definition of genomic coordinates for CNVs when compared with CMA but also provided additional structural information on previously known aberrations.As expected, the concordance reached was similar to that reported in other hematological malignancies, in which OGM detection of clinically relevant known abnormalities ranged between 90 and 100% [35][36][37][40][41][42].The number of missed abnormalities in the present series was low, but the discrepancies found underscore some OGM limitations that should be taken into account.First, although this is a whole-genome view technology, the analysis is compromised at some locations that could not be properly labeled or mapped (such as centromeres, telomeres, or regions with a high number of repetitions).Thus, nearly half of the 30 abnormalities missed in our study involved these regions and were not expected to be detected by OGM.Considered individually, as none had been previously described as clinically relevant in CLL, their misdetection is not assumed to be highly relevant for patient management.However, as suggested by other authors, manual inspection of OGM imaged molecules is highly recommended for pathologies in which aberrations affecting these regions are well-known prognostic biomarkers (e.g., CRLF2 rearrangements involving the PAR1 region on chromosome X in acute lymphoblastic leukemia) [37,41,42].It is noteworthy that these studies were performed against hg19 [GRCh37], as used herein, and some cases would be identified using hg38 [GRCh38] as a reference, which has a more robust assembly for the Xp region [43].Second, OGM specifications describe that at 300x effective coverage, it allows the detection of SVs and CNVs in up to 5% and 10% of the allele fraction, respectively.In our experience, the SV tool was able to detect some translocations at these low frequencies, but the detection of CNVs was not fully guaranteed if present in less than 20% of cells.In this regard, sensitivity was assumed to be the most feasible cause of the discrepancy in 43% of the missed abnormalities.While these variants had been previously detected in a median of 17% of cells, concordant OGM results also included CNVs found in only 12-15% of nuclei by FISH and/or CMA.Although some authors have set the sensitivity threshold for OGM at 10% [35,41], our results were in accordance with Rack et al. and suggested that detection of abnormalities around or below 15% varies among regions or loci [37].It is noteworthy that some of the CNVs not called by OGM were visually identified in the whole-genome CNV view with Bionano Access and could potentially be manually added in future software upgrades, as currently performed for CMA analysis. In contrast to other published OGM studies in hematological malignancies, which focused on the detection of clinically relevant abnormalities, our goal was to evaluate CLL abnormalities genome-wide in order to identify patients with genomic complexity.This strategy allowed us to benefit from the high potential of OGM, underscoring novel aberrations which had not been identified with standard methods and whose clinical relevance deserves further analysis.On the one hand, OGM identified several small SVs (from 3 Kb to 100 Kb) that were considered beyond the scope of this study.Our proof-ofconcept analysis aimed to obtain comparable results with the currently used methodologies; therefore, a cut-off size of 100 Kb was set to generate results with a resolution similar to that of CMA.Nonetheless, these small aberrations missed by standard methods could involve clinically relevant genes (novel or already known) and should be analyzed in more depth in subsequent studies and in larger CLL cohorts.On the other hand, a higher number of chromosomal translocations than initially expected was detected by OGM.Indeed, novel translocations allowed us to better characterize known abnormalities in 54.8% of the patients assessed.These included not only abnormalities involving material of unknown origin by CBA (marker chromosomes or \"add\") but also provided novel structural information related to apparently well-characterized translocations or deletions in the karyotype, as well as CNVs detected by CMA.Notably, not all the novel OGM translocations correlated with the results defined by standard techniques, and nearly 20% of them were classified as false-positive findings.Other authors have also reported similar observations and suggested manual inspection to filter out SV artifacts distinguishable from true translocation calls [35][36][37].Following our experience, those translocation calls based on the mapping of ≤10 labels in one of the translocation partners should be filtered.In contrast, while we were not able to validate two translocations with less than 10 molecules, 22% of the previously known or validated translocations were supported by only five to nine mapped molecules by OGM.Thus, contrarily to some authors that set a minimum of 10 supporting molecules for the analyses [39,40], we recommend not filtering these variants but performing validations with alternative techniques before reporting them.It is remarkable that, since we processed the samples, a new upgrade of Bionano Access has been developed that has improved the filtering of these types of false-positive calls.In this regard, in two of our samples reprocessed with the most recent update of Bionano Access (1.7 version), nine of the ten inter-translocations assumed as potential false-positive results were no longer called, but novel translocations were also identified in this updated version (data not shown).Globally, we have demonstrated that the custom filtering criteria applied herein are useful for OGM analysis, but the distinction between true translocations and technical artifacts is sometimes challenging.Consequently, guidelines for the filtering and interpretation of OGM variants are urgently needed to implement this technique in a routine setting. Concerning genomic complexity assessment by OGM, as expected by its high resolution, our results demonstrated that this novel technique was able to identify an overall higher number of abnormalities than conventional methods.Nonetheless, when data were manually curated, and small SVs (<100 Kb) were excluded from the analysis, a strong correlation between techniques was found.Likewise, we demonstrated that occasional discrepancies in the detection of known aberrations did not hinder the accuracy of the number of abnormalities recorded by OGM in predicting TTFT, which was even better than that achieved by CBA and CMA.Indeed, the higher C-index obtained by OGM indirectly proves the clinical importance of the additional aberrations identified by OGM, which allows a better delineation of the genomic complexity of these patients compared with current methodologies.Apart from its predictive value as a continuous variable, the definition of standard criteria to discriminate those higher-risk patients based on complexity is mandatory to incorporate OGM technology in the standard diagnostic algorithm.In this regard, we were able to demonstrate that 91.7% of patients harboring at least 10 abnormalities by OGM (C-OGM group) were also classified into the highest risk group based on the complexity identified by the combination of CBA and CMA.Moreover, this C-OGM group showed a statistically significantly higher proportion of TP53 del/mut cases and shorter TTFT.It is noteworthy that, as the cohort analyzed herein is limited in size and highly enriched in patients carrying CK, the cut-off used could not be statistically set.Thus, it should only be taken into account as a proof-of-concept of the capability of OGM to stratify patients based on their genomic complexity.Further studies in larger cohorts of CLL patients are required to statistically define robust criteria for genomic complexity stratification by OGM, not only in retrospective series but also to clarify its potential role in the management of patients treated with novel targeted agents. On the other hand, OGM represents a cost-effective approach to substitute CBA combined with multiple FISH tests that reduce global costs and turn-around times in other hematological malignances (e.g., acute lymphoblastic leukemia and acute myeloid leukemia).In contrast, the total expenses of the OGM technique (technician time dedication, reagents, equipment, and other technical infrastructure required for its implementation) are still superior to those of CBA; currently, the standard-of-care method to detect genomic complexity CLL.Thus, major benefits from OGM application in CLL do not rely on the cost savings but on the better genomic characterization achieved and novel abnormalities that could be uncovered from these highly complex genomes. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In summary, we have confirmed that OGM is a valuable tool for the cytogenomic assessment of CLL patients.It not only effectively detects most of the abnormalities defined by the combination of standard methods in a single test but also provides a more comprehensive genomic analysis allowing an enhanced interpretation.Despite a few abnormalities still being missed due to OGM limitations, these are expected to be addressed by the technical and analytical improvements of upcoming Bionano Access upgrades.As for its utility for risk stratification, herein, for the first time, we have demonstrated the association of an increasing number of abnormalities by OGM with a worse clinical evolution of CLL patients.Nonetheless, further studies in larger unselected cohorts are required to define standard genomic complexity criteria by OGM and to define the clinical significance of the novel abnormalities underscored by this technology.In conclusion, OGM is a new methodology that could potentially replace current cytogenomic methodologies for the routine management of CLL patients in the future. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Data Availability Statement: Detailed chromosome banding analyses, chromosomal microarrays and optical genome mapping profiles are provided in Supplemental Tables.Additional data of the study are available from the corresponding authors on reasonable request. No potential conflicts of interest were disclosed. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Data Availability Statement: Detailed chromosome banding analyses, chromosomal microarrays and optical genome mapping profiles are provided in Supplemental Tables.Additional data of the study are available from the corresponding authors on reasonable request. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "No potential conflicts of interest were disclosed. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14143376/s1, Figure S1.Flow diagram for filtering SV and CNV calls to define the final set of OGM abnormalities to be compared with standard techniques.Figure S2.Examples of novel translocations called by OGM that were associated with known CNVs previously identified by CMA. Figure S3.Distribution of the number of abnormalities detected in non-CK and CK groups.Figure S4.Example of two of the high-risk patients by CBA and/or CMA with less than 10 abnormalities detected by OGM. Figure S5.Kaplan-Meier plots for time to first treatment (TTFT) based on the presence of genomic complexity detected by OGM.Table S1.FISH probes tested to validate OGM results.Table S2.Technical performance of OGM in the whole cohort.Table S3.Detailed information on the detection of the 309 known abnormalities by OGM.Table S4.Summary of the interchromosomal translocation calls detected by OGM and comparison with CBA and FISH data.Funding: This work was partly supported by grants from Generalitat de Catalunya (17SGR437), Gilead Sciences Fellowship (GLD17/00282), Ministerio de Universisades of Spain (FPU17/00361) and the \"Xarxa de Bancs de tumors\" sponsored by Pla Director d'Oncologia de Catalunya (XBTC).R. G-S was supported by a predoctoral contract (ACIF/2021/169) from the Consellería de Innovación, Universidades, Ciencia y Sociedad digital (Generalitat Valenciana).Article Processing Charge of this manuscript was funded by Bionano Genomics, Inc. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Drug Research Ethical Committee (CEIm) of the Hospital del Mar, Barcelona (2017/7565/I). Informed Consent Statement: Informed consent was obtained from all patients involved in the study. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14143376/s1, Figure S1.Flow diagram for filtering SV and CNV calls to define the final set of OGM abnormalities to be compared with standard techniques.Figure S2.Examples of novel translocations called by OGM that were associated with known CNVs previously identified by CMA. Figure S3.Distribution of the number of abnormalities detected in non-CK and CK groups.Figure S4.Example of two of the high-risk patients by CBA and/or CMA with less than 10 abnormalities detected by OGM. Figure S5.Kaplan-Meier plots for time to first treatment (TTFT) based on the presence of genomic complexity detected by OGM.Table S1.FISH probes tested to validate OGM results.Table S2.Technical performance of OGM in the whole cohort.Table S3.Detailed information on the detection of the 309 known abnormalities by OGM.Table S4.Summary of the interchromosomal translocation calls detected by OGM and comparison with CBA and FISH data.Funding: This work was partly supported by grants from Generalitat de Catalunya (17SGR437), Gilead Sciences Fellowship (GLD17/00282), Ministerio de Universisades of Spain (FPU17/00361) and the \"Xarxa de Bancs de tumors\" sponsored by Pla Director d'Oncologia de Catalunya (XBTC).R. G-S was supported by a predoctoral contract (ACIF/2021/169) from the Consellería de Innovación, Universidades, Ciencia y Sociedad digital (Generalitat Valenciana).Article Processing Charge of this manuscript was funded by Bionano Genomics, Inc. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Drug Research Ethical Committee (CEIm) of the Hospital del Mar, Barcelona (2017/7565/I). Informed Consent Statement: Informed consent was obtained from all patients involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
}
] |
10.3390/cancers16050980
|
Venetoclax Initiation in Chronic Lymphocytic Leukemia: International Insights and Innovative Approaches for Optimal Patient Care
|
<jats:p>Venetoclax, a highly selective, oral B-cell lymphoma 2 inhibitor, provides a robust targeted-therapy option for the treatment of chronic lymphocytic leukemia (CLL), including patients with high-risk del(17p)/mutated-TP53 and immunoglobulin heavy variable region unmutated CLL and those refractory to chemoimmunotherapy across all age groups. Due to the potent pro-apoptotic effect of venetoclax, treatment initiation carries a risk of tumor lysis syndrome (TLS). Prompt and appropriate management is needed to limit clinical TLS, which may entail serious adverse events and death. Venetoclax ramp-up involves gradual, stepwise increases in daily venetoclax dosing from 20 mg to 400 mg (target dose) over 5 weeks; adherence to on-label scheduling provides a tumor debulking phase, reducing the risk of TLS. The key components of safe venetoclax therapy involve assessment (radiographic evaluation and baseline blood chemistry), preparation (adequate hydration), and initiation (blood chemistry monitoring). In addition to summarizing the evidence for venetoclax’s efficacy and safety, this review uses hypothetical patient scenarios based on risk level for TLS (high, medium, low) to share the authors’ clinical experience with venetoclax initiation and present global approaches utilized in various treatment settings. These hypothetical scenarios highlight the importance of a multidisciplinary approach and shared decision-making, outlining best practices for venetoclax initiation and overall optimal treatment strategies in patients with CLL.</jats:p>
|
[
{
"section_content": "The highly selective, oral B-cell lymphoma 2 (BCL-2) inhibitor venetoclax has been shown to be very effective for the treatment of chronic lymphocytic leukemia (CLL) [1].BCL-2 is highly expressed in CLL cells, and venetoclax blocks antiapoptotic BCL-2 signaling by functioning as a BCL-2 homology domain 3 (BH3) mimetic.Treatment of CLL cells with venetoclax induces the rapid onset of apoptosis in vitro and in vivo via a TP53-independent mechanism [2,3], providing the rationale for evaluating use of venetoclax as targeted therapy in CLL.In clinical trials, the efficacy and safety of venetoclax was demonstrated in both relapsed/refractory (R/R) disease and in the first-line setting.Venetoclax was initially granted accelerated approval by the United States Food and Drug Administration (FDA) in 2016 as continuous monotherapy for treatment of R/R CLL with del (17p) [4][5][6].The label has since expanded, with venetoclax now approved by many international agencies including the FDA, European Medicines Agency (EMA), and other regulatory agencies as fixed-duration combination therapy with either rituximab (for R/R CLL) or obinutuzumab (for treatment-naïve disease).Venetoclax was recently approved by the EMA in combination with ibrutinib (for treatment-naïve disease) [7][8][9].Fixed-duration therapy with venetoclax is an effective alternative to continuous treatment approaches with Bruton tyrosine kinase (BTK) inhibitors [10].The choice of venetoclax-based therapy versus other targeted agents and chemoimmunotherapy depends on multiple factors, including patient age and comorbidities, TP53 status, immunoglobulin heavy variable region (IGHV) mutational status, goals of treatment, tumor bulk, cardiac history and status, renal function, and concomitant medications [10][11][12].Prior therapy (e.g., chemotherapy, BCL-2 inhibitor, BTK inhibitor) and response depth and duration also affect treatment decisions for patients with R/R CLL [10]. Owing to its potent pro-apoptotic effect on malignant cells, venetoclax poses a risk of inducing tumor lysis syndrome (TLS) [13].TLS risk is evaluated before initiating venetoclax, and the determined risk level dictates prophylaxis and administration procedures as per the product information to ensure safe onboarding of this therapy for patients with CLL [7].A gradual, 5-week venetoclax ramp-up with appropriate debulking, prophylaxis, and monitoring has been shown to be safe and effective, and it reduces the risk of TLS and the need for related hospitalization [14].However, this regimen can be challenging to implement in many health care settings, and practitioners are devising real-world solutions to safely onboard patients with venetoclax-examples of which we will highlight in this review. In a review of the literature (January 2010-June 2021), real-world studies reported a significant reduction in TLS incidence when debulking strategies were used in patients with intermediate or high risk of TLS (using venetoclax monotherapy or venetoclax combination regimens) [14].Some variability in reported rates of biochemical changes may stem from a failure to strictly adhere to ramp-up procedures specified in the product labeling, as better adherence to recommended protocols can mitigate TLS risk.Since the initial accelerated approval of venetoclax in 2016, healthcare providers have accrued a wealth of experience with this agent and have developed approaches to implement on-label initiation safely and effectively.This review draws on published data as well as the authors' clinical experience with venetoclax to outline best practices for initiation in patients with CLL.Additionally, this review describes hypothetical patient scenarios to illustrate global approaches used in various treatment settings and emphasizes the importance of a multidisciplinary approach and shared decision-making, with the overall goal of equipping healthcare providers to deliver an optimal experience for patients undergoing treatment with venetoclax. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Clinical Trials and Real-World Studies of Venetoclax in Patients with CLL",
"section_num": "2."
},
{
"section_content": "Results from M13-982 (NCT01889186), a phase 2 trial of venetoclax monotherapy (N = 107) in patients with R/R CLL and del(17p), showed an overall response rate of 79.4% [5,6], leading to the FDA's accelerated approval of venetoclax in the segment of the patient population with at least 1 prior therapy [15].In a randomized phase 3 MURANO trial (NCT02005471; N = 389), the combination of venetoclax plus rituximab (for a fixed duration of 2 years) resulted in a response rate of more than 90% and a significant improvement in progression-free survival (PFS) for patients with R/R CLL, reducing the risk of disease progression or death by 81% compared to bendamustine plus rituximab [16].This resulted in the FDA and EMA approvals of venetoclax plus rituximab in patients with R/R CLL [7,8].These survival benefits of venetoclax were sustained 3 years after treatment cessation (5-year follow-up), with a median PFS of 53.6 months (venetoclax plus rituximab) compared with 17.0 months (bendamustine plus rituximab; p < 0.0001) [17].Compared to the MURANO trial, a real-world analysis from the Polish Adult Leukemia Study group in patients with very high-risk R/R CLL receiving the combination of venetoclax plus rituximab, yielded a shorter median PFS (36.97 months [95% CI 24.5, not reached]) [18].The randomized phase 3 CLL14 study (NCT02242942; N = 432) showed a significant decrease in disease progression or death (hazard ratio [HR], 0.35 [p < 0.001]) and longer PFS at 24-months (88.2% vs. 64.1%)after fixed-duration venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab in patients with previously untreated CLL and coexisting conditions [19], leading to FDA and EMA approvals of venetoclax plus obinutuzumab in first-line therapy [7,8].At 5-year follow-up, PFS remained superior (HR, 0.35 [p < 0.0001]) with a higher PFS rate in the venetoclax plus obinutuzumab group (62.6%) compared to chlorambucil plus obinutuzumab (27.7%) and no significant difference noted in overall survival (OS), although at the end of treatment an undetectable minimal residual disease status with a cutoff at 10 -4 was associated with longer OS [20].Additionally, the re-emergence of detectable disease was significantly slower following venetoclax plus obinutuzumab compared with chlorambucil plus obinutuzumab [21].Results from GLOW (NCT03462719), a phase 3 trial (N = 211) in older patients and/or those with comorbidities with previously untreated CLL, showed that the ibrutinib (3-cycle lead in) plus venetoclax regimen resulted in a significantly longer PFS compared to chlorambucil plus obinutuzumab (HR, 0.216; p < 0.001) [9].This combination is approved by the EMA for adult patients with previously untreated CLL [22].In the CAPTIVATE (NCT02910583) phase 2 trial, fixed-duration ibrutinib (3-cycle lead in) plus venetoclax resulted in a complete response rate of 56% in patients aged ≤70 years with previously untreated CLL (Table 1) [23].In a phase 3 randomized study (NCT0290051), the combination of venetoclax with obinutuzumab or ibrutinib resulted in a significantly higher number of patients with undetectable minimum residual disease at 15 months (venetoclax-obinutuzumab [86.5%]; venetoclax-obinutuzumab-ibrutinib [92.2%] vs. chemoimmunotherapy [52.2%]; p < 0.001 for both comparisons).In addition, at 3 years, the median PFS was 87.7% (HR, 0.42) in the venetoclax-obinutuzumab group, 90.5% (HR, 0.32) in the venetoclax-obinituzumab-ibrutinib group, but only 75.5% following the chemotherapy regimen (p < 0.001 for both comparisons) [24].In large, real-world studies, high response rates were observed in patients with either R/R or treatment-naïve CLL [25][26][27][28][29][30].More specifically, most studies reported response rates between 80%-90% [25-27], with 2-year best overall response rates of more than 90% in patients who received venetoclax as a monotherapy or in combination with rituximab (Table 2) [28]. ",
"section_name": "Efficacy",
"section_num": "2.1."
},
{
"section_content": "Adverse events with venetoclax generally are considered tolerable and manageable.In key trials of patients with CLL with del(17p) and who were treatment-naïve or had R/R disease, neutropenia was the most common grade ≥ 3 adverse event (38-58%) among others, including thrombocytopenia, infections, anemia, and infusion-related reaction (IRR; Table 1) [6,16,17,19,20].In an integrated safety analysis of venetoclax in patients with R/R CLL based on three phase 1/2 trials using continuous single-agent therapy (N = 350) [31], the most common adverse events of any grade were diarrhea, neutropenia, nausea, anemia, fatigue, and upper respiratory tract infections.Discontinuations due to adverse events occurred in 10% of patients, and 8% of patients died, mainly due to disease progression [31].In a retrospective analysis of venetoclax-related adverse events for patients with CLL (N = 297), the most common grade 3 or 4 adverse events were neutropenia, thrombocytopenia, infection, febrile neutropenia, and diarrhea (Table 2) [29].Dose reductions were required in 29% of patients, dose interruptions in 32% of patients, and discontinuations due to adverse events in 7.4% of patients [29]. A retrospective analysis of the MURANO study assessed the impact of early venetoclax discontinuation or dose reduction [32].Treatment interruption due to adverse events occurred in 69% of patients and was most often due to neutropenia; dose reductions were required in 23% of patients, but these did not adversely impact PFS.Only premature discontinuation affected outcomes, suggesting prompt, effective management of venetoclaxrelated adverse events enables resumption of therapy and the maintenance of efficacy [32]. In a real-world pharmacovigilance study of venetoclax-related adverse events in patients with CLL or other hematologic malignancies using the FDA Adverse Event Reporting System database (19,107 cases of adverse events) [33], the median time to occurrence of adverse events was 31 days (range, 7-131 days).Half of the events occurred in the first 30 days and approximately 70% in the first 3 months.Neutropenia was common (40-50%), and the occurrence of grade ≥ 3 neutropenia was associated with risk of pneumonia.Infections accounted for nearly a quarter of venetoclax-related adverse events (pneumonia was the most common), which is consistent with the immunosuppression associated with the underlying CLL [33].Most adverse events can be managed with additional supporting medication (e.g., nausea with ondansetron or prochlorperazine; diarrhea with loperamide).However, careful monitoring for infections and judicious use of venetoclax in patients with pre-existing infections is warranted. Combining venetoclax with antibody therapy or other targeted agents, such as rituximab and BTK inhibitors, may increase the risk of adverse reactions.More specifically, IRRs are frequent when venetoclax is used with obinutuzumab, particularly during the first obinutuzumab dose [19].To reduce risk, patients should be premedicated with corticosteroids, antipyretics, and diphenhydramine the night prior to, the morning of, and immediately prior to infusion initiation, with an initial fractionated dose of obinutuzumab administered on the first day and a very slow infusion rate of obinutuzumab; both can be increased subsequently if no IRRs occur [7].Other obinutuzumab-related adverse events such as neutropenia, thrombocytopenia, and infection can occur.Hepatitis B reactivation and progressive multifocal leukoencephalopathy have also been reported [7], but are rare events; hepatitis B should be tested for prior to therapy.In the phase 3 MURANO study, the most common adverse event of any grade in the venetoclax plus rituximab group was neutropenia, followed by infections and infestations [16]. ",
"section_name": "Safety",
"section_num": "2.2."
},
{
"section_content": "Because venetoclax treatment poses a risk of TLS, great care is required to prevent and manage TLS, especially in patients at high risk.If not managed promptly and appropriately, laboratory TLS (laboratory evidence of metabolic changes, without symptoms such as hyperuricaemia, hyperkalaemia, hyperphosphataemia, secondary hypocalcaemia and uraemia) can worsen to clinical TLS (defined as laboratory TLS with clinical consequences such as acute renal failure, cardiac arrhythmias, seizures, and death [34]).Any evidence of laboratory TLS requires immediate action such as withholding the next day's dose of venetoclax and reassessing after 24-48 h; then resuming at the same dose if the laboratory TLS is resolved and at a reduced dose if the clinical TLS is resolved [34].The importance of adhering to venetoclax administration guidelines has been illustrated by a pooled analysis of venetoclax clinical trials and post-marketing studies (N = 1138) [13].When recommended mitigation measures were followed, the overall TLS incidence was 1.8% (laboratory TLS 1.8% [clinical TLS, <1%]), with all patients resuming therapy after transient TLS, and no irreversible sequelae [13].Key potential causes of TLS were suboptimal blood chemistry monitoring, inappropriate venetoclax dose modification for drug-drug interactions, underestimating the TLS risk level, underestimating the degree of renal dysfunction, failure to adhere to the recommended ramp-up protocol, and failure to follow hydration guidelines [13]. In the MURANO trial (NCT02005471), grade 3/4 TLS was reported in 6/194 patients (3.1%) in the venetoclax-rituximab group and in 2/188 patients (1.1%) in the bendamustinerituximab group; clinical TLS was reported in 1 patient in each treatment group [16].In the phase 3 GLOW study (NCT03462719), no cases of TLS were reported in the ibrutinibvenetoclax group.TLS was reported in 5.7% of the chlorambucil-obinutuzumab group [9]; these cases were likely attributable to the obinutuzumab.In the phase 3 CLL14 study (NCT02242942), TLS was reported in 1.4% of patients in the venetoclax-obinutuzumab group (all cases occurred prior to venetoclax treatment) and 2.3% of patients in the chlorambucil-obinutuzumab group; none of the cases was defined as clinical TLS in either group [19]. In a subset of patients for whom the 5-week ramp-up procedure was followed, the incidence of TLS with venetoclax monotherapy was 1.2% (all laboratory TLS; no TLSrelated deaths) [31].Notably, obinutuzumab administered as a monotherapy [35], or in combination regimens without venetoclax, has been shown to induce TLS.In the pivotal phase 3 CLL11 study (NCT01010061), 4% of patients treated with obinutuzumab plus chlorambucil experienced TLS [36].In an aggregated real-world cohort treated with venetoclax, clinical and laboratory TLS occurred in 2.7% and 5.7% of patients, respectively, and 1 TLS-related death occurred [29].TLS risk could be predicted by pre-treatment TLS risk assessment and a creatinine clearance (CrCl) of < 80 mL/min [29].The occurrence of TLS was dependent on tumor size, the presence of comorbidities (especially impaired renal function), and the venetoclax dose [33].Obinutuzumab, when used as a pre-induction therapy in cycle 1 days 1 and 2, may reduce the risk of TLS if administered before venetoclax [19,37,38]. ",
"section_name": "Risk of Tumor Lysis Syndrome",
"section_num": "2.3."
},
{
"section_content": "Venetoclax ramp-up is designed to reduce the tumor burden gradually (e.g., debulk) by slowly increasing the daily dose to reach the targeted dosing of 400 mg, thus reducing the risk of TLS.Ensuring appropriate prophylaxis, including adequate hydration and anti-hyperuricemia drugs as indicated, can lower TLS risk [7,39].An extended ramp-up schedule has been associated with a measured reduction in peripheral blood lymphocyte count, suggesting a more controlled cytotoxic effect compared to dose escalation [39].Strict adherence to venetoclax on-label ramp-up guidelines results in low rates of TLS, which supports the assertion that treatment can be safely administered using this schedule. Key components of safe venetoclax therapy involve assessment, preparation, and initiation.An assessment of tumor burden (by radiographic evaluation of maximal nodal diameter with reevaluation during ramp-up), and a baseline blood chemistry test are recommended before venetoclax initiation to determine the risk of TLS.The risk level is evaluated based on multiple factors, including reduced renal function (CrCl < 80 mL/min per the label), tumor burden, and the presence of splenomegaly [7].The tumor burden is categorized as low (all lymph nodes < 5 cm, and absolute lymphocyte count < 25 × 10 9 /L), medium (any lymph node 5 to <10 cm, or absolute lymphocyte count ≥ 25 × 10 9 /L), or high (spleen > 6 cm below costal margin, any lymph node ≥ 10 cm, or any lymph node ≥ 5 cm and absolute lymphocyte count ≥ 25 × 10 9 /L) [40,41].Certain comorbidities, such as impaired renal function, may also increase the risk of TLS.Errors in lymph node classification (e.g., due to failure to image or inaccurate interpretation of scans from computed tomography) can result in an inaccurate determination of tumor burden and thus affect the determination of TLS risk and lead to inappropriate management.If a patient receives obinutuzumab prior to venetoclax, imaging should be repeated immediately prior to venetoclax initiation, to ensure a correct assessment of the risk of TLS.In addition, it has been shown that 2 cycles of obinutuzumab prior to initiation of venetoclax was an effective debulking strategy (success rate over 98%) for patients with absolute lymphocyte count > 25 × 10 9 /L and lymph nodes < 5 cm (medium risk TLS) [42]. Preparation consists of ensuring adequate hydration, orally and with an antihyperuricemic such as allopurinol.For those with a prior allopurinol allergy, febuxostat is a safe alternative agent [43].Intravenous hydration during an outpatient stay may be considered for patients with a medium tumor burden and should be administered to all patients with a high tumor burden, possibly with rasburicase if the uric acid level is >8 mg/dL [44].Prior to the administration of rasburicase, glucose-6-phosphate dehydrogenase deficiency may be excluded based on geographical region, as it can lead to methemoglobinemia in susceptible individuals [44].In some institutions, this is routinely established during preparation for venetoclax ramp-up in all patients to avoid the need for rapid testing if rasburicase is urgently needed to treat uncontrolled hyperuricemia.In the case of a high tumor burden and following treatment with rasburicase, patients should start receiving oral allopurinol [45] 48-72 h postdose and continue until at least day 7 post final dose escalation.For low and medium risk cases of TLS, allopurinol should be started 48-72 h prior to the first dose of venetoclax [34].Oral hydration should be initiated ≥ 2 days before the first dose and continued throughout ramp-up.Hydration is especially important on the first day of each dose escalation; however, older patients may struggle to drink enough water (1.5-2.0L/day) or may have fluid restrictions due to heart failure and may require IV hydration under collaborative management with the heart failure care team [7]. Initiation of venetoclax dosing requires the monitoring of blood chemistry (potassium, uric acid, phosphorus, creatinine, lactate dehydrogenase, and calcium) around the first dose at each new level.Hospitalization can be considered for patients with a medium tumor burden and CrCl < 80 mL/min during administration of the initial 20-mg and 50-mg venetoclax doses.Hospitalization during administration of the initial 20-mg and 50-mg venetoclax doses is indicated for all patients with a high tumor burden [7].Venetoclax dose modifications may be required for patients who develop specific grade 3/4 adverse events or have changes in blood chemistry or symptoms suggestive of TLS.Grade 3/4 neutropenia may require dose interruption/reduction, but usually dosing would be continued unaltered for asymptomatic grade 3 neutropenia.Treatment with growth factor support can help maintain absolute neutrophil count during ramp-up and the first several cycles of venetoclax, thus maintaining scheduled dosing [10].Short-acting growth factor and/or intermittent dosing (e.g., once to twice per week) is usually adequate, although long-acting growth factor (e.g., pegfilgrastim) may have prolonged benefit for more than 4-6 weeks.Patients should avoid concomitant use of venetoclax with strong or moderate cytochrome P450, family 3, subfamily A (CYP3A) inhibitors (e.g., ketoconazole, itraconazole, fluconazole) or P-gp inhibitors (e.g., amiodarone, ketoconazole) [7,41] at initiation and during ramp-up because these agents alter venetoclax pharmacokinetics and increase the risk of TLS.Venetoclax dose reductions are required if these agents are unavoidable after the ramp-up period [7,41]. ",
"section_name": "On-Label Venetoclax Initiation",
"section_num": "2.4."
},
{
"section_content": "",
"section_name": "International Insights and Innovative Approaches Illustrated by Hypothetical Patient Scenarios",
"section_num": "3."
},
{
"section_content": "A 70-year-old man with CLL has an extensive medical history that includes ischemic heart disease resulting in an impaired left ventricular ejection fraction of 40%, as well as chronic kidney disease (Crockoff Gault glomerular filtration rate of 30 mL/minute) due to type 2 diabetes (Figure 1).Other medical issues include hypertension and hyperlipidemia.A current molecular and cytogenetic workup revealed an unmutated IGHV gene status, the presence of a TP53 mutation, and a complex karyotype [46].This patient was heavily pre-treated and had received 6 cycles of fludarabine plus cyclophosphamide plus rituximab (prior to approval of targeted agents), which resulted in a partial response with a PFS of 2 years.Subsequently, continuous ibrutinib (420 mg daily) was administered to the patient, which resulted in PFS for 4 years followed by slow disease progression on this BTK inhibitor.At this point, the patient had R/R CLL with a high tumor burden due to a retroperitoneal conglomerate lymph node mass of 12 cm, a splenomegaly of 15 cm, and an absolute lymphocyte count of 80 × 10 9 /L; non-contrast radiographic evaluation was used due to the patient's increased risk of renal damage.As it is recommended to continue on the BTK inhibitor during the transition period to the next line of therapy, especially through venetoclax ramp-up [47], he remained on ibrutinib until the completion of venetoclax ramp-up and the start of continuous venetoclax monotherapy.Before the initiation of continuous venetoclax treatment, the patient received increasing daily doses (20, 50, 100, 200, and 400 mg) of venetoclax every week over a 5-week period (Weeks 1 and 2 were inpatient; Weeks 3, 4, and 5 were at the treatment center as outpatient).Because this hypothetical patient is at high risk of TLS, a more intensive intervention and specialized attention is required with close monitoring of renal function, serial tumor burden, and splenomegaly reduction.Appropriate prophylaxis with adequate hydration (150-200 mL/hr as tolerated prior to first dose with close fluid monitoring [i.e., twice daily weight, urine volume measuring] and diuretic supportive measures to avoid complications such as pulmonary edema), and administration of anti-hyperuricemics/rasburicase at the physician's discretion is critical, along with monitoring and promptly addressing any emerging biochemical laboratory abnormalities, in particular hyperkalemia [34].If available, the utilization of a nurse specialist is essential for continued patient education, the promotion of the patient's adherence to the dosing schedule, the monitoring of blood chemistries, hydration, and supportive medications. ",
"section_name": "Patient Case 1-High Risk for TLS and Renal Failure",
"section_num": "3.1."
},
{
"section_content": "An older woman aged 80 years was diagnosed with CLL (Figure 2).The patient has an extensive medical history which included depression treated with a serotonin selective reuptake inhibitor (i.e., escitalopram).Osteoarthritis was being treated with paracetamol and ibuprofen.In addition, she underwent tumor resection in 2012 for the treatment of Because this hypothetical patient is at high risk of TLS, a more intensive intervention and specialized attention is required with close monitoring of renal function, serial tumor burden, and splenomegaly reduction.Appropriate prophylaxis with adequate hydration (150-200 mL/hr as tolerated prior to first dose with close fluid monitoring [i.e., twice daily weight, urine volume measuring] and diuretic supportive measures to avoid complications such as pulmonary edema), and administration of anti-hyperuricemics/rasburicase at the physician's discretion is critical, along with monitoring and promptly addressing any emerging biochemical laboratory abnormalities, in particular hyperkalemia [34].If available, the utilization of a nurse specialist is essential for continued patient education, the promotion of the patient's adherence to the dosing schedule, the monitoring of blood chemistries, hydration, and supportive medications. ",
"section_name": "Patient Case 2-Medium Risk for TLS with Potential Drug-to-Drug Interactions and Infusion Reactions",
"section_num": "3.2."
},
{
"section_content": "An older woman aged 80 years was diagnosed with CLL (Figure 2).The patient has an extensive medical history which included depression treated with a serotonin selective reuptake inhibitor (i.e., escitalopram).Osteoarthritis was being treated with paracetamol and ibuprofen.In addition, she underwent tumor resection in 2012 for the treatment of colorectal cancer; no ostomy was required.Molecular testing showed a mutated IGHV status and a del(13q) abnormality by fluorescent in-situ hybridization; a TP53 assessment did not show any deletion or mutation.For CLL, there was a medium tumor burden due to mediastinal nodes and intrabdominal nodes (both 4 cm).No splenomegaly was present, the lymphocyte count was 40 × 10 9 /L, the hemoglobin was 95 g/L, and the platelet count was < 100 × 10 9 /L.The patient had not received prior treatment for CLL.For hypertension, this patient had taken verapamil (moderate CYP3A inhibitor), which can interact with venetoclax and is contra-indicated during ramp-up.Verapamil may need to be halted before obinutuzumab infusion to avoid the risk of an increased severity of hypotension with any IRR.Co-administration of verapamil and venetoclax can significantly increase blood levels of venetoclax and increase the risk of TLS and is thereby contra-indicated during ramp-up, so it will need to be stopped.Of note, this patient lives alone, does not drive, and has minimal social support, which could present issues with adherence and/or the ability to attend appointments.the lymphocyte count was 40 × 10 9 /L, the hemoglobin was 95 g/L, and the platelet count was < 100 × 10 9 /L.The patient had not received prior treatment for CLL.For hypertension, this patient had taken verapamil (moderate CYP3A inhibitor), which can interact with venetoclax and is contra-indicated during ramp-up.Verapamil may need to be halted before obinutuzumab infusion to avoid the risk of an increased severity of hypotension with any IRR.Co-administration of verapamil and venetoclax can significantly increase blood levels of venetoclax and increase the risk of TLS and is thereby contra-indicated during ramp-up, so it will need to be stopped.Of note, this patient lives alone, does not drive, and has minimal social support, which could present issues with adherence and/or the ability to attend appointments.This patient will receive the planned fixed-duration treatment with venetoclax plus Obinutuzumab, as the efficacy of this combination is high in patients with this genetic profile.Because the patient will receive obinutuzumab, proper premedications are required to reduce the risk of IRR.The drug regimens and protocols may differ between institutions.Some institutions will give corticosteroids (e.g., dexamethasone, 20 mg) and antihistamines (e.g., diphenhydramine, 25 mg) the night before and the morning of the a Some institutions will give corticosteroids (e.g., dexamethasone, 20 mg) and antihistamines (e.g., diphenhydramine, 25 mg) the night before and morning of the first obinutuzumab infusion, in addition to giving premedications right before the infusion.ALC, absolute lymphocyte count; CLL, chronic lymphocytic leukemia; CrCl, creatinine clearance; CT, computerized tomography; CYP3A, cytochrome P4503A; HCP, healthcare provider; IGHV, immunoglobulin heavy variable; IV, intravenous; IRR, infusion-related reaction; L, liter; LN, lymph node; mg, milligram; SSRI, selective serotonin reuptake inhibitor; TLS, tumor lysis syndrome.This patient will receive the planned fixed-duration treatment with venetoclax plus Obinutuzumab, as the efficacy of this combination is high in patients with this genetic profile.Because the patient will receive obinutuzumab, proper premedications are required to reduce the risk of IRR.The drug regimens and protocols may differ between institutions.Some institutions will give corticosteroids (e.g., dexamethasone, 20 mg) and antihistamines (e.g., diphenhydramine, 25 mg) the night before and the morning of the first obinutuzumab infusion, in addition to giving premedications right before the infusion.At specific hospitals in the United Kingdom, during cycle 1 only (28 days), oral antihistamine (chlorphenamine, 4 mg) and oral acetaminophen (paracetamol, 1 mg) treatments are completed 30 min before obinutuzumab.Additionally, slow intravenous glucocorticoid (dexamethasone, 20 mg) treatment is completed 60 min before intravenous obinutuzumab on Days 1 (100 mg) and 2 (900 mg).In some cases, the above premedications are administered at Days 8 and 15 of cycle 1 prior to a 1000 mg IV of obinutuzumab; however, this hypothetical patient did not receive dexamethasone on Days 8 and 15 because no IRRs were present after Days 1 and 2. Premedications should be reduced and discontinued early when deemed possible by the treating physician.Starting a few days prior to Day 1 of the first cycle, an anti-hyperuricemic (allopurinol, 300 mg orally once daily) is administered for 4 weeks and reviewed after the first cycle is complete.Depending on the institution, other medications that might be administered but are not required per the venetoclax label include an anti-viral (e.g., acyclovir, 400 mg orally twice daily) and an antiemetic metoclopramide (e.g., 10 mg orally 3 times per day as required). One treatment scenario for this patient involves an urban setting (e.g., Limerick, Ireland), where the older patient receives an in-home phlebotomy service that is standard for outpatients during the ramp-up period.Utilizing in-home phlebotomy services not only reduces the burden on the patient by minimizing trips to the clinic and the associated out of pocket costs (e.g., gas, parking), it also reduces patient volume in the clinic.The blood samples are taken in the home and delivered to the hospital laboratory.The laboratory values are expeditiously processed and reviewed with the clinician in real time, and the patient will be updated and given follow-up instructions by phone.This is coordinated by the nurse specialist team.A multidisciplinary team including advanced pharmacists is critical to closely monitor potential drug-drug interactions or complications and care for this patient, who has a long history of medical issues.In this case, verapamil should be avoided; alternative medications should be considered during the initiation and dose-titration phases of venetoclax treatment. A second treatment scenario is in a rural setting (e.g., regional Australia).In regional and remote geographic areas, access to suitable health services may be limited.Similar to the urban setting, education of the patient and clinician is important, along with scheduling and coordinating laboratory workup examinations.Changes in laboratory values (i.e., uric acid, phosphorus, potassium, and calcium) can occur within hours of treatment initiation and thus requires prompt management.To facilitate treatment, patients may require referrals to specialist treatment centers, with appropriate sourcing of local accommodation or inpatient stays.In other cases, patient care may be transferred back to a local hospital for ongoing management and co-managed through telehealth appointments with the referring physician and expert.A multidisciplinary approach with advanced practice nursing or pharmacists can bridge the gap in locations where medical service is limited [48,49].Defining a clear program of roles and responsibilities, establishing escalation pathways, and upskilling/education to outsourced departments (i.e., emergency, day units) is crucial to ensure patient safety.This approach can ensure appropriate prophylaxis and employ more intensive measures such as intravenous hydration if the patient cannot maintain an adequate level of oral hydration, (the recommended volume is 6-8 glasses of water, or 1.5-2 L per day), frequent monitoring of laboratory values (e.g., 6-8 h and 24 h after first dose of venetoclax), and/or potential hospitalization based on assessments (first dose and potential subsequent dose increases of venetoclax) [34]. ",
"section_name": "Patient Case 2-Medium Risk for TLS with Potential Drug-to-Drug Interactions and Infusion Reactions",
"section_num": "3.2."
},
{
"section_content": "A 55-year-old man maintains a healthy lifestyle, has no major comorbidities, works full time, and is raising a young family (Figure 3).Molecular testing showed an IGHV unmutated status and no TP53 abnormalities.He previously received acalabrutinib for 8 years and achieved a partial response.However, CLL slowly progressed, with the next treatment option being ramp-up of continuous venetoclax monotherapy.For this patient, acalabrutinib was ceased the day before initiating venetoclax, although it should be noted that other patients may continue to receive acalabrutinib until the completion of the venetoclax ramp-up, particularly if the patient has aggressive disease progression and there are no issues accessing continued treatment.There was a low tumor burden because all lymph nodes were < 5 cm and the absolute lymphocyte count was < 25 × 10 9 /L.Certain challenges with this patient include work and family commitments, making it difficult to schedule treatment and/or appointments at a medical center.After the 5-week ramp-up treatment, the patient was scheduled to receive a combination of venetoclax plus rituximab. Cancers 2024, 16, x FOR PEER REVIEW 13 of 18 venetoclax ramp-up, particularly if the patient has aggressive disease progression and there are no issues accessing continued treatment.There was a low tumor burden because all lymph nodes were < 5 cm and the absolute lymphocyte count was < 25 × 10 9 /L.Certain challenges with this patient include work and family commitments, making it difficult to schedule treatment and/or appointments at a medical center.After the 5-week ramp-up treatment, the patient was scheduled to receive a combination of venetoclax plus rituximab.To navigate these challenges, the clinician will need to be aware of and understand the necessity of on-label initiation and identify red flags regarding patient stress that pose potential risks to optimal treatment and drug adherence.Careful coordination of dose administration and laboratory tests to accommodate the patient's busy schedule will be extremely important.In addition, the use of electronic tools and applications which contain calendars and reminders can be beneficial to keep track of all scheduled visits, dosing, and tests.For the venetoclax ramp-up, laboratory results will be obtained the day prior to dosing.On Day 1, two 10 mg tablets are taken orally, followed by laboratory tests performed at the medical center or office 6-8 h after the dose for rapid turnaround and action.On Day 2, morning bloodwork is to be completed on-site prior to dosing, with monitoring of potassium, calcium, phosphorus, and uric acid.On Day 7, pre-dose laboratory tests are performed prior to dosing.The same routine is followed for the second week, with the only difference being that the venetoclax dose is increased to 50 mg per day.For Weeks 3 and 4, the venetoclax dose is increased to 100 mg and 200 mg daily, respectively, along with the laboratory tests performed on Day 7 only [34].For the first 2 weeks, during which To navigate these challenges, the clinician will need to be aware of and understand the necessity of on-label initiation and identify red flags regarding patient stress that pose potential risks to optimal treatment and drug adherence.Careful coordination of dose administration and laboratory tests to accommodate the patient's busy schedule will be extremely important.In addition, the use of electronic tools and applications which contain calendars and reminders can be beneficial to keep track of all scheduled visits, dosing, and tests.For the venetoclax ramp-up, laboratory results will be obtained the day prior to dosing.On Day 1, two 10 mg tablets are taken orally, followed by laboratory tests performed at the medical center or office 6-8 h after the dose for rapid turnaround and action.On Day 2, morning bloodwork is to be completed on-site prior to dosing, with monitoring of potassium, calcium, phosphorus, and uric acid.On Day 7, pre-dose laboratory tests are performed prior to dosing.The same routine is followed for the second week, with the only difference being that the venetoclax dose is increased to 50 mg per day.For Weeks 3 and 4, the venetoclax dose is increased to 100 mg and 200 mg daily, respectively, along with the laboratory tests performed on Day 7 only [34].For the first 2 weeks, during which more frequent blood work is collected (Supplementary Figure S1), the patient can be supported for 2-3 days per week to properly accommodate his schedule and ensure that he receives the necessary treatments and testing [34]. ",
"section_name": "Patient Case 3-Low Risk for TLS and Busy Working Patient",
"section_num": "3.3."
},
{
"section_content": "These cases highlight that a multidisciplinary team encompassing the patient, nurse practitioner/coordinator, hematologist, and pharmacist is essential for successful onboarding and facilitating personalized treatment plans [50], with the goal of achieving optimal patient care and treatment outcomes [51].Care team members include primary care physicians, hematologists, pathologists, radiologists, nurses, and pharmacists, all of whom are responsible for different and overlapping roles such as proper diagnosis, (i.e., blood tests and physical examinations), risk assessments (i.e., staging), informative prognostics (e.g., mutational status) and/or therapy determination, and risk-stratified treatment and the management of adverse events (Figure 4).Certain factors and considerations for treatment are administration route(s), length of treatment (which can entail higher or lower costs [52]), insurance coverage, daily lifestyle, age, and potential side effects.It is important for patients to have a voice and share their preferences and concerns about various treatment options [53,54].This will allow patients and providers to agree on a therapy that will fit both the clinical needs to effectively target CLL and the patient's lifestyle for proper accommodation and convenience.Also of importance for treatment management is patient education and supplemental materials, such as diaries for fluid and medication record keeping and other support devices. Cancers 2024, 16, x FOR PEER REVIEW 14 of 18 more frequent blood work is collected (Supplementary Figure S1), the patient can be supported for 2-3 days per week to properly accommodate his schedule and ensure that he receives the necessary treatments and testing [34]. ",
"section_name": "Patient Journey",
"section_num": "4."
},
{
"section_content": "These cases highlight that a multidisciplinary team encompassing the patient, nurse practitioner/coordinator, hematologist, and pharmacist is essential for successful onboarding and facilitating personalized treatment plans [50], with the goal of achieving optimal patient care and treatment outcomes [51].Care team members include primary care physicians, hematologists, pathologists, radiologists, nurses, and pharmacists, all of whom are responsible for different and overlapping roles such as proper diagnosis, (i.e., blood tests and physical examinations), risk assessments (i.e., staging), informative prognostics (e.g., mutational status) and/or therapy determination, and risk-stratified treatment and the management of adverse events (Figure 4).Certain factors and considerations for treatment are administration route(s), length of treatment (which can entail higher or lower costs [52]), insurance coverage, daily lifestyle, age, and potential side effects.It is important for patients to have a voice and share their preferences and concerns about various treatment options [53,54].This will allow patients and providers to agree on a therapy that will fit both the clinical needs to effectively target CLL and the patient's lifestyle for proper accommodation and convenience.Also of importance for treatment management is patient education and supplemental materials, such as diaries for fluid and medication record keeping and other support devices.As described in the hypothetical patient cases, patients may need certain accommodations based on their age, location, level of support from family and society, and busy schedules, further emphasizing that numerous factors must be considered on a patientby-patient basis.Shared decision-making is a continuous process that can change over time and requires constant communication, especially when patients notice changes or worsening of symptoms, regardless of whether they believe it is related to their current treatment regimen. ",
"section_name": "Patient Journey",
"section_num": "4."
},
{
"section_content": "Venetoclax provides a robust targeted-therapy option for the full spectrum of patients with CLL, including those with high-risk del(17p)/mutated-TP53 CLL and those with disease refractory to chemoimmunotherapy.The initiation and ramp-up of venetoclax require proper assessment of the risk of TLS along with risk-stratified monitoring and mitigation measures to allow safe initiation and dose escalation.Venetoclax ramp-up is feasible in a variety of settings with sufficient personnel, on-site clinical laboratories, proper education, adequate planning, and relevant experience with venetoclax.Ambulatory care for moderate or high-risk TLS venetoclax dose escalations is present in many parts of the world and is becoming increasingly popular across specialist centers in the United Kingdom.These centers allow better coordination of logistics, scheduling, administration, and management of adverse events such as IRRs.In academic settings and large clinics, a dedicated nurse champion and/or patient coordinator can facilitate logistics pertaining to venetoclax administration, ramp-up, and the scheduling of patient visits and laboratory tests.Regimens, and specifically premedications as noted in the hypothetical medium-risk case for TLS, may vary based on the institution or practice, all with the goal of avoiding infusion reactions and minimizing the risk of adverse events.Alongside proper education, optimal patient-based treatment strategies can be achieved with a multidisciplinary care team and shared decision-making to navigate potential challenges in this continually evolving treatment landscape.This review summarized 3 hypothetical cases; however, depending on the specific factors described in each case, these treatment approaches can be applied to other cases in this patient population. Lilly and Company, GlaxoSmithKline, Janssen Oncology, Johnson and Johnson, Kite, Loxo Oncology, Merck, Pfizer, TG Therapeutics and Vaniam Group.J.F.S. reports research funding from AbbVie, Celgene, Janssen, and Roche; consultancy or advisory role for AbbVie, AstraZeneca, Celgene, Genentech, Gilead, MEI Pharma, Morphosys, Roche, Sunesis, and Takeda; expert testimony for Celgene and Roche.M.S. reports receiving speaker honoraria from AbbVie.S.S. reports research funding from, consultancy or advisory role for, honoraria from, speakers' bureau participation for, and travel support from AbbVie, Amgen, AstraZeneca, BeiGene, Bristol Myers Squibb, Celgene, Gilead, GlaxoSmithKline, Hoffmann-La Roche, Incyte, Infinity, Janssen, MSD, Novartis, and Sunesis.A.W. reports consultancy or advisory role for and honoraria from AbbVie, AstraZeneca, BeiGene, Roche and Novartis.J.R.B. has served as a consultant for Abbvie, Acerta/Astrazeneca, Alloplex Biotherapeutics, BeiGene, Genentech/Roche, Grifols Worldwide Operations, iOnctura, Kite, Loxo/Lilly, Merck, Numab Therapeutics, Pfizer, Pharmacyclics; received research funding from BeiGene, Gilead, iOnctura, Loxo/Lilly, MEI Pharma, SecuraBio, TG Therapeutics. ",
"section_name": "Conclusions and Future Directions",
"section_num": "5."
}
] |
[
{
"section_content": "Funding: Venetoclax is being developed in collaboration between AbbVie and Genentech.No honoraria or payments were made for authorship.Medical writing support was provided by Phillip Giannopoulos, and Laura Ruhge, of Bio Connections, LLC, funded by AbbVie. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Author Contributions: M.A.A., R.W. and J.R.B. contributed to the conception and design of the paper.M.A.A., R.W., F.H., A.P.K., J.M., S.O., J.F.S., M.S., S.S., A.W. and J.R.B. participated in the drafting, review and approval of this publication.All authors have read and agreed to the published version of the manuscript. Hall Institute that receives milestone payments in relation to venetoclax; has received honoraria from AbbVie, Janssen, AstraZeneca, BeiGene, Roche, Novartis, Gilead Sciences, CSL, Takeda.R.W. reports receiving speaker fees, advisory board fees, and meeting sponsorship from AbbVie, BeiGene and Janssen; speaker fees and advisory board fees from AstraZeneca; and advisory board fees from Secura Bio.F.H. reports receiving speaker fees, advisory board fees and honoraria from AbbVie, AstraZeneca, BeiGene, Janssen and Takeda.A.P.K. reports research grants from AbbVie, Astra Zeneca, Bristol Meyers Squibb, Janssen, and Roche Genentech and has performed advisory board activities for AbbVie, Astra Zeneca, Bristol Meyers Squibb, Janssen, LAVA, and Roche Genentech.J.M. reports advisory board activities for AbbVie and BeiGene.S.O.reports research support from Alliance, AstraZeneca, Caribou Biosciences, Gilead, Kite, Mustang Bio, Nurix Therapeutics, Pfizer, Pharmacyclics, Regeneron; consultancy or advisory role for AbbVie, AstraZeneca, Autolus, BeiGene, Bristol Meyers Squibb, Caribou Biosciences, Ely ",
"section_name": "Conflicts of Interest: M.A.A. is an employee of the Walter and Eliza",
"section_num": null
},
{
"section_content": "Author Contributions: M.A.A., R.W. and J.R.B. contributed to the conception and design of the paper.M.A.A., R.W., F.H., A.P.K., J.M., S.O., J.F.S., M.S., S.S., A.W. and J.R.B. participated in the drafting, review and approval of this publication.All authors have read and agreed to the published version of the manuscript. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Hall Institute that receives milestone payments in relation to venetoclax; has received honoraria from AbbVie, Janssen, AstraZeneca, BeiGene, Roche, Novartis, Gilead Sciences, CSL, Takeda.R.W. reports receiving speaker fees, advisory board fees, and meeting sponsorship from AbbVie, BeiGene and Janssen; speaker fees and advisory board fees from AstraZeneca; and advisory board fees from Secura Bio.F.H. reports receiving speaker fees, advisory board fees and honoraria from AbbVie, AstraZeneca, BeiGene, Janssen and Takeda.A.P.K. reports research grants from AbbVie, Astra Zeneca, Bristol Meyers Squibb, Janssen, and Roche Genentech and has performed advisory board activities for AbbVie, Astra Zeneca, Bristol Meyers Squibb, Janssen, LAVA, and Roche Genentech.J.M. reports advisory board activities for AbbVie and BeiGene.S.O.reports research support from Alliance, AstraZeneca, Caribou Biosciences, Gilead, Kite, Mustang Bio, Nurix Therapeutics, Pfizer, Pharmacyclics, Regeneron; consultancy or advisory role for AbbVie, AstraZeneca, Autolus, BeiGene, Bristol Meyers Squibb, Caribou Biosciences, Ely ",
"section_name": "Conflicts of Interest: M.A.A. is an employee of the Walter and Eliza",
"section_num": null
}
] |
10.1371/journal.pone.0034347
|
Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia
|
Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38-/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).
|
[
{
"section_content": "Accumulating epidemiological and genetic evidence indicates that disruption of circadian rhythms may increase the susceptibility for developing cancer including non-Hodgkin lymphoma (NHL) and also adversely affects tumor progression [1][2][3][4][5].At the molecular level, several genes constituting the clock machinery have been found to establish functional interplays with regulators of the cell cycle, and disrupted expression of these genes has been shown to result in aberrant cell proliferation [2,3,6,7].In a previous study we first described disturbances in the molecular circadian machinery of CLL cells and hypothesized that these alterations may play a role in the molecular pathogenesis of the disease [8].In particular, we found that the core circadian gene CRY1 is up-regulated in samples from high-risk ZAP-70+/CD38+ CLL patients as compared to their ZAP-702/CD382 counterparts which are characterized by a more benign clinical course [8,9].Therefore, based on these data we and others have proposed that CRY1 may serve as a novel prognostic factor which could be useful for the clinical management of CLL patients [8][9][10][11].Functionally, CRY1 has been shown to be essential to the maintenance of circadian rhythm because of its role in the negative arm of the circadian feedback loop [12].However, independent of its circadian function it may have an additional role as a transcriptional regulator of a number of genes involved in cell metabolism and proliferation [4,6,12,13].In the current study we further investigated the role of CRY1 by comparing its expression pattern in molecularly defined CLL subgroups to that of B cells obtained from the peripheral blood of normal donors.Furthermore, we aimed to determine the molecular mechanism(s) underlying deregulated CRY1 expression in CLL. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Peripheral blood samples from 76 CLL patients and 35 normal donors (ND) were analyzed after obtaining written consent according to our institutional guidelines, approved by the Ethics Commission of the University of Essen-Duisburg.The diagnosis of CLL required a persistent lymphocytosis of more than 5.0610 9 /l and a typical CD19 + , CD20 + , CD5 + , CD23 + , Ig light chain (k or l light chain) restricted immunophenotype as revealed by flow cytometry of peripheral blood cells [14].Blood samples from CLL patients and ND were drawn in the morning hours and immediately processed.Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep density gradient centrifugation (Invitrogen, Karlsruhe; Germany) and cryopreserved until further analysis.Patient selection for this study was based on the availability of viably frozen DMSO preserved PBMC and/or freshly isolated total RNA stored in our CLL cell bank.Clinical and laboratory data of the study population are shown in Table 1. Five healthy donors and a subgroup of 57 CLL patients were subjected to DNA methylation analysis.Patient characteristics of this subgroup are shown separately in Table S1.For this set of experiments CD19+ cells were positively selected from PBMCs employing the EasySep Human CD19 Selection Kit (StemCell Technologies, Canada) according to the manufacturer's instruction resulting in a .90%purity of CD19+ B cells.DNA was isolated from the immunomagnetically purified CD19+ cell fraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). ",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "Cell surface expression of CD38 was examined by flow cytometry using a previously described panel of fluorochromelabeled monoclonal antibodies (CD38 PE, clone HB7) in a standard three-color flow cytometry approach using a 20% cut-off to define CD38 positivity [15,16]. ",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "Prognostically relevant anomalies of chromosomal regions 11q, 13q, and 17p, and of chromosome 12 were assessed by fluorescence in situ hybridization, as described previously [16]. ",
"section_name": "Fluorescence in situ hybridization (FISH)",
"section_num": null
},
{
"section_content": "Total RNA from PBMC was extracted with RNeasy Midi Kit (Qiagen, Hilden, Germany) and spectrophotometrically quantified as previously described [8].First strand DNA was synthesized from 1 mg of RNA using oligo(dT) primers employing a commercially available kit (RT-PCR Amplimers, Becton Dickinson, Heidelberg, Germany) according to the manufacturer's instructions.Real-Time PCR was performed with the ABI Prism 7900HT Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the TaqMan Universal PCR Master Mix protocol (Applied Biosystems).Specific assays for CRY1 (Hs00172734_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), BMAL1 (Hs00154147_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), CLOCK (Hs00231857_m1, Assayson-Demand Gene Expression System, Applied Biosystems), PER1 (Hs00242988_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), PER2 (Hs00256144_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) and GAPDH (Hs99999905_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) were used.All reactions were carried out in a 10 ml final volume containing 5 ml Master Mix, 0.5 ml of the specific assay and 4.5 ml of 1:2 diluted cDNA.The amplification was performed under following conditions: 95uC for 10 min followed by 40 cycles of denaturation at 95uC for 15 s and annealing/elongation at 60uC for 1 min.Standard curves for all assays show similar gradients (coefficient of variation 6.8%, data not shown).CRY1 mRNA expression was normalized against the housekeeping gene glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous reference by computing the difference between the respective Ct values (DCt = C t [gene]2C t [GAPDH]).All PCR reactions were performed in duplicate (mean coefficient of variation for all target genes was below 1%).The mean threshold cycle number (C t ) for each tested mRNA was used to quantify the relative expression of each gene: 2 2DCt .DNA methylation analysis of the CRY1 gene by bisulphite genomic sequencing.Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction.The primers for amplifying bisulphite-modified DNA were: CRY1forward: 59-TTTGTGAGGGAAGGTTTAGTTT-39, CRY1reverse: 59-AACAATTTCCAAACCCTCC-39. For possible sequencing of the PCR product we attached a tag (forward 59-CTTGCTTCCTGGCACGAG-39, reverse 59-CAGGAAACAG-CTATGAC-39). The PCR was carried out as follows: denaturation at 95uC for 10 minutes followed by 35 cycles comprising a second denaturation of 30 seconds at 94uC, annealing at 61uC for 30 seconds and extension at 72uC for 45 seconds, followed by 7 minutes elongation at 72uC.The sequence of the PCR product is depicted in Figure S1.PCR products were separated by agarose gel electrophoresis, excised and purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany).PCR products were ligated into pGEMH-T Easy Vector (Promega, Madison, USA) and transformed in XL1-Blue Competent Cells (Stratagene, La Jolla, CA, USA).Plasmid DNA isolated from multiple colonies derived from each PCR product were sequenced using the CRY1 reverse primer on an ABI 3130 Genetic Analyzer. ",
"section_name": "Real-time reverse-transcriptase-PCR (qRT-PCR)",
"section_num": null
},
{
"section_content": "MassArray assays.Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction.The reverse primer contained a T7-promoter tag for in vitro transcription (59-cagtaatacgactcactatagggagaaggct-39) and the forward primer was tagged with a 10 mer (59-aggaagagag-39).Bisulphite-treated DNA was PCR amplified (denaturation at 95uC for 10 minutes followed by 40 cycles comprising a second denaturation of 30 seconds at 95uC, annealing at 61uC for 30 seconds and extension at 74uC for 30 seconds, followed by 10 minutes elongation at 74uC), PCR products were purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany).5 mL of the PCR product were used for the assay.Bisulphite MassArray assays were performed by the Genomics Core Facility, Albert Einstein College of Medicine.The data were analyzed using the analytical pipeline previously published [17].DNA quality and no-template controls, 0%, and 100% methylated DNA were included in all assays. 50 K SNP array analysis of CRY1 copy number at chromosome 12q23.For SNP array studies, genomic DNA was extracted from CD19 positively selected PBMCs using the QIAamp blood kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.Array experiments were performed according to the standard protocol for Affymetrix GeneChip Mapping 50 K arrays (Affymetrix).Briefly, a 250 ng sample of DNA was digested with XbaI, ligated to adaptors, amplified by PCR, fragmented with DNAse I, and biotin-labeled.The labeled samples were hybridized to the Affymetrix 50 K SNP XbaI mapping array (Affymetrix Inc., Santa Clara, CA) followed by washing, staining and scanning.The acquired signal data was normalized with the dChip [18] program, using model-based expression, perfect match (PM)-only background subtraction and quantiles as probe-selection method.The normalized signal was then used as raw copy number per SNP and further analyzed by the GLAD algorithm [19] included in the GenePattern [20] suite, which segmented the data and assigned aggregated copy numbers to segments.A segment was defined as aberrant if its copy number was below 1.7 (loss) or above 2.3 (gain).The resulting list of aberrant regions was in addition filtered for regions consisting of more than 10 SNPs to exclude regions resulting from random noise in the copy number signal. ",
"section_name": "DNA methylation analysis of the CRY1 gene by Bisulphite",
"section_num": null
},
{
"section_content": "Comparisons of clinical and biological parameters between subgroups were carried out using the Mann-Whitney-U test for continuous variables and Fisher's exact test for categorical data.Correlation between CRY1 delta CT and percentage of promoter methylation was tested using Spearman correlation.The Wilcoxon test for paired samples was used to compare CRY1 delta CT analysed in B and T cells from ND. Survival analysis was carried out with the Kaplan-Meier method and differences in treatment free survival between risk groups were tested with the log-rank test.All analyses were performed using R statistical software version 2.10.1 (R Development Core Team, Vienna, Austria, http:// www.r-project.org) and GraphPad Prism Version 5.04 (GraphPad Software). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We measured the mRNA expression of the circadian gene CRY1 in peripheral blood mononuclear cells (PBMC) containing more than 80% CD19+CD5+ leukemic B cells as determined by multiparameter flow cytometry in a cohort of 76 CLL patients (Table 1).In line with our previously published work [8,9] we observed significantly higher CRY1 mRNA levels in high-risk(HR) patients defined by the expression of CD38 and/or unmutated IgVH genes as compared to their CD38 negative and/or IgVH mutated low risk (LR) counterparts (Figure 1A,1B).We could also confirm the prognostic value of CRY1 expression in CLL by comparing the clinical outcome of CLL patients with high vs. low CRY1 expression using the median DCt value as a cut-off.Significant differences in treatment free survival (TFS) were observed between the two groups (Figure S2).Of note, comparative analysis with PBMC derived from age matched normal blood donors revealed that these expression differences can be attributed to an under-expression of CRY1 in LR CLL cases rather than over-expression in the HR group (Figure 1A,1B).As the cellular composition of normal PBMC and PBMC isolated from CLL patients is known to vastly differ with regard to the content of B and T cells we compared mRNA expression levels of CRY1 in immunomagnetically purified CD19+ B cells and CD3+ T cells from normal donors (Figure S3).CRY1 expression was very similar in these normal lymphocyte subsets suggesting that under-expression in LR CLL samples relative to ND controls reflected true down-regulation of the gene in the leukemic cells rather than differences in the cellular composition of the PBMC samples. ",
"section_name": "Analysis of CRY1 expression in normal donor derived vs. CLL PBMC",
"section_num": null
},
{
"section_content": "Aiming at investigating the molecular mechanism(s) underlying disrupted CRY1 expression in CLL cells we then speculated that this phenomenon may be explained by aberration of this gene at chromosome 12q23-q24.1.Exploiting a panel of CLL cases which had been previously investigated for the presence of structural chromosomal abnormalities [21] by high resolution SNP array profiling we could not detect loss of chromosomal material at the CRY1 locus in any of the 55 individual CLL samples analyzed (data not shown).Moreover, an elevated copy number at this position is only detected for samples harbouring a trisomy 12, all other samples exhibit the regular two copies of this gene.The CLL patient cohort of this study (n = 76) comprises 12 cases with a trisomy 12.Among the CD38+ population 10 patients had a trisomy 12 whereas 14 were negative.Among the CD382 patients 2 had a trisomy 12 whereas 28 subjects proved to be negative.No differences in CRY1 mRNA expression were noted neither in the CD38+ nor the CD382 subgroups defined by the presence or absence of trisomy 12 (p = 0.54 and 0.49, respectively). ",
"section_name": "Dysregulation of CRY1 in CLL cannot be explained by chromosomal aberrations",
"section_num": null
},
{
"section_content": "As CRY1 has been previously shown to undergo aberrant DNA methylation events in various human cancers [7,22], we then examined whether epigenetic silencing could explain CRY1 mRNA expression differences in CLL.DNA methylation was studied within the promoter region of the CRY1 gene (Figure S1) employing sequencing of cloned PCR products generated from bisulphite-modified DNA extracted from immunomagnetically purified CD19+ B cells from the peripheral blood of CLL (N = 14) and normal donors (N = 4).We observed that patients with low CRY1 expression show hypermethylation of the analyzed region in comparison to patients with a high CRY1 expression and normal donors (Figure 2A).Using the Sequenom MassArray Compact System for methylation analysis in the same CRY1 promoter region we then confirmed these results in a cohort of 58 CLL samples and 5 normal donors (Figures 3A andS4, Table S3).LR CLL patients either defined by CD38 or IgVH mutational status showed a significantly higher degree of methylation of the CRY1 promoter in comparison to the HR group.In all, there was a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression as detected by qRT-PCR (Figure 2B and3B).Comparing both methods for methylation analysis a high correlation (r = 0.89, mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = 20.62,p = 0.006, Spearman correlation).The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable (open circles indicate ND).doi:10.1371/journal.pone.0034347.g002p,0.0001) proves the consistency and robustness of each of the applied methods (Figure 3C). ",
"section_name": "DNA methylation analysis of the CRY1 gene",
"section_num": null
},
{
"section_content": "To assess the prognostic value of the methylation profile of the CRY1 promoter we defined two groups, i.e. patients with lowly methylated and highly methylated promoter region.The threshold value of 9% was derived from the median value of the CLL samples subjected to MassArray methylation analysis.On survival analysis patients with a highly methylated CRY1 promoter region exhibited a significantly longer treatment-free survival as compared to the hypomethylated patient subgroup (Figure 4). ",
"section_name": "Prognostic value of CRY1 methylation pattern in CLL",
"section_num": null
},
{
"section_content": "Twenty-seven patients were studied at two or more time points using the qRT-PCR assay.As illustrated in Figure S5, CRY1 mRNA expression was relatively stable over time in the majority of patients with stable disease (10/13 (77%), Figure S5, left panel) as well as in patients with progressive disease (12/14 (86%), right panel).The fact that three of the five patients with increasing CRY1 mRNA levels had a comparably benign course of disease may suggest that changes in CRY1 expression over time are not stringently related to disease progression.Moreover, in 6 of the 14 patients (marked by arrows in Figure S5) with a progressive course of the disease first treatment was initiated after collection of the baseline sample in this longitudinal analysis.Although all these patients responded to therapy, no reduction in CRY1 mRNA expression could be documented.It is noteworthy that we did not observe a single case with decreasing CRY1 mRNA levels during follow-up in the entire cohort of patients with samples available for sequential analysis. Interestingly, one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation experienced disease progression as evidenced by rising lymphocyte counts and transition from Binet stage A to stage C disease.Increasing CRY1 expression in this particular patient was shown to be associated with loss of CRY1 promoter hypermethylation (Figure S6). ",
"section_name": "Analysis of CRY1 expression over time",
"section_num": null
},
{
"section_content": "At the molecular level, circadian rhythms are encoded by an autoregulatory loop composed of a set of transcription activators (CLOCK/BMAL1) that induce expression of PER and CRY.Accumulated PER and CRY proteins in turn inhibit BMAL1/ CLOCK transcriptional activity and repress their expression.Thus, aberrant silencing of CRY1 expression in LR CLL may be associated with dysregulated expression of other circadian genes.Indeed, we could observe increased PER2 and CLOCK mRNA levels in CLL as compared to ND derived PBMC, whereas mRNA levels of BMAL1 and PER1 were similar in both groups (Figure Unpaired two-tailed t-test was used to compute p-values.B Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis with the bisulphite MassArray assay.mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = 20.63,p,0.0001,Spearman correlation).The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable.C Correlation between the methylation data resulting from bisulphite genomic sequencing and the MassArray method showed high consistency (r = 0.86, p,0.0001,Spearman correlation).doi:10.1371/journal.pone.0034347.g003S7).However, changes in PER2 and CLOCK expression were noted in both LR and HR CLL samples and could not be related to alterations in CRY1 expression (Figure S7) suggesting that these abnormalities may occur independently of each other. ",
"section_name": "Analysis of the role of CRY1 within the circadian clock's transcription/translation-based feedback loop in CLL",
"section_num": null
},
{
"section_content": "From a cancer biology standpoint we next wanted to test whether deregulated CRY1 expression may also be observed in other lymphoid malignancies than CLL.To this end we employed the CRY1 qRT-PCR assay to screen RNA samples isolated from a wide range of lymphoproliferative disorders including T prolymphocytic leukemia (T-PLL, n = 10), mantle cell lymphoma (MCL, n = 6), hairy cell leukemia (HCL, n = 3), multiple myeloma (MM, n = 8), plasma cell leukemia (PCL, n = 2) and B and T lineage acute lymphoid leukemia (n = 29 resp.n = 19).In disease entities with more than 5 observations, statistical comparisons with ND derived PBMC did not reveal significant expression differences (Figure 5A).However, this finding needs to be interpreted with caution because of the small numbers of samples tested in this series.The availability of a comparably large number of ALL samples (n = 48) allowed for a more detailed analysis of this disease group.The clinical characteristics of this patient cohort are given in Table S2.Similar to its distribution in CLL, CRY1 expression in ALL was found to be very heterogeneous where one patient with a mature B-ALL exhibited a markedly low CRY1 transcript level (Figure 5A).This particular sample demonstrated a high degree of CRY1 promoter methylation while two other ALL samples with a higher CRY1 expression were nearly completely demethylated (Figure 5B). Next we investigated the prognostic value of CRY1 expression in ALL.To this end we compared the clinical outcome of ALL patients with high vs. low CRY1 expression using the median DCt value as a cut-off.No significant differences in overall survival (OS) were observed between the two groups (Figure S8). ",
"section_name": "Analysis of CRY1 expression and CpG island hypermethylation in other hematologic malignancies",
"section_num": null
},
{
"section_content": "In this study, we analyzed the expression of CRY1 mRNA, which encodes a key component of the central and peripheral circadian oscillator, in the PBMC from normal blood donors and patients with CLL [8,11,23,24].In line with our previous work [8,9] and that of others [10,11] we detected elevated CRY1 transcript levels in patients with high risk disease defined by the expression of CD38 and/or unmutated IgVH (UM) genes as compared to their CD38 negative and/or IgVH mutated (M) low risk (LR) counterparts.Lewintre et al. confirmed these results in a recently published microarray-based gene expression profiling study including 36 patients with early-stage CLL [11].Therefore, determination of CRY1 may have potential as a novel prognostic marker in CLL and should be tested in comparison to other established molecular risk factors in the setting of prospective randomized trials. We now uncover that HR CLL cases and ND derived B cells exhibit comparable levels of CRY1 mRNA expression.Thus, disrupted CRY1 expression in CLL can be attributed to downmodulation of CRY1 in LR CLL cases rather than over- expression in the HR group.We then aimed to investigate the molecular mechanisms underlying down-regulation of CRY1 in LR CLL.As CRY1 had been previously shown to undergo aberrant DNA methylation events in various solid human malignancies including breast and ovarian cancers [3,7,22], we examined whether epigenetic silencing could also explain the observed CRY1 mRNA expression differences in CLL subgroups.To this end we performed comparative DNA methylation analysis of highly purified CD19+ B cells from the peripheral blood of 57 CLL patients and normal donors.Indeed, our results show that CRY1 is transcriptionally silenced by promoter hypermethylation in LR CLL cases while HR cases and ND derived B cells exhibit hypomethylated CRY1 promoter regions.Importantly, DNA methylation analysis was complemented by qRT-PCR performed on the same samples revealing a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression in individual cases, suggesting a direct regulation of CRY1 expression through methylation of its promoter.We could show that the methylation status of the CRY1 promoter predicts clinical outcome in CLL patients; where aberrant hypermethylation was associated with a more benign course of the disease.To date, only few reports describe an aberrant methylation phenotype as a predictor of outcome in haematological diseases.Recently, Irving et al reported a panel of methylation markers (CD38, HOXA4, BTG4) in which an overall methylation score was significantly associated with time to first treatment in CLL [25].Olk-Batz et al [26] described that a high methylation profile is associated with an aggressive biological variant of juvenile myelomonocytic leukemia. As stability of CRY1 expression by the leukemic cell clone over time is an important prerequisite for its reliable use as a prognostic marker [27], we sequentially analyzed CRY1 expression in 27 patients.CRY1 transcript levels were remarkably stable in the majority of patients and no consistent changes were noted in relation to alterations in disease activity and treatment history of individual patients.Significant expression changes during followup occurred in 19% of the patients, which could limit the value of this marker under routine clinical conditions.However, these findings are limited by the comparably small number of CLL cases analyzed in this study and thus need to be validated in a larger patient cohort.In one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation disease progression was linked to increasing CRY1 expression and hypomethylation of the CRY1 promoter region.In aggregate, these findings raise the possibility that epigenetic silencing of CRY1 occurs early in the disease and may be lost during disease progression at least in rare cases.Conversely, it appears unlikely that the leukemic cells acquire epigenetic silencing of CRY1 during the course of the disease as none of the 27 patients in the longitudinal analysis showed a decrease of CRY1 expression.It would be interesting to further investigate this hypothesis by systematically comparing the prevalence of CRY1 methylation events in individuals with monoclonal B cell lymphocytosis with Binet stage A, B and C CLL patients. While differential CRY1 expression in HR vs. LR CLL subgroups is now well established [8][9][10][11]28], the functional consequences of CRY1 down-modulation in the leukemic cells are currently unknown.It is tempting to speculate that epigenetic silencing of CRY1 may contribute to the benign clinical behaviour of LR CLL cases.At first sight this notion appears counterintuitive to the general conception that the core components of the molecular clock machinery may function as tumor suppressor genes [29][30][31][32].This view is mainly based on epidemiological and genetic evidence indicating that disruption of circadian rhythms might be directly linked to cancer development including non-Hodgkin-lymphoma [2,5,[33][34][35].While the circadian genes PER1 and PER2 have been clearly shown to function as tumor suppressors in the mouse model [30], a recent study showed that epidermal deletion of BMAL1 in a transgenic mouse model which spontaneously develops squamous tumours leads to significantly fewer neoplastic lesions [36].Along the same line transgenic CRY deficient mice do not show a predisposition to cancer [6].Furthermore and somewhat unexpectedly, ablation in the mouse of both CRY genes in a TP53 2/2 background delays the onset of cancer [13].This latter observation supports our hypothesis that epigenetic silencing of CRY1 may functionally contribute to the indolent clinical behaviour of LR CLL.It is currently unclear whether abrogation of one CRY gene, i.e.CRY1 or CRY2 suffices to fully block the circadian rhythmicity of an individual cell.Studies comparing the biologic characteristics of transgenic mice lacking either one or both CRY genes indicate a certain degree of functional redundancy [37]. Another matter of controversy is whether circadian rhythmicity per se or only certain core clock components are involved in tumorigenesis [4].To address this issue we correlated CRY1 mRNA expression with other components of the circadian clock's transcription/translation-based feedback loop in individual CLL samples.Recently, a number of in vitro and animal studies have further elucidated the important functional role of CRY proteins for the regulation of the molecular clock.For example, work by Ye et al [38] demonstrated that CRY directly interacts with the BMAL1:CLOCK:E-box complex independent of PER resulting in inhibition of the transactivator function of CLOCK:BMAL1.Furthermore, Busino et al. [39] showed in an in vitro model that Cry1 2/2 Cry2 2/2 mouse embryonic fibroblasts are characterized by a loss of oscillation in Per1 and Per2 and exhibit increased Per2 but not Per1 mRNA transcript levels in comparison to control fibroblasts isolated from CRY wild type animals. Here, we found that the expression of PER2 and CLOCK but not BMAL1 and PER1 are also disrupted in CLL as compared to ND derived control samples.However, contrary to what could be expected from above described findings [38,39] these abnormalities could neither be correlated with changes in CRY1 expression nor the clinical course of the disease in individual CLL cases.In aggregate, these latter results clearly suggest that defects in the circadian molecular machinery may be a common phenomenon in CLL cells and are not restricted to CRY1.Ongoing work in our laboratory is aimed at determining the molecular and cellular consequences of silencing different circadian genes in CLL cells in vitro using siRNA oligonucleotide technology. Finally, we wanted to determine whether CRY1 expression may also be disturbed in other lymphoid malignancies than CLL.To this end we measured CRY1 transcript levels in PBMC samples from a range of different disease entities including T-PLL, MCL, HCL, MM, PCL and B and T cell ALL.In a subgroup analysis focussing on ALL we found a heterogeneous expression pattern comparable to that observed in CLL.One patient with a mature B-ALL exhibited a particularly low CRY1 expression which correlated with a high degree of promoter methylation, suggesting that aberrant epigenetic silencing of this gene may also occur in other lymphoid disease entities.However, contrary to CLL survival analysis did not show a significant difference between ALL patients with high and low expression of CRY1, respectively. In conclusion, our data indicate that the previously reported CRY1 gene expression differences in LR vs. HR CLL patients [8,11] are caused by aberrant methylation of the CRY1 promoter in the LR patient subgroup.To our knowledge this is the first report in CLL research linking epigenetic silencing of a specific gene to an indolent clinical course of the disease. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
[
{
"section_content": "We thank Sima Vagaie, Michael Mo ¨llmann, Sana Mohamad and Olga Rempel for excellent technical assistance, Anja Fu ¨hrer and Sabrina Kieruzel for maintaining the CLL biobank and Ute Schmu ¨cker for FACS analyses. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "indicates a uniquely assayable site.Fragmentation patterns are shown in corresponding colors, yellow highlights primer sequences.Methylation data are shown as an average from duplicates.Bar height denotes percent methylation on a scale from 0% (low) to 100% (high), error bars indicate median absolute deviation.CG sites that are putatively outside the usable mass window are indicated as boxes with gray background.(TIF) ",
"section_name": "",
"section_num": ""
}
] |
10.1186/s13046-019-1458-7
|
Notch1 signaling in NOTCH1-mutated mantle cell lymphoma depends on Delta-Like ligand 4 and is a potential target for specific antibody therapy
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p><jats:italic>NOTCH1</jats:italic>gene mutations in mantle cell lymphoma (MCL) have been described in about 5–10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in<jats:italic>NOTCH1</jats:italic>-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of<jats:italic>Delta-like ligand 4</jats:italic>(DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Notch1 expression can be effectively stimulated in<jats:italic>NOTCH1</jats:italic>-mutated Mino cells by DLL4, whereas in the<jats:italic>NOTCH1-</jats:italic>unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both,<jats:italic>NOTCH1</jats:italic>-mutated and –unmutated MCL lymph nodes. Treatment of<jats:italic>NOTCH1</jats:italic>-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with<jats:italic>NOTCH1/2</jats:italic>mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of<jats:italic>NOTCH1</jats:italic>-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for<jats:italic>NOTCH1</jats:italic>-mutated cells as they did not occur in the<jats:italic>NOTCH1</jats:italic>-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>DLL4 effectively stimulates Notch1 signaling in<jats:italic>NOTCH1</jats:italic>-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.</jats:p></jats:sec>
|
[
{
"section_content": "Although with current standard therapy high initial response rates can be achieved, early relapses and rapid disease progression determine the clinical course of most mantle cell lymphoma (MCL) patients [1].In the last years, new therapies were approved targeting the proteasome, the PI3K-Akt-mTOR pathway, the B-cell receptor (BCR) signaling pathway and the anti-apoptotic Bcl-2 protein family.Yet, MCL remains an incurable disease [2].The t (11;14)(q13;q32) translocation leading to Cyclin D1 overexpression is the primary oncogenic event in MCL pathogenesis [3].Furthermore, constitutive activation of BCR signaling plays an important role in disease development [4].Additionally, genomic profiling revealed a high number of secondary genetic alterations and recurrent mutations affecting cell cycle, DNA damage response and apoptosis pathways that contribute to pathogenesis and aggressiveness of MCL [3].Among them, NOTCH1 gene mutations have been described with a frequency of 5-10% and were shown to be associated with shorter survival rates [5,6].Therefore, further investigation of the biological effect of this mutation in MCL and its potential as a therapeutic target is of great interest. The majority of the previously described NOTCH1mutations in MCL consist of either small frameshiftcausing indels or nonsense mutations in exon 34 [6].These mutations lead to truncation of the C-terminal PEST-domain, thereby removing the recognition site from the ubiquitin ligase degradation complex, resulting in a more stable and transcriptionally active form of Notch1-intracellular domain (NICD).The Notch signaling pathway operates in a context-and tissue-dependent way participating in diverse cellular processes, such as cancer stem cell biology, angiogenesis, cell proliferation and survival [7].In addition to the well-known Notch target genes HES1 and HEY1, two basic-helix-loop-helix (Bhlh)-class of transcription factors, numerous genes have been identified as directly regulated by activated Notch1 [8].Some cancer-related target genes of Notch include NF-κB family members, CYCLIN D1, p21, GATA3, MYC and DTX1 [9].In mammals, Notch signaling is usually activated upon interactions with Delta-like ligands (DLL1, DLL3, DLL4) and Jagged ligands (JAG1, JAG2), resulting in a series of proteolytic cleavage events that finally release NICD from its membrane receptor and lead to its nuclear translocation [7]. Targeting Notch signaling has been studied in various cancer types and particularly using gamma-secretase inhibitors (GSI) in hematological malignancies [6,10,11].However, the clinical applicability of GSI is limited as it can cause severe diarrhea resulting from simultaneous inhibition of Notch1 and Notch2 signaling in gut epithelial stem cells [12,13].Thus, alternative strategies for therapeutic targeting of Notch1 are highly warranted.Recently, antibodies that inhibit signaling of both, wildtype and mutated Notch1 receptors have been characterized [14].OMP-52M51 (brontictuzumab) is a full length IgG2 humanized monoclonal antibody that selectively binds the negative regulatory region of the Notch1 receptor leading to inhibition of Notch1 signaling [15].A phase I study has been conducted in subjects with solid tumors showing efficacy in cases with Notch1 pathway activation [16] .In this study, we investigated the role of the Notch ligands in activating Notch1 signaling in NOTCH1-mutated and -unmutated MCL cell lines and evaluated the effects of OMP-52M51 in these cell lines. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "MCL cell lines Mino (CRL-3000), JeKo-1 (CRL-3006) and REC-1 (CRL-3004), were obtained from American Type Culture Collection (ATCC).To avoid Mycoplasma contamination, cell lines were routinely tested for Mycoplasma infection by PCR.The identity of all cell lines was verified by using GenePrint® kit (Promega, Madison, WI, USA).MCL cell lines were cultured in RPMI 1640 complemented with 10-20% fetal bovine serum (FBS), 2 mM L-glutamine and 50 μg/mL penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) and grown in a humidified atmosphere at 37 °C with 5% CO 2 .The murine bone-marrow derived stromal cell line OP9 (CRL-2749; ATCC) overexpressing DLL4 (OP9-DLL4) was generated and grown as described [17,18].Primary cells from MCL patients were isolated and cultured as described [19] and conserved within the Hematopathology collection of our institution registered at the Biobank from Hospital Clínic-IDIBAPS (R121004-094).The ethical approval for this project including informed patient consent was granted following guidelines of the Hospital Clínic Ethics Committee. ",
"section_name": "Cell lines and primary MCL cells",
"section_num": null
},
{
"section_content": "Four micrograms per milliliter of the recombinant Notch-receptor-ligands DLL1, DLL4, JAG1 and JAG2 (R&D Systems, Minneapolis, MN, USA) were resuspended in phosphate buffered saline (PBS) and stored in culture plates for 4 h at 4 °C to let them attach to the plates.MCL cells were treated with 25 μg/mL of OMP-52M51 or human IgG2 (both kindly provided by Oncomed Pharmaceuticals, Redwood City, CA, USA) and incubated at 37 °C for 2 h prior to adding them to the ligand-coated plates for stimulation.For coculture experiments, OP9 cells were plated overnight, and then medium was replaced by MCL cells (0.5 × 10 6 cells/mL) previously treated for 2 h with OMP-52M51.After 24 h of coculture, MCL cells were collected by carefully rinsing the wells without disturbing the stroma monolayer and processed as required. ",
"section_name": "Ligand stimulation and cell treatment",
"section_num": null
},
{
"section_content": "Whole-cell protein extracts were obtained using Triton containing lysis buffer (Tris-HCl pH 7.6 20 mmol/L, NaCl 150 mmol/L, EDTA 1 mmol/L, 1% Triton X-100) supplemented with protease and phosphatase inhibitors (10 μg/ mL leupeptin, 10 μg/mL aprotinin, 1 mmol/L phenylmethanesulfonyl fluoride, 5 mmol/L NaF, 2 mmol/L Na 3 VO 4 ).Solubilized proteins were quantified by Bradford protein assay and 50 μg of protein was analyzed by Western Blotting.The following primary antibodies were used: cleaved-NOTCH1 (Val1744)(D3B8), MEK1/2, phospho-MEK1/2 (Ser217/221)(41G9) (Cell Signaling, Boston, MA, USA), ERK1 (K-23), phospho-ERK (Thy202/204) (E-4) (Santa Cruz, Dallas, TX, USA), α-tubulin and β-actin (Sigma Aldrich, Saint Louis, MI, USA).Chemiluminescence was detected with ECL substrate (Pierce Biotechnology Waltham, MA, USA) on a mini-LAS4000 Fujifilm device (Fujifilm, Valhalla, NY, USA).Protein expression was densitometrically quantified using the Image Gauge software (Fujifilm). ",
"section_name": "Protein analysis",
"section_num": null
},
{
"section_content": "Lymph node (LN) biopsies from MCL cases were obtained from the Hematopathology collection of our institution registered at the Biobank from Hospital Clínic-IDIBAPS (R121004-094).Formalin-fixed paraffin-embedded (FFPE) tissue slides (serial 8 μm sections) were dewaxed and tissue antigens were retrieved by boiling for 10-15 min in sodium citrate (10 mM, pH 6.0).Slides were allowed to cool down to room temperature (RT) and then washed in distilled water and PBS.Quenching was carried out using 1% H 2 O 2 in 100% methanol (40 min, RT) and permeabilization with 0,3% Triton-X-100 (20 min, Sigma-Aldrich) in PBS.Sections were incubated overnight with anti-human DLL4 (H-70) (Santa Cruz) and anti-human CD68 (Dako, Glostrup, Germany).Background staining was determined by incubating with irrelevant antibodies.Unspecific fluorescence was quenched by incubating with avidin/biotin blocking solutions (Vector Lab, Burlingame, CA, USA).For DLL4 detection, tissue slides were incubated with anti-rabbit IgG-HRP antibody (Dako).Signal was amplified using the Cyanine-3 Tyramide Signal Amplification Kit (TSA; NEL 744, Perkin Elmer, Waltham, MA).For CD68 detection, a biotinylated anti-mouse IgG (Vector Lab) was added following incubation with avidin/ biotin complex (Elite Vectastain ABC Complex kit, Vector Lab).Signal was developed by adding Alexa-488-conjugated streptavidin.Nuclei were stained with Topro-3 (Invitrogen, Carlsbad, CA, USA) and mounted with Fluoromount-G (Southern Biotech., Birmingham, AL, USA).Images were acquired using a LSM510 laser scan confocal microscope (Zeiss, Oberkochen, Germany) coupled to an Axiovert200 (Zeiss) microscope, using 63x Plan-Neofluar magnification. ",
"section_name": "Immunohistochemistry and confocal microscopy",
"section_num": null
},
{
"section_content": "A total of 3 × 10 5 MCL cells were collected after stimulation with DLL4 and incubated with OMP-52M51 for 48 h.Then cells were fixed in ice-cold 70% ethanol while being gently vortexed, incubated at -20 °C for 24 h, washed in PBS and resuspended in 500 μL of staining solution containing 20 μg/mL propidium iodide (Invitrogen) and 100 μg/mL RNase A (Thermofisher) in PBS.Cells were incubated during 30 min at 37 °C and analysed using BD LSRFortessa 4 L cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).Cell cycle analysis was performed using FlowJo software (Becton Dickinson). ",
"section_name": "Cell cycle assay",
"section_num": null
},
{
"section_content": "MCL cells were stimulated with DLL4 and incubated with OMP-52M51 for 48 h.Transwell culture polycarbonate inserts (6.5 mm diameter, 8 μm of pore size) (Corning, NY, USA) were transferred to wells containing 600 μL of RPMI supplemented with 0.5% BSA with 200 ng/mL of human recombinant CXCL12 (Peprotech).One hundred microliter of cell suspension (0.5 x 10 6 cells) was then added into the transwell inserts.Input cell count was obtained from adding 100 μL of cell suspension to wells containing 600 μL of 0.5% BSA in RPMI 1640.After 3 h, 100 μL were collected in triplicates from each lower chamber and input well, viable cells were counted on an Attune cytometer (Thermofisher, Waltham, MA, USA) under constant flow rate.Migration is represented as the ratio between migrated cells and total viable input cells. ",
"section_name": "Chemotaxis assay",
"section_num": null
},
{
"section_content": "HUVEC, kindly provided by Dr. MC Cid (IDIBAPS), were cultured as described [20].Supernatants from MCL cells (1 × 10 6 cells/mL) were collected after 48 h of OMP-52M51 treatment of DLL4-stimulated cells.24well plates were coated with 300 μL of Matrigel (Becton Dickinson) before 500 μL of HUVEC (0.5 × 10 5 cells/mL] and 500 μL of the supernatants were added.After 24 h, number of branch points was quantified as a measure of in vitro angiogenesis as the mean of five randomly chosen fields from each well.Images were taken with a DM IL LED microscope coupled to a DFC295 camera (magnification 100x) with Leica Application Suite v 3.7 software (Leica, Wetzlar, Germany). ",
"section_name": "Human umbilical vein endothelial cells (HUVEC) tube formation assay",
"section_num": null
},
{
"section_content": "Total RNA was extracted using the TRIzol method (Life technologies) according to manufacturer's instructions.RNA integrity was examined with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and only high quality RNA samples were further processed.RNA from MCL cell lines was hybridized to a Gene Chip HT HG-U219 array, according to Affymetrix (Santa Clara, CA, USA) standard protocols.RNA from MCL lymph nodes was hybridized to a Gene Chip Human Genome U133 plus 2.0 arrays, according to Affymetrix standard protocols.Determination of the detection call for each probe set of the array was obtained with GeneChip® Command Console® Software (AGCC) (Affymetrix).Raw data was normalized using the Robust Multichip Analysis (RMA) algorithm of the BioConductor Affy Package.Differential expression data analysis was carried out using the Multiexperiment Viewer Platform (TM4-MEV) [21].The number of statistically significant up-and down-regulated genes was determined using Rank Products methodology [22] for cell lines, and Volcano plot [23] for MCL lymph nodes, both setting up a false discovery rate (FDR) < 0.2 and an absolute fold change (FC) > 1.75.PANTHER (http:// pantherdb.org/)was used to perform gene ontology (GO) pathways analysis [24] to visualize the relationships between the significantly modulated genes of NOTCH-mutated MCL lymph nodes and proteins in known pathways of biological processes [25].Primary microarray data of MCL cell lines and primary MCL lymph nodes are available at the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information under accession Nos.GSE125349, GSE36000 and GSE46969. ",
"section_name": "Gene expression profiling",
"section_num": null
},
{
"section_content": "For GSEA, the desktop application version 2.0 (GSEA, Broad Institute at MIT, Cambridge, MA; http://www.broadinstitute.org/gsea/)was applied using experimentally derived custom genes.The \"Custom MCL\" gene set was designed by manually grouping gene sets involved in pathways of Notch-activated genes in MCL according to the results described by Ryan et al. [26].Briefly, we searched molecular signatures (http://software.broadinstitute.org/gsea/msigdb/)corresponding to the pathways described in the GO canonical pathways analysis and GSEA analysis of transcripts increased by Notch in Mino cells (except MYC), and the significant genes were grouped into specific pathways (Additional file 1: Table S1).The \"NOTCH1 direct targets\" gene set was also constructed based on the 79 genes described by Ryan et al. [26].Similarly, the \"NOTCH1 custom\" gene set was designed by manually selecting significant Notch1 related genes found in the literature [6,11,15,[27][28][29][30]. Two-class analysis with 1000 permutations of gene sets and a weighted metric was used for all cases.The resulting gene sets with a false discovery rate (FDR) < 0.05 were considered to be significant, except for the \"NOTCH1\" gene set, where a FDR < 0.12 was used.GSEA analysis was performed of MCL cell lines stimulated or not with DLL4 and treated or not with OMP-52M51 and of MCL lymph node tissues with or without mutations in a NOTCH gene.HeatMaps were created using the Morpheus software (https://software.broadinstitute.org/morpheus/)followed by hierarchical clustering using one minus Pearson correlation of the average of gene expression in order to illustrate the differential expression of those genes significantly modulated by DLL4 stimulation and OMP-52M51 treatment in the MCL cell lines and by NOTCH gene mutation in MCL lymph node tissues for the all custom gene sets analysis performed. In vivo mouse model NSG (NOD-scid-gamma) mice were injected intravenously (i.v.) with 10 × 10 6 Mino cells.MCL cell engraftment was periodically monitored over a 3 months period.After 3 months, mice presented infiltration in several organs.Tumor cells from lymph nodes were collected, cultured in RPMI 1640 + 10% FBS as described above and cryopreserved after several passages.We next confirmed that these Mino cells engraft faster in a secondary transplant (45-60 days).Again, these cells obtained from lymph nodes were collected and cryopreserved.These \"fast engrafting\" tumor cells were then thawed and expanded to get enough cells for in vivo studies.225 × 10 6 Mino cells were then stimulated ex vivo by coculturing them with OP9-DLL4 cells (7.5 × 10 6 Mino cells/plate 100 × 20 mm 2 (Corning).After 24 h of incubation, 15 × 10 6 stimulated Mino cells were injected into the intraperitoneal cavity (IC) of 12 female NSG mice at the age of 10 weeks.Mice were treated intraperitoneally 1 day prior to injection of cells and then every 4 days with 20 mg/kg of OMP-52M51 or control antibody human IgG2 (6 mice/group).After 10 days, mice were sacrificed and a peritoneal lavage (PL) was done by injecting the cavity with 5 mL of cold PBS.Human B-cells were purified by using human CD19 beads.Protein extracts were obtained and expression of cleaved Notch1 was analyzed by Western Blot.Procedures involving animals and their care are conforming to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86/ 609, OJ L 358, 12 December, 1987) and were authorized by the local ethical committee. ",
"section_name": "Gene set enrichment analysis (GSEA)",
"section_num": null
},
{
"section_content": "Data is represented as the mean ± SD of 3 independent experiments.All statistical analyses were done by using GraphPad Prism 6.01 software (GraphPad Software, La Jolla, CA, USA).Volcano plot of P values as a function of weighted FC for mRNA was performed by using Multiplot Studio v1.5.20 software (Benooist-Mathis Lab, Harvard Medical School, MA, USA).Comparisons between 2 groups of samples were analyzed with Kruskall-Wallis nonparametric test followed by Mann-Whitney U test.Results were considered statistically significant when P < 0.05 (*P < 0.05; **P < 0.01; ***P < 0.001). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Activation of Notch1 signaling can effectively be achieved by stimulation with DLL4 in NOTCH1-mutated MCL cells A NOTCH1-mutation affecting the PEST-domain in exon 34 (p.Q2487*) was described in the established MCL cell line Mino [5,6].The effect of this mutation is considered to be ligand-dependent.In contrast, the MCL cell line REC-1 presented an intragenic deletion in exon 28 encoding for a truncated Notch1 protein with increased activity in a ligand-independent fashion [26,31]. First, we analyzed which Notch ligand was the most effective to stimulate Notch1 signaling in MCL.NOTCH1mutated cell lines Mino and REC-1 and the NOTCH1-wt cell line JeKo-1 were stimulated with the recombinant ligands DLL1, DLL4, JAG1 and JAG2 and the Notch activation status in these samples was determined by Western Blot analysis of cleaved Notch1.As shown in Fig. 1a, DLL4 and DLL1 activated the expression of cleaved Notch1 in Mino cells, DLL4 being the most potent ligand.In contrast, these ligands induced only a minor Notch1 activation in unmutated JeKo-1 cells.We confirmed that REC-1 cells overexpressed cleaved Notch1 independently of the ligands.The effect of DLL4 was then confirmed by coculturing of Mino cells with the bone-marrow derived mesenchymal stem cell line OP9 overexpressing DLL4 (OP9-DLL4), which represents a cell culture model better reflecting the situation in vivo, where Notch1 ligands are usually presented by microenvironmental cells [26,31].Increased expression of cleaved Notch1 in Mino cells upon coculture with OP9-DLL4 cells was detected by Western Blot (Fig. 1b).In view of the remarkable in vitro effect of DLL4 in NOTCH1-mutated MCL, we sought to characterize which cells could express this ligand and trigger Notch activation in vivo.We therefore analyzed the expression of DLL4 in MCL lymph nodes (LN) using immunofluorescence staining and confocal microscopy.We found that DLL4 was widely expressed in the vascular endothelium (data not shown) and, importantly, it was expressed by some histiocytic (CD68+) cells in both NOTCH1-mutated and -unmutated cases (Fig. 1c). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We next investigated the effect of the humanized monoclonal Notch1 antibody OMP-52M51 in NOTCH1-mutated (Mino) and unmutated (JeKo-1) MCL cell lines.Since the recognition-binding site of OMP-52M51 in the EGF-like domain is missing in the REC-1 cell line due a deletion in this site [26], this cell line was not suitable for further investigation. We observed that treatment of DLL4-stimulated Mino cells with OMP-52M51 for 24 and 48 h resulted in inhibition of DLL4-mediated cleaved-Notch1 overexpression.This effect could also be observed in primary cells from a NOTCH1-mutated MCL case [MCL#1] carrying the typical 2-bp deletion in exon 34 (c.7541_7542delCT) (Fig. 2a).We confirmed the potential of OMP-52M51 to inhibit DLL-stimulated induction of Notch1 signaling also in the coculture system of Mino cells with OP9-DLL4 (Fig. 2b).Again, no effect was detected in the NOTCH1-wt JeKo-1 cell line. To evaluate the effect of OMP-52M51 on downstream signaling of Notch1, we performed a gene expression profile analysis in JeKo-1 and Mino cells stimulated with DLL4 and treated with OMP-52M51 for 48 h.Using the GSEA software analyzing a customized NOTCH1 set of genes (NOTCH1 custom) [6,11,15,[27][28][29][30], we detected 44 significantly upregulated leading edge genes (Fig. 2c, Additional file 2: Table S2) upon DLL4-treatment in Mino cells (FDR < 0,123; p < 0,005, NES > 1.48).As displayed in the Z score heatmap, the significantly modulated genes upon DLL4 stimulation in Mino cells were effectively downregulated by treatment with OMP-52M51 (Fig. 2c). When we applied a signature of NOTCH1 target genes described in MCL cells (NOTCH1 direct targets) [26], a strong upregulation of these genes was detected in Mino cells stimulated with DLL4 (FDR < 0.001; p < 0.001, NES = 3.00).A good correlation was also observed in JeKo-1 stimulated with DLL4 (FDR = 0.025; p = 0.025, NES = 1.40) (Additional file 3: Table S3 and Figure S1), confirming again that in NOTCH1-unmutated cells the activation of Notch signaling is less potent than in mutated cells.This may be due to the fact that the signaling would not be sustained enough as the wild type Notch1 protein has a rapid turnover [7].Again, OMP-52M51 was able to revert the expression of these direct Notchtarget genes in Mino cells (Fig. 2d). ",
"section_name": "Treatment of NOTCH1-mutated MCL cells with OMP-52M51 effectively prevents DLL4-dependent activation of Notch1",
"section_num": null
},
{
"section_content": "Our gene expression results were analyzed by GSEA using a custom gene set (Custom MCL) generated with data obtained from an integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes and genome conformation data in MCL cell lines [26].We observed that DLL4 upregulated genes related to angiogenesis, apoptosis, migration and adhesion, cell cycle, cytokine signaling, DNA damage and repair, MTOR and MAPK signaling, leukocyte proliferation and defense response (Fig. 3a).OMP-52M51 was able to modulate all these gene sets only in the NOTCH1-mutated Mino cell line (Additional file 4: Table S4). Next, we analyzed the gene expression profile of lymph nodes from primary MCL cases (n = 21), 3 of them harboring NOTCH1/2 mutations.By using the same GSEA analysis, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4stimulated Mino cells (Fig. 3b, Additional file 5: Table S5).In these cases, to maintain Notch signaling, a crosstalk between tumor MCL cells and accessory cells, probably histiocytes, is required.In addition, the PAN-THER GO analysis of the 55 genes differentially expressed in MCL lymph nodes carrying NOTCH gene mutations compared to NOTCH-unmutated MCL lymph nodes, filtered by Volcano plot (Fig. 3c, Additional file 6: Table S6), revealed 818 signaling pathways associated with leukocyte biology primarily enriched for biological processes associated with disease progression such as regulation of tethering/rolling, adhesion and migration of leukocytes (Fig. 3d). Given that most of these gene sets were related to B cell activation, regulation of leukocyte tethering or rolling and leukocyte adhesion and migration, we selected these signatures for further functional validation.As the activation of ERK is an important integration point for B cell activation [32], we analyzed phosphorylation of ERK1/2 and MEK by Western Blot after stimulation of Mino and JeKo-1 cells with DLL4 and treatment with OMP-52M51.As displayed in Fig. 4a, DLL4 stimulation increased phosphorylation of both, MEK and ERK in Mino cells, indicating that aberrant Notch1 signaling stimulates B cell activation in MCL.This effect could be reduced by treatment with OMP-52M51, a process that was not observed in NOTCH1-unmutated JeKo-1 cells (Fig. 4a).To corroborate whether the upregulation of genes related with cell cycle is effectively dependent of aberrant expression of NOTCH1 genes, we analyzed the modulation of cell cycle in NOTCH1-mutated Mino cells and NOTCH1-unmutated JeKo-1 cells treated or not with DLL4 and/or OMP-52M51.We observed that DLL4-stimulated Mino cells showed a significant increase of cell proportion in G 2 -G M phase that was partially reverted (p = 0.057) by incubation with OMP-52M51 for 48 h (Fig. 4b). We next studied the effect of aberrant Notch1 signaling on cell migration by means of chemotaxis assays.As shown in Fig. 4c, DLL4 stimulation of Notch1 significantly increased CXCL12-induced migratory capacity of Mino cells that could be abrogated by treatment with OMP-52M51.In contrast, no effect could be observed in unmutated JeKo-1 cells (Fig. 4c). The DLL4-Notch1 signaling pathway plays an important role in regulating blood vessel formation during physiological and pathological angiogenesis [33,34].Furthermore, data from our gene expression arrays revealed that DLL4 stimulation induces several genes related to angiogenesis.This prompted us to investigate the impact of activating NOTCH1 mutations on tumor angiogenesis.We therefore used supernatants of Mino and JeKo-1 cells stimulated or not with DLL4 and treated or not with OMP-52M51 in a tube formation assay with HUVEC.Supernatants of DLL4 stimulated NOTCH1mutated Mino cells significantly increased number of branch points as a measure of in vitro angiogenesis compared to those of unstimulated cells.Importantly, the proangiogenic effect of these supernatants could be effectively abolished by treatment of cells with OMP-52M51.Again, no differences were detected in unmutated JeKo-1 cells (Fig. 4d). ",
"section_name": "OMP-52M51 significantly impedes DLL4-induced upregulation of genes involved in lymphoid biology, lymphomagenesis and disease progression",
"section_num": null
},
{
"section_content": "Finally, we aimed to confirm the activity of OMP-52M51 in an in vivo MCL model.As NOTCH1-mutated Mino cells are dependent on ligand activation, these cells were stimulated ex vivo with OP9-DLL4 cells to ensure human Notch1 activation.After stimulation of Mino cells for 24 h, cells were injected into the IC of NSG mice and treated every 4 days with 20 mg/kg of OMP-52M51.After 10 days, mice were sacrificed and cells were recollected from the IC (Fig. 5a).Total recovery of the peritoneal cells was evaluated by flow cytometry after staining with huCD45/CD19/antibodies.This short-term treatment with the anti-Notch1 antibody did not affect cell viability or tumor cell counts (data not shown).Protein extracts were obtained and Western Blot analysis confirmed that OMP-52M51 was able to inhibit cleaved Notch1 in vivo (Fig. 5b), although this short-term OMP-52M51 treatment was not enough to cause a significant efficacy in tumor growth. ",
"section_name": "OMP-52 M51 effectively inhibits DLL4 induced activation of Notch1 in an in vivo model",
"section_num": null
},
{
"section_content": "Activating NOTCH1 mutations are among the most frequent secondary genetic alterations in MCL detected in 5-10% of cases [5,6,35].Similar to CLL, the majority of NOTCH1 alterations described in MCL are nonsense truncating mutations and small frame-shift indels located in exon 34 which encodes the PEST domain, resulting in enhanced Notch1 signaling [7,30].The clinical significance of this mutation is highlighted by the fact that patients harboring a NOTCH1-mutation had significantly shorter overall survival rates [5,6].Yet, the molecular impact of NOTCH1 mutations in MCL is not well understood.Recently, it has been postulated that Notch signaling regulates genes involved in BCR and cytokine signaling as well as the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines [26].Targeting Notch signaling has been studied in various cancer types [6,10,36], and particularly in hematological malignancies carrying NOTCH1 mutations [10,37].It has been reported that GSI-mediated inhibition of Notch signaling in NOTCH1-mutated MCL cells resulted in reduction of cell proliferation and apoptosis induction [6].However, the clinical applicability of GSI is limited due to severe gastrointestinal toxicities [12,13].Thus, alternative strategies for targeting Notch signaling are highly warranted.In this way, we analyzed the effect of a humanized IgG2 monoclonal antibody OMP-52M51 that showed encouraging antitumor efficacy in T-acute lymphoblastic leukemia (ALL) xenograft models [15].Recently, phenotypic and molecular features of resistance after long-term treatment with OMP-52M51 have been reported, that are highly heterogeneous, suggesting that leukemia cells can adopt several strategies to evade Notch inhibition according to the therapeutic drug used [38]. In Non-Hodgkin lymphomas, only little is known about the dependency of Notch1 signaling activation upon a certain ligand.Candidate cellular sources include (See figure on previous page.)Fig. 4 Activating NOTCH1 mutations promote BCR signaling, proliferation, angiogenesis and enhance tumor cell migration upon DLL4-stimulation that can be abolished by treatment with OMP-52M51.a Effect of DLL4-stimulation and treatment with OMP-52M51 on ERK and MEK phosphorylation.Mino and JeKo-1 MCL cells were stimulated with DLL4 (4 μg/mL) and treated with OMP-52M51 or IgG2.After 48 h, protein expression of p-ERK, total ERK1, p-MEK1/2 and total MEK1/2 was assessed by Western Blot [n = 3; one representative experiment is shown].b Relative cell proportion in G 2 -G M phase of cell cycle in Mino and JeKo-1 cells stimulated or not with DLL4 and treated with OMP-52M51 or IgG2 for 48 h.Cell cycle phases were measured by flow cytometry using propidium iodide and analysed using FlowJo software (n = 4, *p = 0.0286, bars represent the mean ± SD). c Mino and JeKo-1 cells were stimulated with DLL4 and treated with OMP-52M51 or IgG2 for 48 h.Migration of cells was assessed in a transwell system with inserts of 8 μm pore size.Migration was defined by counting the cells that migrated to the lower chambers containing medium with the chemoattractant CXCL12 (200 ng/mL) (n = 5, **p = 0.0079, bars represent the mean ± SD). d Cells were stimulated with DLL4 and treated with OMP-52M51 or IgG2 for 48 h.Supernatants were then harvested and added to HUVEC.After 24 h, the number of branch points was quantified as the mean of five randomly chosen fields from each well.Pictures were taken with a DM IL LED microscope coupled to a DFC295 camera (magnification 100x) (n = 5, bars represent the mean ± SD, *p = 0.05; ***p = 0.001).Microscope images from one representative experiment are shown Fig. 5 OMP-52M51 effectively inhibits DLL4 induced activation of Notch1 in an in vivo model.a Mino cells were stimulated ex vivo by coculturing them with DLL4 expressing stromal cells.After stimulation for 24 h, cells were injected into the intraperitoneal cavity of NSG mice.Mice were treated IP 1 day prior to injection of cells and then every 4 days with 20 mg/kg of OMP-52M51.After 10 days, mice were sacrificed and cells were recollected from the intraperitoneal cavity.b Expression of cleaved Notch1 was analyzed by Western Blot other hematopoietic cells; endothelial cells, which are capable of inducing Notch signaling in B lymphoma cells [39] and DLL1/4-expressing fibroblastic cells in secondary lymphoid organs [40].In this study, we show for the first time that DLL4 is a potent ligand to activate Notch1 signaling in MCL cells harboring a NOTCH1 mutation in the PEST domain, whereas JAG1 and JAG2 could not sufficiently stimulate Notch1 activity.DLL4 was shown to be constitutively expressed in some lymphoid organs, where it influences regulation of Notch signaling during hematopoiesis [41].In T-ALL cells, where mutations in NOTCH1 are frequent and well characterized, DLL4 plays an important role as part of the tumor microenvironment contributing to early steps of T-ALL cell growth [42].Although immobilized DLL1 was also used to model liganddependent Notch1 activation in MCL [26], the effect of DLL1 in MCL cells is lower than of DLL4.Only a minor effect upon any ligand stimulation was observed in NOTCH1-wt cells corroborating the fact that NOTCH1 mutations in the PEST domain lead to a more stabilized protein due to loss of the recognition site from the ubiquitin ligase degradation complex [7].Furthermore, we provide evidence that DLL4 is expressed in the lymph node MCL compartment, where it could promote Notch activation.In particular, we detected DLL4 to be highly expressed in the vascular endothelium and by some CD68-positive cells, indicating that DLL4 expressed by the LN microenvironment might provide a specific niche for Notch activation. Furthermore, our results confirm that stimulation of Notch signaling by Delta-like ligands could have a critical role in MCL pathogenesis as we observed upregulation of several direct Notch target genes [26] involved in angiogenesis, apoptosis, migration and adhesion, cell cycle, cytokine signaling, DNA damage and repair, MTOR and MAPK signaling, leukocyte proliferation and defense response of B cell activation, cell cycle progression and oncogenesis both in DLL4-stimulated Mino cells and in lymph nodes from primary MCL cases carrying NOTCH mutations.In this way, we detected the induction of transcription factors genes implicated in B-cell differentiation and activation, PAX5 and IRF8 [43][44][45][46] as well as of MYBL2, a transcription factor participating in cell cycle progression [47,48] and recently described as a direct Notch1 target in B cell lymphomas [26].We confirmed a possible interaction between Notch and BCR signaling in B cell malignancies [26] as we observed the upregulation of several genes related to B cell activation (FYN, FGR, NEDD9 and SH2B2) and an increase of cell proliferation after DLL4 stimulation in NOTCH1-mutated MCL cells.Moreover, the results obtained for NOTCH -mutated MCL lymph nodes could be considered a proof-of-concept for those observed in DLL4-stimulated Mino cells, and are in concordance with the poor overall survival [6] and prognosis [5] associated with NOTCH mutations in MCL.Our next aim was to analyze the potential effect of the anti-Notch1 antibody OMP-52M51 on blocking the Delta-like ligandinduced signal in MCL.Treatment of Mino cells with OMP-52M51 could effectively inhibit DLL4-dependent Notch1-activation and suppress transcriptional expression of several direct Notch target genes described in MCL [26].These findings are in line with results observed in T-ALL, where OMP-52M51 treatment of T-ALL patient samples harboring mutations in the NOTCH1-PEST-domain caused strong inhibitory effects on the expression of Notch-target genes [15].Furthermore, we confirmed that in our model anti-Notch1 therapy attenuated the expression levels of the four well known Notch-target genes that have been described as a marker of the effect of OMP-52M51 in T-ALL (CR2, DTX1, HES1 and HES4) [15].Moreover, we elucidated a functional relationship between Notch1 signaling and microenvironment processes related to MCL aggressiveness such as cell proliferation, cell migration and angiogenesis.Notch1 signaling has been shown to play a role in CCL19-driven homing of CLL cells [49] and Notch1 signaling inhibition in multiple myeloma was described to prevent tumor cell migration [50].Accordingly, we showed that OMP-52M51 reverts the strong induction of gene signatures related to tumor cell migration and adhesion upon Notch1 activation and prevents DLL4stimulated migratory capacity of MCL cells. The DLL4-Notch1 axis is known to play an important role in regulating angiogenesis.Previous studies have shown that productive tumor angiogenesis requires cooperation between VEGF-A, which induces proliferation of endothelial 'tip' cells and expression of DLL4 in 'stalk' cells [51].In this context, DLL4 inhibits endothelial proliferation and promotes branching morphogenesis, and the balance between proliferation and branching is key to the formation of a functional capillary network.As such, treatment with anti-DLL4 antibodies resulted in disorganized angiogenesis, characterized by endothelial proliferation without formation of functional capillaries [52].In line with this, we showed that supernatants of NOTCH1-mutated MCL cells stimulated with DLL4 increased HUVEC tube formation, whereas OMP-52M51 blocked this proangiogenic effect.We therefore postulate that by promoting pronounced vasculature development, DLL4stimulated Notch1 signaling might contribute to the clinically observed aggressive behavior of NOTCH1-mutated MCL.Interestingly, NRARP, one of the genes with the strongest induction upon Notch1 stimulation in our gene expression array, has been described to be related to Notch signaling [53] and shown to act as a molecular link between Notch and Wnt signaling in endothelial cells to control stability of new vessel connections [54].In this sense, our results suggest that in MCL, the link between Notch1 and NRARP might promote angiogenesis and needs to be further explored. Importantly, we showed that even if DLL4 could potentially activate Notch1 signaling irrespective of the mutational status of NOTCH1, its functional effects are specific for NOTCH1-mutated MCL cells.This might be due to the fact that in NOTCH1-unmutated MCL, the weak expression of intracellular cleaved Notch1 upon ligand-activation seems to be very unstable due to rapid proteasomal degradation and might not be potent enough to cause functionally relevant transcriptional effects.This observation is of clinical relevance as a specific Notch1-antibody therapy might be a promising therapeutic alternative for the subgroup of patients with NOTCH1-PEST-mutations. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "We show for the first time that DLL4 is a potent stimulator of Notch1 signaling in NOTCH1-mutated MCL and that expression of this ligand observed in histiocytic cells from MCL lymph nodes might provide a specific niche for Notch activation.We propose a link between Notch1-induced expression of tumor-promoting genes and activation of processes contributing to a more aggressive MCL phenotype.Furthermore, our findings indicate that specific inhibition of the Notch1-ligandreceptor interaction provides an efficient and specific therapeutic alternative for a subset of MCL patients. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Jocabed Roldan and the Genomics Unit of IDIBAPS for their technical support. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Primary microarray data are available at the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information Nos.GSE125349, GSE36000 and GSE46969. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1186/s13046-019-1458-7. Additional file 1: Table S1.Gene sets included in the \"Custom MCL\" set of genes. Additional file 2: Table S2.Significantly regulated NOTCH1 target genes upon DLL4 stimulation using a customized NOTCH1 set of genes (NOTCH1 custom) [6,11,15,[27][28][29][30] Additional file 3: Table S3.Significantly regulated direct NOTCH1 target genes upon DLL4 stimulation using a customized set of genes according to Ryan et al. (NOTCH1 direct targets) [26].Figure S1.GSEA Enrichment plots upon DLL4 stimulation in Mino and JeKo-1 cells using a customized set of genes according to Ryan et al. (NOTCH1 direct targets) [26] Additional file 4: Table S4.Modulated gene sets upon treatment with OMP-52M51 of DLL4-stimulated Mino cells using a customized set of genes (Custom MCL) Additional file 5: Table S5.Modulated gene sets comparing NOTCHmutated and wild type lymph nodes from MCL patients using a customized set of genes (Custom MCL) Additional file 6: Table S6 Primary cells from MCL patients conserved within the Hematopathology collection of our institution registered at the Biobank from Hospital Clínic-IDIBAPS (R121004-094).The ethical approval for this project including informed patient consent was granted following guidelines of the Hospital Clínic Ethics Committee.Procedures involving animals and their care are conforming to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 12 December, 1987) and were authorized by the local ethical committee. 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/s13046-019-1458-7. Additional file 1: Table S1.Gene sets included in the \"Custom MCL\" set of genes. Additional file 2: Table S2.Significantly regulated NOTCH1 target genes upon DLL4 stimulation using a customized NOTCH1 set of genes (NOTCH1 custom) [6,11,15,[27][28][29][30] Additional file 3: Table S3.Significantly regulated direct NOTCH1 target genes upon DLL4 stimulation using a customized set of genes according to Ryan et al. (NOTCH1 direct targets) [26].Figure S1.GSEA Enrichment plots upon DLL4 stimulation in Mino and JeKo-1 cells using a customized set of genes according to Ryan et al. (NOTCH1 direct targets) [26] Additional file 4: Table S4.Modulated gene sets upon treatment with OMP-52M51 of DLL4-stimulated Mino cells using a customized set of genes (Custom MCL) Additional file 5: Table S5.Modulated gene sets comparing NOTCHmutated and wild type lymph nodes from MCL patients using a customized set of genes (Custom MCL) Additional file 6: Table S6 ",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "Primary cells from MCL patients conserved within the Hematopathology collection of our institution registered at the Biobank from Hospital Clínic-IDIBAPS (R121004-094).The ethical approval for this project including informed patient consent was granted following guidelines of the Hospital Clínic Ethics Committee.Procedures involving animals and their care are conforming to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 12 December, 1987) and were authorized by the local ethical committee. ",
"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
}
] |
10.5507/bp.2012.031
|
Array-based karyotyping in chronic lymphocytic leukemia (CLL) detects new unbalanced abnormalities that escape conventional cytogenetics and CLL FISH panel
|
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia with a very heterogeneous course. Progress in molecular genetic characterization of CLL has confirmed the prognostic role of unbalanced chromosomal abnormalities currently defined by molecular cytogenetic methods: conventional karyotyping and FISH. However, a significant percentage of genomic abnormalities escapes routine investigation due to the limitations of these methods. It is presently clear that some of these aberrations have impact on prognosis and disease progression.We examined copy number changes in the tumor genomes of 50 CLL patients using bacterial artificial chromosome (BAC) and/or oligonucleotide array platforms. We compared the results of arrayCGH with those obtained by FISH and conventional cytogenetics and evaluated their clinical importance.A total of 111 copy number changes were detected in 43 patients (86%) with clonal abnormalities present in at least 23% of the cells. Moreover, 14 patients (28%) were found to have 39 genomic changes that had not been detected by standard cytogenetic and/or FISH analyses. These included possibly prognostically important recurrent 2p and 8q24 gains. The most frequent unbalanced changes involved chromosomes 18, 7, 3, 9 and 17. We also determined the minimal deleted region on chromosome 6q in 7 cases by chromosome 6/7 specific array.The results showed that a subset of potentially significant genomic aberrations in CLL is being missed by the current routine techniques. Further, we clearly demonstrated the robustness, high sensitivity and specificity of the arrayCGH analysis as well as its potential for use in routine screening of CLL.
|
[
{
"section_content": "The editors and the authors of the papers submitted to the Journal are grateful for the help of our reviewers. • Abdellatif Abdelmohsen ",
"section_name": "",
"section_num": ""
}
] |
[] |
10.7892/boris.111254
|
Guidelines for the use of flow cytometry and cell sorting in immunological studies<sup>*</sup>
|
No abstract available.
|
[
{
"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."
}
] |
[] |
10.3390/ijms25063234
|
Targeting BTK in B Cell Malignancies: From Mode of Action to Resistance Mechanisms
|
<jats:p>The B cell receptor (BCR) signaling pathway plays a crucial role in B cell development and contributes to the pathogenesis of B cell neoplasms. In B cell malignancies, the BCR is constitutively active through both ligand-dependent and ligand-independent mechanisms, resulting in continuous Bruton tyrosine kinase (BTK) signaling activation, which provides a survival and proliferation advantage to the neoplastic clone. Among B cell malignancies, those in which the most significant results were obtained by treatment with BTK inhibitors (BTKi) include chronic lymphocytic leukemia, mantle cell lymphoma, lymphoplasmacytic lymphoma, and diffuse large B cell lymphoma. Covalent BTKi (namely ibrutinib, acalabrutinib, and zanubrutinib) functions by irreversibly blocking BTK through covalent binding to the cysteine residue 481 (Cys-481) in the ATP-binding domain. Despite the high efficacy and safety of BTKi treatment, a significant fraction of patients affected by B cell malignancies who are treated with these drugs experience disease relapse. Several mechanisms of resistance to covalent BTKi, including Cys-481 mutations of BTK, have been investigated in B cell malignancies. Non-covalent BTKi, such as pirtobrutinib, have been developed and proven effective in patients carrying both Cys-481-mutated and unmutated BTK. Moreover, targeting BTK with proteolysis-targeting chimeras (PROTACs) represents a promising strategy to overcome resistance to BTKi in B cell neoplasms.</jats:p>
|
[
{
"section_content": "B cell malignancies are a heterogeneous group of hematologic neoplasms characterized by the abnormal proliferation of malignant B lymphocytes and include the majority of lymphomas, several types of leukemia, and multiple myeloma (MM) [1,2].Although chemoimmunotherapy (CIT) has represented the standard of care in B cell malignancies, in recent years, targeting Bruton tyrosine kinase (BTK) with innovative drugs, namely BTK inhibitors (BTKi), has revolutionized the therapeutic landscape for these diseases [3].Currently, BTKi can be distinguished into covalent and non-covalent BTKi, according to the precise mode of action [4,5]. The B cell receptor (BCR) signaling pathway plays a crucial role in B cell development and contributes to the pathogenesis of B cell neoplasms [6].The activation of BCR signaling occurs when an antigen binds to the surface immunoglobulin (sIg), leading to the coupling and autophosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) on the cytoplasmic tails of CD79A (Igα) and CD79B (Igβ) by the protein kinase LYN, which 2 of 16 belongs to the Src family [7].Subsequently, ITAM phosphorylation creates docking sites for the tyrosine kinase SYK, which activates the B cell linker scaffold protein BLNK [8].As a consequence, BTK is activated through phosphorylation at its aminoacidic Y551 residue by either LYN or SYK [8].Upon activation, BTK initiates several downstream signaling pathways, such as the phosphoinositide 3-kinase (PI3K)-AKT pathway and phospholipase Cγ2 (PLCγ2) pathway, leading to the activation and nuclear migration of various transcription factors, including mTOR, NF-κB, ERK1/2, and NFAT [8,9].These pathways are involved in switching on cellular programs relevant for the survival, differentiation, and proliferation of B cells [10].Additionally, BTK can be triggered by other receptors, such as growth factors, cytokine receptors, and G-protein-coupled receptors (GPCRs), including chemokine receptors and integrins [11]. In B cell malignancies, the BCR is constitutively active through both ligand-dependent and ligand-independent mechanisms, resulting in continuous BTK signaling activation [12].This persistent activation provides a survival and proliferation advantage to the neoplastic clone of B cell neoplasms [13].Among B cell malignancies, those in which the most significant results were obtained by BTK targeting are chronic lymphocytic leukemia/small lymphoma (CLL/SLL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL), and diffuse large B cell lymphoma (DLBCL) [3,[14][15][16]. CLL is a hematologic neoplasm marked by the clonal proliferation of mature B cells [1,2].The clinical diagnosis of CLL requires a lymphocyte blood count ≥ 5 × 10 9 /L, along with a specific immunophenotypic profile that includes the clonal expression of CD19, CD5, CD20, CD23, and sIg [2].CLL is the most common leukemia in adults [17].The disease has a low risk of progression and relatively low lethality, with a 5-year relative survival rate approaching 90% [18].Importantly, CLL predominantly affects elderly individuals, with a median age at diagnosis of 72 years in high-income countries (HICs) [17,18].Despite its indolent clinical course and extended overall survival (OS), CLL is not completely curable, and patients refractory to currently available therapies face the risk of disease progression and, eventually, disease-related mortality [13].This emphasizes the need to address treatment refractoriness in CLL and to develop novel therapeutic approaches.The current standard of care for CLL includes a variety of treatment options with pathway inhibitors, targeting either the BTK or the BCL2 pathways, to manage the disease and improve patient outcomes [14].Ongoing research efforts aim to uncover new treatment modalities, understand the underlying biology of CLL, and explore innovative therapeutic strategies to enhance the management of this hematologic malignancy. MCL is a distinct and relatively aggressive subtype of B cell non-Hodgkin lymphoma (NHL) that accounts for approximately 5% of all NHL cases [1,2,19,20].MCL typically affects individuals in their mid-60s and is characterized by the t(11;14)(q13;q32) translocation, leading to the overexpression of cyclin D1, which is a major regulator of cell cycle progression from the G 1 to S phase [21,22].This genetic alteration results in uncontrolled cell cycle progression, contributing to the aggressive nature of MCL [21].The clinical presentation of MCL often involves lymph node enlargement, splenomegaly, and bone marrow involvement [1].MCL can exhibit systemic dissemination, with extranodal involvement commonly observed in the gastrointestinal tract [23].Treatment approaches for MCL are tailored based on various factors, including the patient's age, performance status and comorbidities, and disease characteristics [24].Intensive chemotherapy regimens, immunotherapy, and autologous stem cell transplantation (ASCT) are commonly employed in young and fit transplant-eligible patients.Notably, the introduction of novel agents has transformed the treatment landscape for MCL; in particular, BTKi have demonstrated significant efficacy, providing a targeted therapy for MCL patients [25][26][27][28]. LPL, also known as Waldenström's macroglobulinemia (WM), is a B cell neoplasm characterized by the clonal expansion of small lymphocytes, plasma cells, and plasmacytoid lymphocytes in the lymphoid tissues, particularly in the bone marrow [1,2].This indolent NHL is characterized by the presence of a monoclonal immunoglobulin M (IgM) in the serum, which is a key diagnostic feature [29].More than 90% of LPL cases harbor the L625P activating mutation of the MYD88 gene [30].The pivotal involvement of MYD88 in LPL pathogenesis is evident in its enhancement of Toll-like receptor signaling, ultimately activating transcription factors belonging to the NF-κB family and associated with the growth and survival of both normal and neoplastic B cells [30,31].LPL commonly involves the bone marrow, leading to cytopenias and bone marrow failure, and might manifest systemically, affecting various organs such as the spleen and liver [29].Common clinical manifestations of LPL include B symptoms, bruising, fatigue, malaise, and clinical manifestations related to blood hyperviscosity [32].Management strategies for LPL depend on the clinical presentation and disease burden [33].Asymptomatic or minimally symptomatic cases may be subjected to watchful waiting with periodic assessments.Symptomatic cases or those with progressive disease may require systemic therapies, including chemotherapeutic agents, immunomodulatory drugs, or targeted therapies like BTKi [29,34,35]. DLBCL is an aggressive, highly heterogeneous non-Hodgkin lymphoma (NHL) that represents the most common subtype of lymphoma [2].Most patients present with generalized lymphadenopathy.Also, extranodal involvement is found in about 30% of patients, most commonly involving the gastrointestinal tract, bone, testes, spleen, central nervous system, and other sites [36].DLBCL is a potentially curable disease with an OS of 60-70%, with the currently used first-line CIT consisting of R-CHOP [37].Nevertheless, 30-40% of the patients are either refractory to the first-line treatment or experience relapse (R/R). DLBCL, based on gene expression profiles, is divided into activated B cell-like (ABC-DLBCL) and germinal center B cell (GCB-DLBCL) types, with each group representing a differentiation state [38].The ABC subtype is characterized by persistent activation of the NF-κB signaling cascade following stimulation of the BCR pathway, thus providing a rationale for experimental BTKi therapy targeting.In contrast, GCB-DLBCL shows an increased dependence on the PI3K and BCL2 signaling pathways [39,40].In the era of precision medicine, extensive efforts have been dedicated to subclassifying DLBCL based on genetic and biological characteristics, which holds promise for targeted therapeutic interventions.Among the notable molecular subgroups within this framework are the MYD88-driven subtype (MCD), characterized by the enrichment of gain-of-function mutations in MYD88 L265P and/or CD79B, and the N1 subtype distinguished by NOTCH1 mutations [41][42][43]. The extensive use of BTKi in multiple B cell malignancies over the years has also led to clinical refractoriness in a fraction of cases, prompting the search for the molecular mechanisms underlying clinical refractoriness [13,44].On these grounds, the aim of this review is to summarize the mode of action of BTKi and the mechanisms of resistance to BTK targeting in B cell malignancies, with a particular focus on CLL, MCL, LPL, and DLBCL. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "The first-in-class covalent BTKi is ibrutinib, a highly potent, first-generation BTKi approved by the Food and Drug Administration (FDA) in 2013 for the treatment of MCL [45].Covalent BTKi function by irreversibly blocking BTK through covalent binding to the cysteine residue 481 (Cys-481) in the ATP-binding domain [13,46].This action results in the occupation of the ATP-binding site, preventing the phosphorylation of downstream targets like Akt and PLCγ2 [13].Consequently, BTK signaling is hindered, leading to the inhibition of the BCR pathway observed both in vitro and in vivo [47].Apart from this intended on-target effect, ibrutinib also deactivates several off-targets, including EGFR, ErbB2, ITK, and TEC [48,49].While contributing to the anti-tumor effect, these off-target effects are associated with adverse events, such as atrial fibrillation, bleeding, and the impairment of macrophage phagocytosis [48][49][50]. More recently, second-generation BTKi, namely acalabrutinib and zanubrutinib, were developed and approved for the treatment of B cell malignancies [51][52][53].Acalabrutinib and zanubrutinib are irreversible and highly effective BTKi, exhibiting greater selectivity than ibrutinib for the Cys-481 residue in the binding site.This results in the reduced off-target inhibition of other kinases in the TEC family, including EGFR and ITK, leading to fewer adverse events [54,55].Compared to ibrutinib-based therapy, treatment with second-generation BTKi is burdened by a lower incidence of cardiovascular adverse events, such as atrial fibrillation/flutter and bleeding [54][55][56].In particular, a recent meta-analysis that compared standard treatment versus second-generation BTKi therapy in B cell malignancies reported no difference in death rate due to cardiovascular adverse events [57]. ",
"section_name": "Covalent BTK Inhibitors",
"section_num": "2."
},
{
"section_content": "The introduction of ibrutinib for CLL treatment marked the beginning of the era of kinase-targeted drugs for this disease [58].In 2014, approval for the use of ibrutinib for the treatment of CLL was granted based on the outcomes of the pivotal Phase 1b/2 trial PCYC 1102, which highlighted its effectiveness in individuals with untreated and relapsed/refractory (R/R) CLL/SLL [58,59].With a median follow-up of 26 months, the estimated 7-year progression-free survival (PFS) reached 83% for treatment-naïve patients and 34% for R/R patients, with an overall response rate (ORR) of 89% [59].In the context of R/R disease, ibrutinib demonstrated superiority (in terms of PFS and OS) over ofatumumab in the RESONATE trial [60].Additionally, as a frontline therapy, ibrutinib outperformed chlorambucil in terms of PFS and OS in the RESONATE-2 trial [61].Further validation of ibrutinib efficacy was provided by the Alliance trial (A041202), which assessed the efficacy of ibrutinib (alone and in combination with rituximab) against chemoimmunotherapy (bendamustine-rituximab, BR) [62].Similarly, the E1912 trial compared the ibrutinib-rituximab regimen with chemoimmunotherapy (fludarabine, cyclophosphamide, and rituximab, FCR) in young and fit patients [63].Both studies indicated prolonged PFS for patients treated with ibrutinib, with the ibrutinib-treated patients in E1912 experiencing extended OS [62,63]. Acalabrutinib, with or without the anti-CD20 monoclonal antibody (mAb) obinutuzumab, demonstrated superior performance compared to CIT regimens in treatmentnaïve and R/R CLL [64,65].Notably, this efficacy superiority was maintained in IGHV unmutated patients and TP53-disrupted patients, representing disease categories associated with biomarkers of poor prognosis [64].The SEQUOIA trial demonstrated the superiority of zanubrutinib over BR, with significantly improved PFS in non-del(17p) patients.The ALPINE trial, a head-to-head comparison of zanubrutinib and ibrutinib, revealed a higher overall response rate (ORR) (78.3% vs. 62.5%) and 12-month OS rates (97% vs. 92.7%)[55,66]. ",
"section_name": "Covalent BTK Inhibitors in CLL",
"section_num": "2.1."
},
{
"section_content": "In recent years, BTKi, notably ibrutinib, have emerged as promising treatment options for MCL [67].A single-arm phase II trial explored the efficacy of ibrutinib and rituximab (IR) induction followed by R-HyperCVAD/MA (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone/methotrexate, cytarabine) in 131 patients, reporting a 98% ORR with 87% complete response (CR) after a median follow-up of 42 months [68].The phase III TRIANGLE trial evaluated ibrutinib in induction and maintenance therapy in 870 patients randomly assigned to three arms [69]: (i) arm A, in which patients received an induction regimen comprising three cycles of R-CHOP/R-DHAP followed by ASCT; (ii) arm A + I, consisting in induction therapy with the incorporation of ibrutinib into three cycles of R-CHOP/R-DHAP, followed by a two-year maintenance phase with ibrutinib; (iii) arm I, which involved a combination approach, utilizing ibrutinib alongside the induction therapy of R-CHOP/R-DHAP, followed by a two-year maintenance period with ibrutinib, omitting ASCT [69].Arm A + I showed an improved failure-free survival (FFS) reaching 88%, compared to 72% in arm A, after a median follow-up of 31 months.Three-year OS rates were 86%, 91%, and 92% for arms A, A + I, and I, respectively.Notably, ibrutinib-containing treatments without ASCT demonstrated a safety profile comparable to standard regimens [69]. Recently, the covalent BTKi zanubrutinib was approved for the treatment of MCL based on the promising results obtained in phase II trials, and phase III trials with this drug are now ongoing [28,70].The ongoing phase III study NCT04002297 is comparing zanubrutinib plus rituximab followed by zanubrutinib monotherapy versus BR, followed by observation in transplant-ineligible, previously untreated MCL patients [71].Moreover, in a phase II trial involving R/R MCL patients, promising results were obtained with acalabrutinib monotherapy, displaying an ORR and CR rate of 81.5% and 47.6%, respectively [27].Over a median follow-up duration of 38.1 months, the median PFS was 22.0 months, and the estimated median OS was 59.2 months.Remarkably, patients with blastoid/pleomorphic histology, which represents a risk factor for a more aggressive and refractory disease, achieved an ORR of 80.8%, similar to that of the general population [27]. ",
"section_name": "Covalent BTK Inhibitors in MCL",
"section_num": "2.2."
},
{
"section_content": "The approval of ibrutinib has changed the treatment landscape for LPL, ushering in a chemotherapy-free approach [72].Several clinical trials highlight ibrutinib's efficacy as a single agent in LPL.In a phase II study dedicated to previously treated patients, ibrutinib achieved a 91% ORR and 73% major response rate (MRR), with rapid minor and major responses in 1 and 2 months, respectively [73].Additionally, the INNOVATE study, a phase II trial combining ibrutinib with rituximab, demonstrated superior ORR and major response rates compared to rituximab alone [74]. Although the advent of ibrutinib marked a paradigm shift in LPL treatment, resulting in notable improvements in patients' quality of life, adverse events (AEs), including atrial fibrillation, have raised concerns [75,76].In addition to the lower cardiovascular toxicity displayed by zanubrutinib, its pharmacokinetics are unaffected by renal dysfunction and hepatic impairment, ensuring flexibility in cases with severe hepato-renal alterations [77].Monotherapy studies in treatment-naïve and R/R patients underscored its tolerability and deep, durable responses across molecular subtypes, even in MYD88 wild-type (MYD88 wt ) cases that otherwise responded poorly to ibrutinib monotherapy [73,74,77,78].The phase III ASPEN study compared zanubrutinib and ibrutinib in LPL, emphasizing zanubrutinib's superior safety, with lower atrial fibrillation and improved overall tolerability [78]. In a phase II trial that enrolled 106 LPL patients, comprising 14 treatment-naïve and 92 previously treated cases, acalabrutinib monotherapy obtained an ORR of 93% [79].MRR was 79% for treatment-naïve and 78% for previously treated patients.Similarly to zanubrutinib, acalabrutinib achieved a higher ORR and MRR in MYD88 wt LPL patients compared to ibrutinib.Their favorable safety and efficacy profile position zanubrutinib and acalabrutinib as compelling, long-term single-agent options for the treatment of LPL, providing a significant therapeutic advancement in this challenging disease. ",
"section_name": "Covalent BTK Inhibitors in LPL",
"section_num": "2.3."
},
{
"section_content": "Patients diagnosed with ABC-DLBCL have significantly poorer survival outcomes when treated with standard R-CHOP therapy compared to those presenting with the GCB subtype [39].Given the chronic activation of the BCR signaling pathway in ABC-DLBCL, clinical studies commenced with the stratification of DLBCL based on subtype determination.In a pivotal phase 1/2 trial (NCT00849654, NCT01325701), ibrutinib displayed notable efficacy, primarily in the ABC subtype, demonstrating an overall response rate (ORR) of 37% as opposed to 5% in individuals with the GCB subtype [80].Based on these grounds, the phase III PHOENIX trial investigated the efficacy of ibrutinib compared to placebo in combination with first-line R-CHOP in patients with non-GCB subtype disease.However, adding ibrutinib to R-CHOP did not demonstrate an OS benefit in the study population as a whole [81].A subgroup analysis revealed improvements in event-free survival (EFS), PFS, and OS that were limited to the <60-year-old cohort.The study showed that EFS and OS exhibited increases of 10.8% and 12.3%, respectively, in the ibrutinib arm compared to the control arm.The lack of benefit observed in older patients was partly attributed to increased treatment-related toxicity with the combination therapy, leading to a suboptimal dosing of CIT. A subsequent subgroup analysis focusing on younger patients (age ≤ 60) revealed that patients with ABC-DLBCL of the MCD and N1 subtypes experienced a notable 3-year EFS 6 of 16 and OS rate of 100% when treated with ibrutinib plus R-CHOP, in contrast to 42.9% and 50%, respectively, in the R-CHOP-alone arm [82].Although this study was not statistically designed to compare responses or survival outcomes among these subgroups, the results are hypothesis-generating and underscore the need for larger studies to identify genetic subgroups that may derive greater benefits from BTKi-containing therapy.According to a recent study, the high sensitivity of the MCD-DLBCL subtype to ibrutinib is attributed to a non-canonical form of chronic selective autophagy [83].Specifically, this autophagic process targets ubiquitinated MYD88 L265P for degradation in a tank-binding kinase 1 (TBK1)-dependent manner within MCD-DLBCL.TBK1 is a crucial serine/threonine kinase involved in various physiological cellular processes, including selective autophagy and innate immunity regulation.However, MCD tumors undergo genetic and epigenetic alterations that attenuate this autophagic tumor-suppressive pathway.Conversely, BTKi promote the autophagic degradation of MYD88 L265P, thereby elucidating their clinical benefit in MCD-DLBCL and suggesting the evaluation of autophagy inhibitors as future therapeutic agents. ",
"section_name": "Covalent BTK Inhibitors in DLBCL",
"section_num": "2.4."
},
{
"section_content": "Despite the high efficacy and safety of BTKi treatment, a significant fraction of patients affected by B cell malignancies treated with these drugs experience disease relapse [13,44].The mechanisms of resistance have been investigated in CLL, MCL, and LPL, while little is known about BTKi resistance in other B cell malignancies.However, different primary and acquired mechanisms have been described to cause resistance to both covalent and non-covalent BTKi. Baseline characteristics, such as del(17p), TP53 mutation, and complex karyotype, carry a higher risk of progression in ibrutinib-treated CLL [13,84].Moreover, the acquired mechanisms of resistance were determined during treatment with BTKi or at relapse [13,85].In this respect, acquired mutations of the BTK and phospholipase C gamma 2 (PLCG2) genes represent the most frequently reported resistance mechanisms in patients receiving covalent BTKi-based therapy (Figure 1).Mutations occur frequently at Cys-481, resulting in the replacement of cysteine by other amino acids (e.g., C481S, and C481R) [13].Mutations at this site lead to the abrogation of covalent binding of ibrutinib, acalabrutinib, and zanubrutinib, with only transient inhibition of the mutant protein.Additionally, less frequent aminoacidic substitutions of PLCγ2 generally lead to gain-of-function of downstream signaling and promote BCR signaling despite BTK inhibition [85].In a multicenter international retrospective study, the enrichment of 8p loss has been reported in BTKirefractory patients in the absence of mutations of the BTK or PLCG2 genes (Figure 1) [86].This genetic aberration leads to haploinsufficiency of the TRAIL receptor (TRAIL-R), which causes leukemic cells to become resistant to TRAIL-induced apoptosis, independent of the mutational status of BTK and/or PLCG2.Moreover, BIRC3 and NFKBIE mutations have been detected exclusively in ibrutinib relapsing patients carrying a wild-type BTK gene, suggesting that the aberrant activation of the canonical/noncanonical NF-κB pathway might be a possible mechanism of drug evasion [86].A longer follow-up is needed to determine whether the presence of these mutations is associated with subsequent resistance to treatment with covalent BTKi.Other genetic alterations may complement BTK mutations in inducing BTKi resistance.For example, the transcription factor EGR2 was found to be almost exclusively mutated in relapsed patients carrying BTK mutations [86].EGR2 is activated by ERK phosphorylation upon BCR stimulation, suggesting that EGR2 mutations may lead to constitutively dysregulated BCR signaling that, together with the existing BTK/PLCG2 mutations, results in resistance to covalent BTKi (Figure 1).factor EGR2 was found to be almost exclusively mutated in relapsed patients carrying BTK mutations [86].EGR2 is activated by ERK phosphorylation upon BCR stimulation, suggesting that EGR2 mutations may lead to constitutively dysregulated BCR signaling that, together with the existing BTK/PLCG2 mutations, results in resistance to covalent BTKi (Figure 1).Mutations of BTK and PLCG2 play a role in acquired resistance to covalent BTKi also in MCL [87].On the other hand, primary resistance to covalent BTKi in MCL may be due to elevated cyclin D1 levels, which can also arise from genomic deletions or point mutations in the 3'-untranslated region, producing shorter and more stable cyclin D1 transcripts (Figure 1) [88].In addition, specific mutations (E36K, Y44D, or C47S) in CCND1 increase cyclin D1 protein levels, causing defective proteolysis and promoting resistance to ibrutinib in MCL cell lines (Figure 1) [89].Both mutant CCND1 and overexpression of the wild-type gene have been shown to confer increased resistance to ibrutinib.Notably, the Y44D mutation of CCND1 is associated with resistance even at high ibrutinib concentrations (5-10 μmol/L).Mutations of BTK and PLCG2 play a role in acquired resistance to covalent BTKi also in MCL [87].On the other hand, primary resistance to covalent BTKi in MCL may be due to elevated cyclin D1 levels, which can also arise from genomic deletions or point mutations in the 3'-untranslated region, producing shorter and more stable cyclin D1 transcripts (Figure 1) [88].In addition, specific mutations (E36K, Y44D, or C47S) in CCND1 increase cyclin D1 protein levels, causing defective proteolysis and promoting resistance to ibrutinib in MCL cell lines (Figure 1) [89].Both mutant CCND1 and overexpression of the wildtype gene have been shown to confer increased resistance to ibrutinib.Notably, the Y44D mutation of CCND1 is associated with resistance even at high ibrutinib concentrations (5-10 µmol/L). Moreover, MCL cell lines responsive to ibrutinib exhibit continuous BCR signaling, which leads to the activation of the classical NF-κB pathway via BTK [90].In contrast, a possible mechanism of resistance to ibrutinib in MCL is represented by the hyper-activation of the alternative NF-κB pathway, which is independent of BTK signaling [90,91].Somatic mutations identified in resistant MCL cell lines, including nonsense mutations in tumor necrosis factor receptor-associated factor 2 (TRAF2) and deletions in TRAF3, indicate a shift to the alternative NF-κB pathway (Figure 1) [90].These mutations lead to loss-of-function in negative regulators of the alternative NF-κB pathway, promoting stabilization of the MAP3K14 enzyme.This stabilization facilitates the processing of p100 to nuclear factor kappa B subunit 2 (NFKB2), resulting in continuous activation of the alternative NF-κB pathway.The genomic profiling of archived MCL tumor samples has identified recurrent mutations in the TRAF2, BIRC3, and MAP3K14 genes, suggesting a dependence on either the BCR-BTK-NF-κB (classical) or MAP3K14-NF-κB (alternative) pathways for MCL pathogenesis [90]. Recently, whole-exome sequencing of MCL samples has revealed recurrent mutations in various genes, including those already known to be mutated in MCL, such as ATM, MEF2B, and KMT2D [92].Furthermore, this approach has identified novel mutated genes, including CARD11, encoding a scaffold protein required for BCR-induced NF-κB activation [92,93].CARD11 mutations were found in 5-15% of additional MCL cases and, when overexpressed in vitro, conferred resistance to ibrutinib and lenalidomide, indicating continuous activation of the NF-κB pathway, irrespective of BTKi (Figure 1) [92].These findings offer new insights into ibrutinib resistance mechanisms in MCL. Acquired resistance mechanisms to covalent BTKi in LPL include 8p loss, as well as aminoacidic substitutions of Cys-481 of BTK and PLCG2 mutations [94].Furthermore, CXCR4 is the second most frequently mutated gene in LPL, with mutations occurring in up to 40% of patients [95].In a physiological context, the interaction between the surface receptor CXCR4 and the chemokine CXCL12 leads to the activation of Akt signaling, initiating the transduction of anti-apoptotic stimuli [96].Truncating mutations of the C terminal domain of CXCR4 cause defective internalization of the receptor upon binding CXCL12, resulting in a gain-of-function of CXCR4 [97].Consistently, gain-of-function mutations of CXCR4 lead to the constitutive activation of Akt signaling, resulting in sustained survival signals for cancer cells [98].Based on the mutation profile of MYD88 and CXCR4, LPL patients may be divided into four subgroups, to predict their response to covalent BTKi [73,99]: (i) MYD88 mut CXCR4 wt , which is the most prevalent subtype and is characterized by LPL bearing the MYD88 mutation without CXCR4 mutation.These patients typically exhibit favorable responses to covalent BTK inhibition, with a major response rate (MRR) of more than 90% [95]; (ii) MYD88 mut CXCR4 mut , which identifies LPL harboring both mutations, which are more prone to presenting with hyperviscosity and bone marrow involvement.In comparison to CXCR4 wild-type tumors, MYD88 mut CXCR4 mut display an extended time to major response, reduced PFS, and reduced response to covalent BTKi.Response to BTKi in this subgroup also depends on the type of CXCR4 mutation: frameshift mutations lead to an MRR of ~80%, while nonsense mutations display a worse predictive value, with an MRR of 55% [95]; (iii) MYD88 wt CXCR4 wt , which, in contrast to tumors with MYD88 mutations, identifies LPL with wild-type MYD88 and CXCR4, follows a more aggressive course and is associated with decreased OS.Additionally, MYD88 wt CXCR4 wt cases exhibit poor responsiveness to covalent BTKi (MRR to ibrutinib: near to 0%) [95]; (iv) MYD88 wt CXCR4 mut , a rare combination, which likely has the same prognosis as MYD88 wt CXCR4 wt cases.DLBCL primary resistance to ibrutinib is associated with the presence of activating mutations in CARD11 and inactivating mutations in TNFAIP3 (also known as A20), a negative regulator of NF-κB.These genes function downstream of BTK and thus enhance NF-κB activity independent of upstream BTK activity [39,40,100,101].The KLHL14 gene frequently undergoes inactivating mutations in mature B cell malignancies, particularly in the MYD88 L265P, CD79B mutant (MCD) subtype of DLBCL, which heavily relies on BCR signaling for survival [102].Despite its unclear pathogenic role and molecular function in DLBCL, recent evidence reveals that KLHL14 facilitates the turnover of immature glycoforms of BCR subunits, leading to reduced total cellular BCR levels.Loss of KLHL14 confers resistance to ibrutinib and enhances the assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, thereby promoting prosurvival NF-κB activation. Investigations of secondary resistance to ibrutinib in DLBCL primarily rely on cell line models due to the lack of comprehensive long-term follow-up data.Interestingly, mutations in BTK and PLCG2 were not identified; however, resistant cases showed upregulation of the PI3K/AKT/mTOR signaling pathway, leading to increased tumor cell survival [103].Additionally, recent studies have revealed the role of epigenetic mechanisms in ibrutinib resistance, particularly in ABC DLBCL cell lines [104].In this respect, RAC2, a small GTPase, is upregulated through the increased accessibility of its enhancer domain.RAC2 activates PLCγ2 independent of BTK, thereby resulting in the activation of NF-κB. ",
"section_name": "Mechanisms of Resistance to Covalent BTK Inhibitors",
"section_num": "3."
},
{
"section_content": "Reversible and non-covalent BTKi have been developed and proven effective in both C481-mutated and unmutated BTK in preclinical and clinical studies (Table 1).Examples include vecabrutinib, fenebrutinib, nemtabrutinib (ARQ 531), and pirtobrutinib (LOXO-305) [105][106][107].Pirtobrutinib, a highly selective and noncovalent reversible BTKi, received approval in January 2023 for the treatment of adult patients with R/R MCL in the US [108].Pirtobrutinib has demonstrated enduring efficacy and a positive safety profile in heavily pretreated patients with R/R MCL, especially those who had previously received covalent BTKi therapy (Table 1) [109].Remarkably, the median ORR in patients who experienced progressive disease on a prior covalent BTKi was 43%, leading to the approval via the Accelerated Approval Program by FDA.Additionally, patients with high-risk disease features, such as blastoid/pleomorphic variants, elevated Ki-67 index, and TP53 mutations, also exhibited favorable responses to pirtobrutinib [109].In addition, the BRUIN study demonstrated that pirtobrutinib is able to achieve an ORR of 62% in R/R CLL cases after multiple lines of treatment, with the majority of cases previously being treated with a covalent BTKi [110].A recent update of the BRUIN study reported a high efficacy profile of pirtobrutinib in LPL, with a remarkable MRR of 64% in patients who previously received at least one line of therapy with covalent BTKi [111].Remarkably, recent reports have identified various mutations causing acquired resistance to both noncovalent and certain covalent BTK inhibitors in CLL [13].These mutations, such as V416L, A428D, M437R, T474I, and L528W in the tyrosine kinase domain of BTK, have been shown to impair binding to both non-covalent and covalent BTKi in vitro [112].Interestingly, enrichment of the BTK-L528W mutation was observed in CLL patients receiving zanubrutinib compared to ibrutinib in observational studies, suggesting the potential for cross-resistance with reversible BTK inhibitors [113].Despite this evidence, a recent update of the results of the BRUIN study reported that although the L528W and T474x BTK mutations occur more frequently in patients who underwent treatment with covalent BTKi, response to pirtobrutinib is high even in those who display such mutations [114]. Targeting BTK with proteolysis-targeting chimeras (PROTACs) represents a promising strategy to overcome resistance to BTKi in B cell neoplasms (Table 2) [13,115].PROTACs represent a novel category of small molecules that employ two covalently linked ligands [115].These ligands work in tandem to recruit both the target protein and E3 ubiquitin ligase, initiating and facilitating the proteasomal degradation of the target protein [116].Several preclinical studies have demonstrated the effectiveness of PROTACs against in vitro mutant BTK-C481 cells, inducing BTK degradation through ubiquitin-mediated protein breakdown.Notably, NX-2127, the pioneering targeted protein degrader of BTK, exhibited its efficacy in preclinical studies by promoting the degradation of both wild-type and mutant BTK [117].In addition to BTK degradation, NX-2127 also demonstrated a preclinical efficacy comparable to that of immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide [118,119].NX-2127 achieves this effect by catalyzing the ubiquitination of Ikaros (IKZF1) and Aiolos (IKZF3), thus leading to heightened T-cell activation [118,120,121].While further research is required, the increased T-cell activity facilitated by NX-2127 has potential for addressing the immune dysfunction commonly associated with CLL [122].Recently reported clinical results from a first-in-human phase I trial on NX-2127 involved 23 R/R CLL patients, with a median of six prior therapies (2)(3)(4)(5)(6)(7)(8)(9)(10)(11), all of whom had previously undergone treatment with a covalent BTKi and/or BCL2 inhibitor venetoclax [117].In this challenging patient cohort with limited therapeutic options, NX-2127 demonstrated an overall response rate (ORR) of 33% in 12 evaluable patients, with a median follow-up of 5.6 months [117].These findings underscore the potential utility of BTK degraders, such as NX-2127, in patients refractory to multiple lines of therapy, irrespective of their BTK mutation status.Phase I clinical trials of BTK degraders in the treatment of B cell malignancies have been initiated and are currently ongoing (Table 2).Abbreviations: LPL, lymphoplasmacytic lymphoma; CLL, chronic lymphocytic leukemia; R/R, relapsed/refractory; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; NHL, non-Hodgkin lymphoma; DLBCL, diffuse large B cell lymphoma; FL, follicular lymphoma; RS, Richter syndrome; PCNSL, primary central nervous system lymphoma. ",
"section_name": "BTK Targeting Approaches to Overcome Covalent BTKi Resistance",
"section_num": "4."
},
{
"section_content": "Great progress has been made in the treatment of B cell neoplasms, particularly in targeting BTK in CLL, MCL, and LPL.The first drugs developed to inhibit BTK activity were covalent BTKi, namely first-generation (ibrutinib) and second-generation (acalabrutinib, zanubrutinib) BTKi.The mechanism of action of covalent BTKi involves inhibiting the kinase activity of the molecule via the binding of the drugs to the amino acid residue Cys-481, located in the ATP-binding pocket.Despite the significant efficacy demonstrated in CLL, MCL, and LPL, primary and secondary resistance phenomena have emerged in patients treated with covalent BTKi. Third-generation inhibitors, such as pirtobrutinib, can overcome the resistance to covalent inhibitors developed by B cell neoplasms because they have a non-covalent mode of action, featuring a reversible binding to BTK.Pirtobrutinib has indeed shown excellent results in terms of efficacy in CLL, MCL, and LPL, even in diseases refractory to treatment with second-generation inhibitors.Furthermore, as a perspective for the future, BTK degraders have demonstrated their effectiveness in CLL, and several studies are underway to test these drugs in other B cell neoplasms. ",
"section_name": "Conclusions and Perspectives",
"section_num": "5."
}
] |
[
{
"section_content": "Funding: Work by the authors described in this review has been supported by: Molecular bases of disease dissemination in lymphoid malignancies to optimize curative therapeutic strategies, (5 × 1000 No. 21198), Associazione Italiana per la Ricerca sul Cancro Foundation Milan, Italy; PNRR-MAD-2022-12375673 (Next Generation EU, M6/C2_CALL 2022), Italian MoH, Rome, Italy; the AGING Project-Department of Excellence-DIMET, Università del Piemonte Orientale, Novara, Italy; Ricerca Finalizzata 2018 (project RF-2018-12365790), MoH, Rome, Italy; and AIL Novara VCO OdV, Novara, Italy.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable. ",
"section_name": "",
"section_num": ""
}
] |
10.31557/apjcp.2020.21.12.3517
|
Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia
|
Objective: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. Methodology: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. Results: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. Conclusion: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second generation and third generation TKIs.
|
[
{
"section_content": "Abstract leukemia in adults (Izzo et al., 2019).BCR-ABL, an oncoprotein central to the pathogenesis of CML, is fusion product of Abelson murine leukemia (ABL) and breakpoint cluster region (BCR) genes located on chromosome 9 and 22 respectively (Rowley, 1973).BCR-ABL, a tyrosine kinase, is constitutively expressed to promote growth and replication (Himburg et al., 2019).Genomic instability in CML is well established that is primarily caused by BCR-ABL itself, and it leads to acquisition of further mutations in BCR-ABL itself as well as other genes with the clinical course of the disease (Slupianek et al., 2011;Wu et al., 2018). Imatinib mesylate (Gleevec ® USA; Glivec-Europe/ Australia; formerly STI571) is a BCR-ABL TKI which has intensely improved the treatment outcomes in chronic-phase CML patients (CML-CP).The drug inhibits BCR-ABL by binding to an inactive conformation of the kinase, preventing signal transduction and cellular proliferation (Druker et al., 2001;Druker et al., 1996).Regardless of the efficacy of imatinib in patients with Ph-positive (Ph+) CML-CP, approximately 15% of patients display resistance to imatinib or relapse after an initial therapeutic response and thus have less favorable long-term outcomes (Hochhaus et al., 2009).A simple mechanism of resistance to TKI treatment is owing to mutations in the kinase domain of BCR-ABL.Such mutations have been reported in 40% to 60% of imatinibresistant patients (Jabbour et al., 2006).More than 90 distinct mutations in BCR-ABL have been recognized, each conferring variable degrees of resistance to imatinib treatment (Soverini et al., 2010). Nilotinib, a small rationally designed TKI by Tasigna; Novartis Pharmaceuticals Corporation, is good inhibitor of BCR-ABL and is approved for imatinib-resistant CML. in vitro study shown wide inhibitory activity of nilotinib against most of the imatinib-resistant mutants of BCR-ABL (Weisberg et al., 2006).Previous data have demonstrated that imatinib-resistant patients with CML-CP or CML-accelerated phase (AP) treated with nilotinib had high and durable rates of response for all mutations except T315I, Y253H, E255K/V, and F359C/V (Hughes et al., 2009;Kantarjian et al., 2011).In last few years, compound BCR-ABL mutations (multiple BCR-ABL mutations in a single molecule) have posed a new threat by causing resistance to all FDA approved TKIs including T315Imutation specific drug ponatinib (Kang et al., 2018).Studies related to compound BCR-ABL mutations can help in early determination of drug resistance, to find out complex mechanisms of multi-drug resistance and help in designing new drugs for multiple / compound mutant CML cases (Hochhaus et al., 2020;Soverini et al., 2020). Here we report a novel compound BCR-ABL mutation causing multi-TKI resistance CML as well as structural BCR-ABL changes induced by this mutant associated with multiple kinase inhibitor resistance. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "A total of 10 CML imatinib resistant patients who had received nilotinib treatment for at least 12 months or more, from Allied Hospital Faisalabad, Pakistan, were included in this study.Number of patients was low as very few patients could afford nilotinib, a very expensive drug.Three ml blood was taken in labeled EDTA tubes per manufacturer's instructions.Blood was processed within 24 hours to ensure minimum degradation of mRNA, otherwise stored at -40°C.Randomization was stratified according to the Sokal risk score at the time of diagnosis.This study was approved by the local ethics committee or review board and was conducted in accordance with the Declaration of Helsinki with written consent form each patient. ",
"section_name": "Patients and blood sample collection",
"section_num": null
},
{
"section_content": "A complete clinical history since the diagnosis of CML were collected in patient data forms and required permission and consent were taken from hospital authorities and participating patient.Patients were routinely followed-up to record hematologic and cytogenetic response on monthly basis.We followed European Leukemia Net guidelines for categorizing CML patient samples into drug sensitive or resistant on the basis of their response to therapy (Baccarani et al., 2014). ",
"section_name": "Clinical history and patient's data",
"section_num": null
},
{
"section_content": "RNA was extracted from patient blood using protocols described previously (Chomczynski et al., 1987;Liedtke et al., 1994).All RNA extraction steps were carried out on crushed ice to evade degradation of nucleic acid.Isolated RNA was stored immediately at -40˚C till further processing. ",
"section_name": "RNA extraction from blood samples",
"section_num": null
},
{
"section_content": "Quality of RNA was assessed by performing native agarose gel electrophoresis (Biocom and PowerPac Basic, BIO-RAD) and RNA was observed as two discrete fluorescent bands (28S and 18S) (UVP PhotoDoc-ItTM Imaging System). ",
"section_name": "Qualitative analysis of RNA",
"section_num": null
},
{
"section_content": "cDNA was synthesized by reverse transcription of RNA previously described protocol with slight variations (Thermo scientific™, USA) (van Dongen et al., 1999). ",
"section_name": "Complementary DNA synthesis",
"section_num": null
},
{
"section_content": "Integrity of cDNA was assessed by amplification of housekeeping gene, glyceraldehyde 3-phosphate d e h y d r o g e n a s e ( G A P D H ) u s i n g f o r w a r d : 5ˊ-ACCACAGTCCATGCCATCA-3ˊ and reverse: 5ˊ-TCCACCACCCTGTTGCTGTA-3ˊ primers (Thermo Scientific™, USA and Applied Biosystems 2720 thermal cycler) (Asad et al., 2012). ",
"section_name": "cDNA integrity check",
"section_num": null
},
{
"section_content": "Amplification of BCR-ABL fusion oncogene was performed by nRT-PCR (Applied Biosystems 2720 thermal cycler) using long range enzyme (Thermo ScientificTM, USA).For comparing CML patients (n =10) with control, we amplified the kinase domain of ABL gene from healthy population (n = 20).Primer sequences and PCR reaction conditions were followed from Willis et al., DOI:10.31557/APJCP.2020.21.12.3517 Novel BCR-ABL Compound Mutations and Multiple TKI Resistance in CML kinase protein structure in complex with nilotinib was retrieved from worldwide archive of structural data of biological macromolecules, RCSB's PDB (PDB ID: 3cs9) and viewed using Delano Scientific's PyMOL (DeLano, 2002).Further modeling studies were done to find out structural changes responsible for drug resistance.Modeling studies for structure and function prediction of the mutant structure was done using I-Tasser server with alignment generated using MUSTER threading program (Laskowski and Swindells, 2011).Detailed structural visualization and comparative alignment was done for the wild type and mutant modeled using PyMOL.LigPlot+ v.1.4.5 program (EBI, Cambridge, UK) was used to check and schematically plot the polar and hydrophobic interactions between the protein BCR-ABL kinase and the inhibitor nilotinib (Cortes et al., 2012).The 3-D structure of the protein-ligand complex was given as an input in PDB format and the software displayed their interacting residues and bonds. ",
"section_name": "Nested reverse transcription PCR (nRT-PCR)",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Peripheral-blood samples were collected from imatinib resistant CML patients aged ≥18 years who were receiving nilotinib (400 mg twice daily) for at least 12 months.Clinical characteristics of CML patients, wherein all were in chronic phase CML have been summarized in Table 2.The response to nilotinib was determined by examining the clinical end points: complete hematological response (CHR), and complete cytogenetic response (CCyR, n=2).Number of male patients was more than females with male: female ratio of 8:2.Splenomegaly (80%) was the most common symptom followed by fever, weight loss (40%), fatigue and hepatomegaly (20%).The mean age of patients in the present study was 37.2 years (age range: 28-50 years).White cell count ranging within normal values (0.5-1.0 x 10 9 /L) was observed in 2 patient, while 8 individuals had more.Eight patients had platelet count between 100-450 x 10 9 /L, while 2 had more than 450 x 109/L.Six patients had blast cells less than 5% and four patients had their hemoglobin level < 10g/dl).Sokal risk scores were low of six, intermediate for two and high 2005 (Willis et al., 2005), with slight variations (Table 1). ",
"section_name": "Patient demographics, clinical and lab oratory findings",
"section_num": null
},
{
"section_content": "To avoid any contamination of non-specific DNA fragments during sequencing, PCR products were purified from agarose gel (Quick gel extraction Kit, Invitrogen) and visualized (UVP PhotoDoc-ItTM Imaging System).Needed PCR products of specific size were carefully excised, and purified PCR products were stored at -20˚C for sequencing or further use. ",
"section_name": "Purification (Gel extraction) of amplified PCR products",
"section_num": null
},
{
"section_content": "To detect any likely point mutation in ABL-kinase domain of CML patients, we directly sequenced the amplified products by Sanger's method using primer pair (ABI-3130XL and ABI-3730 genetic analyzers, Applied Biosystems, USA) (Willis et al., 2005). ",
"section_name": "Direct Sequencing analysis of PCR amplicons",
"section_num": null
},
{
"section_content": "To find out any polymorphic variations in our population, the ABL kinase domain DNA sequences of healthy individuals were aligned against the reference sequence (NCBI's GenBank Accession Number: M14752.1).Polymorphic variations were excluded while detecting point mutations in CML patient's sequences (Geneious R7).Entire kinase domain sequence including P-loop, C-Helix, SH2 contact and activation loop was explored to identify any probable nucleotide substitutions responsible for altered response to imatinib treatment. ",
"section_name": "Analysis of sequencing data",
"section_num": null
},
{
"section_content": "Fisher's exact test (Chi-square test) was done for comparing patient's response (sensitivity or resistance) towards therapy and mutation status among different clinical categories or predictors of CML patients (IBM SPSS version 19, Chicago, IL, USA.). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Structural bioinformatics analysis was done for the BCR-ABL protein for structurally validating our findings.Crystallographically determined human ABL ",
"section_name": "Bioinformatics Analysis",
"section_num": null
},
{
"section_content": "The total RNA isolated from blood samples of CML patients was run on 1.5% agarose gel to check the integrity of extracted RNA (Figure 1).The PCR quality of cDNA was evaluated by running the amplification product of GAPDH housekeeping gene on 1.5% agarose gel (Figure 2).The nested PCR of the BCR-ABL gene was successfully amplified in all samples at the end of second round nested RT-PCR.Nested PCR products of nilotinib sensitive wild type (a) nilotinib sensitive mutant (b) CML patients were run on 1% agarose gel.As CML patients had two common variants of fusion oncogene, the resulting products in different samples were 1306 bp (b2a2) and 1380 bp (b3a2) in size (Figure 3). ",
"section_name": "RNA extraction and Nested RT-PCR amplification of BCR-ABL kinase domain",
"section_num": null
},
{
"section_content": "Direct sequencing analysis of the amplified PCR products was performed to detect point mutations in the ABL-kinase domain of CML cases.The electropherogram showing compound mutations, including a novel BCR-ABL mutation associated with primary nilotinib resistance in CML patient (Figure 4).Molecular Structural Studies: Molecular modeling were done to find out structural changes induced in wild type (Lys245, Gly250) mutated to mutant type (Asp245, Trp250) responsible for conferring drug resistance.Our residues of interest lies within the glycine-rich phosphate loop (P-loop) traversing from 242-256 constitute (Figure 5, 6, and7).Ligplot analysis was carried to check the schematic 2-D molecular interactions between kinase protein and the bound ligand nilotinib (Figure 8).The primarily interacting residues were Glu286, Thr315, Met318 and Asp381.In the current study all CP-CML patients who were receiving nilotinib (≥ 12 months) second line TKI therapy after imatinib resistance were included and investigated for any mutations related to nilotinib resistance.Male to female ratio was 4:1, respectively.In previous literature incidence of CML with male-to-female ratio of 1.1:1 to 1.74:1 had been reported (Aziz et al., 2010;Jacobs et al., 2018).The altered ratio in present study is owing to small number of available patients on nilotinib therapy.Enlargement of spleen was the most commonly observed symptom in the patients during clinical examination (80%).This finding is in concordance with the conclusions that massive splenomegaly is often associated with CML (Efficace et al., 2011).The incidence of other signs and symptoms; including fever, hepatomegaly and fatigue etc. varies in literature and is stated to be 40-64.4%,21.7-100% and 22-100%, respectively (Aguayo et al., 2008;Karimi et al., 2008). ",
"section_name": "Direct DNA Sequencing",
"section_num": null
},
{
"section_content": "Among the patients included in the present studies, 80% showed a massive over-production of WBCs ranging between 12-670 x 10 9 /L at the time of diagnosis.In literature, leukocytosis (>10 x 10 9 /L) has been stated between 40.7--77.3% of patients with CML [29,35].The platelet counts ranged between 105-694 x 10 9 /L.Only one patient had platelet count >450 x 10 9 /L i.e. 694 x 10 9 /L (20%) which is in agreement to the reported range of platelet count in CML patents (10.2-21.4%)(Jabbour et al., 2011;Jacobs et al., 2018).Hemoglobin level lower 10g/dl was evident in 2 patients (40%) In cancer patient's low Hb level has been reported in range of 15-59.3% of CML patients (Jacobs et al., 2018;Jabbour et al., 2017). The monitoring of cytogenetic response (CyR) in CML has a prognostic value and CCyR achievement has been linked with prolonged survival (Druker et al., 2006).In the present study, patients exhibiting CCyR, partial CyR and minor CyR was 40%, 40% and 20%, respectively. Similarly, HR is also important in CML response monitoring.In the present study, CP-CML patients showed CHR to be 80%.In literature, HR has been reported to be 90% 53 and 93.75% (Gupta and Prasad, 2007;Hochhaus and Kantarjian, 2013). In the present study, 1 out 5 CP-CML patients treated with nilotinib 400 mg twice daily, compound mutations, including a novel K245N mutation and G250W mutation in BCR-ABL domain were detected.The presence of this compound mutations most likely have contributed to nilotinib resistance in this patient.This is the first report of such novel mutation occurring in BCR-ABL.The nilotinib resistant patient progressed to accelerated phase (AP), however, mutations alone are not a cause of disease progress to advanced stage or treatment failure and alternative mechanisms may be involved (Melo and Barnes, 2007).Furthermore, we cannot confirm whether treatment-emergent mutation in our case is solely a driver mutation and cause of disease progress to advance stage.The remaining patients (n=4) of nilotinib responders did not show any mutations.G250E (P-loop) mutation has been reported in CML patients treated with either imatinib or nilotinib (Melo and Barnes, 2007). Although BCR-ABL inhibitor based treatment helps majority of CML patients to lead normal life till treatment continue but unfortunately few patients i.e. 20-30% develop TKI resistance, either primary or secondary and are prone to progress toward the AP or BP of the disease (Kang et al., 2006).BCR-ABL mutations are the major background players in manifestation of resistance to all FDA approved TKIs including imatinib, dasatinib and nilotinib (Hochhaus et al., 2020;Baccarani et al., 2014).According to European or North American clinical guidelines, detection of mutations is a vital during drug resistance or drug switching, because nature of mutation determine the resistance or sensitivity for different drugs.So in case of resistance or unsatisfactory response to In summary, novel compound mutations resulted due to innate genomic instability in BCR-ABL+ CML cells leading to nilotinib resistance have been reported in our studies.Protein modelling studies showed that mutations caused structural changes in BCR-ABL protein which may have impacted binding of drug to the BCR-ABL target and led to nilotinib resistance.It is important to perform mutational analysis prior to drug switching and distinction between compound versus single mutation because it may influence the clinical selection of the most suitable TKI to overcome resistance.Studies on novel compound BCR-ABL mutations associated with multiple TKI resistance will help understand mechanism of drug resistance in CML in more depth and will lead to better drug resistance management strategies to improve CML survival, possibly leading to a potential cure ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This work was partially supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.This Work was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul-Aziz City for Science and Technology, Kingdom of Saudi Arabia, grant Number 14-Med2817-02. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Novel BCR-ABL Compound Mutations and Multiple TKI Resistance in CML BCR-ABL inhibitors, mutational information can help in deciding better alternative TKI.Till date four main mutational regions for TKIs resistance have been reported: P-loop, SH3 contact (3) SH2 contact and (4) A-loop (Kang et al., 2006;Druker etal., 2001). Nilotinib, a second-generation inhibitor, has greater potency and specificity (approximately 20 fold) for wild type BCR-ABL than imatinib (Blay et al., 2011).It binds to ABL tyrosine kinase and disrupt the ATP-phosphatebinding site causing inhibition of catalytic activity of enzyme.Other than T315I mutation, it inhibits all imatinib-resistant mutants (Hamad et al., 2013).Clinically, nilotinib based treatment is recommended by ELN for all newly diagnosed Ph+ CML-CP and imatinib intolerant Ph+ CML in CP or AP patients.Better understanding of reasons and mechanism of resistance will improve the treatment strategy (Hamad et al., 2013).Nilotinib is good suppressor of overall resistance causing BCR-ABL mutations (Marchesi et al., 2013), and found to sensitive against D816V mutant variant, which is resistant to imatinib (Weisberg et al., 2007).Blast crisis stage of patients need more attention as about 40-90% of BCR-ABL mutation in imatinib resistant patients detected here (Soverini et al., 2020). Coexistence of two or more mutations in the same BCR-ABL mRNA, is termed as a \"compound mutation\".Patients harboring compound mutations like V299L, T315A, F317L/V/I/C17 (Soverini et al., 2006;Burgess et al., 2005) were found to be less sensitive to dasatinib therapy and nilotinib treatment failure has been reported in several studies in patients with compound mutations like E255K/V,Y253H, F359V/C/I [Hughes et al., 2009;Soverini et al., 2009;Khorashad et al., 2013).Khorashad et al., (2013) analyzed BCR-ABL kinase domain mutations in 1,700 samples by direct DNA sequencing and found that 11.4% CML patient samples harbored ≥2 mutations.In total, 30 different compound mutations were observed, with the most frequent mutation being T315I among all compound mutants.These mutations existed in two forms: compound or polyclonal.Compound mutations (multiple mutations in the same BCR-ABL molecule) accounted for 70.2% and 29.8% were polyclonal mutations (mutations occurring in multiple BCR-ABL molecules) .Therefore, the distinction between compound versus single mutations is clinically important because it may influence the selection of the most suitable TKI to overcome resistance.Several compound mutations have been shown to confer resistance to ponatinib and this is likely to apply to other third-line TKIs as well (O'Hare et al., 2009;Yang et al., 2014).Yang et al., (2014), evaluated the effect of nilotinib on two CML imatinib resistant patients with V299L mutation.Nilotinib treatment successfully turned V299L mutation of both cases into negative.Acquired BCR-ABL mutations related to nilotinib treatment in patients with CML was initially available from two meeting abstracts (Hughes et al., 2007).The first of these described F349V, E255K/V, E355G, G250E, M244V and T315I.The second report described Y253F, E255V, T315I, and F359V as emerging mutations.Many mutations developing in response to nilotinib have now been described in literature allowing improved interpretation.These mutations include: T315I, Y253C, Y253H, E255V, L248V, K285N, E282K, E255K, F359C, F359V, K247N, W430L, T406I, E255R, A380S, Y253F, Q252H, L387F, F311V, L273F, H375P, H396P, E292K, D276G, E275K, L387M, G250E, E431G, V379I, Q252H, F317L, F311L, N297T, E355A, F317V, H396R, G250A, E355G, F486S, V289L, M244V, D325N, M388L, F317C, G250V, M237I, S348L, E255D, E281K, M351T, E453K, E344G, E459Q, F311I, A433T, F349V, E292V, L384M, E274K, L370P, M388I, E450K, S438C, E459G, E459K (Khorashad et al., 2013;Hughes et al., 2007), and F359I/V, E279K, L248L/V (Ursan et al., 2015). A systematic literature search was performed by Ursan et al., (2015) between January 1966 and May 2013, regarding use of nilotinib as first line TKI in CML patients.A single study of nilotinib was found in this search as firstline treatment in CML that included 61 patients, in whom the mutation incidence was 3.3% (95% CI = 0.0%-7.7%).In these patients (n=61), E255K and F359C mutations were reported and experienced disease progression (Branford et al., 2009).Clinically relevant mutations to second-generation inhibitors were F317L/I/C/V, V299L, and T315A for dasatinib; Y253H, E255K/V, and F359V/C for nilotinib; and T315I for both TKIs (Goodrich, 2014). A 3 rd generation molecule such as ponatinib, has been approved by FDA and can inhibit majority of mutations in kinase domain.However, it's unlikely that ponatinib will be used for treatment because of drug dosing issue (Yang et al., 2014).Additionally, ponatinib had been found to be vulnerable to couple of compound mutations like G250E/ T315I and E255V/T315I.These BCR-ABL1 compound mutants causing high-level resistance against ponatinib is next significant challenge to 2 nd and 3 rd generation TKIs in CML patients (Marchesi, 2013;Eide et al., 2011;Azam et al., 2009). Protein structure modeling of Human ABL kinase -nilotinib complex was done to find out structural changes responsible for resistance.The residues spanning from 242-256 constitute the glycine-rich phosphate loop (P-loop) that facilitates the transphosphorylation reaction.Residues of present interest (245 and 250) are not directly involved in binding with the drug.However, may contribute to the development of resistance against nilotinib in the mutated protein.Substitution mutation (glycine, the smallest amino acid to the bulkiest aromatic tryptophan) will potentially hinder the smooth entry of the drug into the binding cavity.Additionally, the mutation in the surface residue from basic Lys to acidic Asn might also interfere with inhibitor binding.In past, BCR-ABL protein modelling studies have led to unravel mechanisms of TKI resistance in CML and to design novel drugs to treat drugresistant CML (Ozkan et al., 2018;Altıntop et al., 2017).We hope that reported compound BCR-ABL mutations and its protein modelling studies will help unravel complex mechanism of drug resistance and to design more effective drugs for CML.Further studies on compound BCR-ABL mutations, specifically resistance to 2 nd and 3 rd generation TKI resistance, by employing genomic techniques will lead to better clinical management of recent challenges in CML treatment (Hochhaus et al., 2020;Soverini et al., 2020). Authors declare no conflict of interest. ",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "Novel BCR-ABL Compound Mutations and Multiple TKI Resistance in CML BCR-ABL inhibitors, mutational information can help in deciding better alternative TKI.Till date four main mutational regions for TKIs resistance have been reported: P-loop, SH3 contact (3) SH2 contact and (4) A-loop (Kang et al., 2006;Druker etal., 2001). Nilotinib, a second-generation inhibitor, has greater potency and specificity (approximately 20 fold) for wild type BCR-ABL than imatinib (Blay et al., 2011).It binds to ABL tyrosine kinase and disrupt the ATP-phosphatebinding site causing inhibition of catalytic activity of enzyme.Other than T315I mutation, it inhibits all imatinib-resistant mutants (Hamad et al., 2013).Clinically, nilotinib based treatment is recommended by ELN for all newly diagnosed Ph+ CML-CP and imatinib intolerant Ph+ CML in CP or AP patients.Better understanding of reasons and mechanism of resistance will improve the treatment strategy (Hamad et al., 2013).Nilotinib is good suppressor of overall resistance causing BCR-ABL mutations (Marchesi et al., 2013), and found to sensitive against D816V mutant variant, which is resistant to imatinib (Weisberg et al., 2007).Blast crisis stage of patients need more attention as about 40-90% of BCR-ABL mutation in imatinib resistant patients detected here (Soverini et al., 2020). Coexistence of two or more mutations in the same BCR-ABL mRNA, is termed as a \"compound mutation\".Patients harboring compound mutations like V299L, T315A, F317L/V/I/C17 (Soverini et al., 2006;Burgess et al., 2005) were found to be less sensitive to dasatinib therapy and nilotinib treatment failure has been reported in several studies in patients with compound mutations like E255K/V,Y253H, F359V/C/I [Hughes et al., 2009;Soverini et al., 2009;Khorashad et al., 2013).Khorashad et al., (2013) analyzed BCR-ABL kinase domain mutations in 1,700 samples by direct DNA sequencing and found that 11.4% CML patient samples harbored ≥2 mutations.In total, 30 different compound mutations were observed, with the most frequent mutation being T315I among all compound mutants.These mutations existed in two forms: compound or polyclonal.Compound mutations (multiple mutations in the same BCR-ABL molecule) accounted for 70.2% and 29.8% were polyclonal mutations (mutations occurring in multiple BCR-ABL molecules) .Therefore, the distinction between compound versus single mutations is clinically important because it may influence the selection of the most suitable TKI to overcome resistance.Several compound mutations have been shown to confer resistance to ponatinib and this is likely to apply to other third-line TKIs as well (O'Hare et al., 2009;Yang et al., 2014).Yang et al., (2014), evaluated the effect of nilotinib on two CML imatinib resistant patients with V299L mutation.Nilotinib treatment successfully turned V299L mutation of both cases into negative.Acquired BCR-ABL mutations related to nilotinib treatment in patients with CML was initially available from two meeting abstracts (Hughes et al., 2007).The first of these described F349V, E255K/V, E355G, G250E, M244V and T315I.The second report described Y253F, E255V, T315I, and F359V as emerging mutations.Many mutations developing in response to nilotinib have now been described in literature allowing improved interpretation.These mutations include: T315I, Y253C, Y253H, E255V, L248V, K285N, E282K, E255K, F359C, F359V, K247N, W430L, T406I, E255R, A380S, Y253F, Q252H, L387F, F311V, L273F, H375P, H396P, E292K, D276G, E275K, L387M, G250E, E431G, V379I, Q252H, F317L, F311L, N297T, E355A, F317V, H396R, G250A, E355G, F486S, V289L, M244V, D325N, M388L, F317C, G250V, M237I, S348L, E255D, E281K, M351T, E453K, E344G, E459Q, F311I, A433T, F349V, E292V, L384M, E274K, L370P, M388I, E450K, S438C, E459G, E459K (Khorashad et al., 2013;Hughes et al., 2007), and F359I/V, E279K, L248L/V (Ursan et al., 2015). A systematic literature search was performed by Ursan et al., (2015) between January 1966 and May 2013, regarding use of nilotinib as first line TKI in CML patients.A single study of nilotinib was found in this search as firstline treatment in CML that included 61 patients, in whom the mutation incidence was 3.3% (95% CI = 0.0%-7.7%).In these patients (n=61), E255K and F359C mutations were reported and experienced disease progression (Branford et al., 2009).Clinically relevant mutations to second-generation inhibitors were F317L/I/C/V, V299L, and T315A for dasatinib; Y253H, E255K/V, and F359V/C for nilotinib; and T315I for both TKIs (Goodrich, 2014). A 3 rd generation molecule such as ponatinib, has been approved by FDA and can inhibit majority of mutations in kinase domain.However, it's unlikely that ponatinib will be used for treatment because of drug dosing issue (Yang et al., 2014).Additionally, ponatinib had been found to be vulnerable to couple of compound mutations like G250E/ T315I and E255V/T315I.These BCR-ABL1 compound mutants causing high-level resistance against ponatinib is next significant challenge to 2 nd and 3 rd generation TKIs in CML patients (Marchesi, 2013;Eide et al., 2011;Azam et al., 2009). Protein structure modeling of Human ABL kinase -nilotinib complex was done to find out structural changes responsible for resistance.The residues spanning from 242-256 constitute the glycine-rich phosphate loop (P-loop) that facilitates the transphosphorylation reaction.Residues of present interest (245 and 250) are not directly involved in binding with the drug.However, may contribute to the development of resistance against nilotinib in the mutated protein.Substitution mutation (glycine, the smallest amino acid to the bulkiest aromatic tryptophan) will potentially hinder the smooth entry of the drug into the binding cavity.Additionally, the mutation in the surface residue from basic Lys to acidic Asn might also interfere with inhibitor binding.In past, BCR-ABL protein modelling studies have led to unravel mechanisms of TKI resistance in CML and to design novel drugs to treat drugresistant CML (Ozkan et al., 2018;Altıntop et al., 2017).We hope that reported compound BCR-ABL mutations and its protein modelling studies will help unravel complex mechanism of drug resistance and to design more effective drugs for CML.Further studies on compound BCR-ABL mutations, specifically resistance to 2 nd and 3 rd generation TKI resistance, by employing genomic techniques will lead to better clinical management of recent challenges in CML treatment (Hochhaus et al., 2020;Soverini et al., 2020). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Authors declare no conflict of interest. ",
"section_name": "Conflict of interest",
"section_num": null
}
] |
10.18632/oncotarget.26043
|
Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia
|
Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is classified as a lymphoproliferative disorder characterized by the accumulation of a clonally expanded lymphocytic population with resistance to apoptosis and coexpression of CD5, CD19, and CD23 in B lymphocytes in the peripheral blood, lymph nodes, bone marrow, spleen and liver [1][2][3][4].Cytogenetic analysis indicates that CLL has many different genetic mutations which are heterogeneous in terms of progression, therapeutic response and outcome [5][6][7][8].Several biological markers related to CLL outcome have been identified such as deletion of chromosome 17p13, 11q23 and 13q14, trisomy 12, expression of ZAP70, IgVH genomic rearrangement, and aberration of tumor protein 53 gene [5][6][7][8].These cytogenetic markers allow the stratification of broad prognostic groups of CLL patients; however, underlined mechanisms of drug insensitivity (primary drug refractory and chemo-caused drug-relapse) and the regulation to overcome drug-resistance remain poorly understood. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Since 1995, fludarabine (flu) has been used as one of the chemotherapy agents to treat CLL [9].Later, rituximab, a monoclonal antibody against B-cell marker CD20, and alemtuzumab, a monoclonal antibody that binds to CD52, were also developed and have been used for immunotherapy in chemo naïve patients [10,11].More recently, in order to reduce the relapse rate and increase complete responses, a combination of multiple therapeutic agents such as the combination of flu, cyclophosphamide and rituximab (FCR) has been developed [12][13][14].FCR has been established as one of the current standard first line treatment for medicallyfit CLL patients.The medium survival of FCR treatment is longer than 10 years, however, the survival for flu-refractory patients is only 0-3 years [10,12].Furthermore, most of the treated patients will eventually relapse, and about 10% of CLL patients are primary flu-refractory [10,12].It is clear that flu-insensitivity (primary flu-refractory and flu-caused relapse) is associated with poor survival, and represents a big challenge for treatment used flu and other purine analogue drug containing regimens.Therefore, it is very important to understand molecular mechanisms of fluresistance, to identify the novel targets, to develop rational therapeutic strategies for overcoming flu-resistance and to provide new therapeutic options. Drug-resistance is still one of the most pressing problems in treating cancer.Overexpression of some proteins [such as P-glycoprotein (P-gp), also known as ATP-binding cassette sub-family B member, multidrug resistance protein or cluster of differentiation 243 (CD243)] or alteration of some genes (such as p53) leads to the aberrant cell signaling and dysregulation of cell function [15,16].Sphingolipids are a class of lipids with important functions involved in a variety of cellular processes such as growth, proliferation, differentiation, senescence, apoptosis, survival and drug-resistance [17][18][19][20].The metabolism of sphingolipids is one of the important signaling pathways that regulate apoptotic (chemotherapy), survival (drug resistance) and proliferative (cancer progression) activities [17][18][19][20].Deregulation of sphingolipid metabolism is reflected in various pathophysiological conditions including metabolic disorders and cancers [17][18][19][20].Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative and pro-apoptotic functions, and has important therapeutic potential [21].A number of anticancer drugs or cytotoxic agents can significantly induce the accumulation of ceramide in response to treatment [19].On the contrary, ceramide can also be converted to glucosylceramide by glucosylceramide synthase (GCS) which transfers the glucose from uridine diphosphate glucose to ceramide, promptly decreasing ceramide levels and consequently promoting cell survival [18,19].It is very important to understand how ceramide metabolism is associated with drug-resistance. In the present study, we isolated the peripheral blood mononuclear cells (PBMCs) from 34 CLL patients, treated them with flu, and analyzed cell viability to identify primary flu-refractory and flu-relapsed patients.We used MEC-2 cells, a CLL cell line established from the peripheral blood of a patient with B-chronic lymphocytic leukemia [22], to establish flu-resistant clonal cells and demonstrated that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell (LSC)-like cells, and that the modulation of ceramide metabolism can enhance flu sensitivity and reverse flu resistance. ",
"section_name": "Research Paper",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We isolated the PBMCs from 34 CLL patients: 14 patients are chemo-naïve and 20 patients were treated with either single drug (alemtuzumab, rituximab, ofatumumab, pembrolizumab, bendamustine, ibrutinib or idelalisib) or combinations (bendamustine and rituximab; flu and rituximab; cyclphosphamide, vincristine and prednisone or FCR).The isolated PBMCs were treated with 10 µM flu for 72 hrs and then measured cell viability.Table 1 showed patient prognostic, pretreatment characteristics and cell viability.We found four flu-insensitive patients which cell viability is over 85%.Two are chemo-naïve patients (P7 and P21), one is bendamustine-rituximab-treating patient (P3) and the other is FCR-treating patient (P19).Due to the limited amount of patient blood samples and most of the PBMCs only survive but do not proliferate in vitro, we used MEC-2 cells, a CLL cell line, to establish flu-resistant clonal cells and to study molecular mechanism of flu-resistance. ",
"section_name": "Effect of flu on PBMC viability",
"section_num": null
},
{
"section_content": "Using escalating concentrations of flu (from 30 µM up to 200 µM), we have established multiple MEC-2 fluresistant clones (Figure 1A).Flu-resistant clonal cells did not display any obvious morphological changes, except they grew in large clumps (Figure 1B).To determine the B cell lineage of flu-resistant clonal cells, we performed immunoblotting to compare expression of CD20, a B-cell marker, in MEC-2 cells and flu-resistant clonal cells (clones 13A and 18A).Figure 1C illustrated that expression of CD20 levels in MEC-2 cells and flu-resistant clonal cells was similar.Next, we determined the effect of flu concentrations on cell viability of MEC-2 cells and flu-resistant clonal cells.Figure 1D shows that the clonal cells are clearly resistant to flu-treatment.The lethal dose 50 (LD 50 ) in MEC-2 cells is 13.5 ± 2.1 µM, but >400 µM in flu-resistant clonal cells.To confirm flu-resistance, we analyzed expression of P-gp which is a drug-resistant marker and can pump drug out of cells [15].Expression of P-gp was significantly up-regulated in flu-resistant clonal cells (Figure 1E).These two lines of evidence demonstrate that our selected clonal cells are flu-resistant cells. ",
"section_name": "Establishment and characteristics of flu-resistant clones",
"section_num": null
},
{
"section_content": "Earlier studies showed the involvement of caspase activation and ceramide accumulation in flu-induced apoptosis of B-cell leukemia cell lines (WSU and JVM-2 cells) and Jurkat lymphoblastic leukemia cells [23,24].In order to investigate whether flu-resistance is associated with ceramide metabolism, we firstly determined whether flu induces MEC-2 cell apoptosis ",
"section_name": "Flu-treatment induces apoptosis in MEC-2 cells but not in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Ceramide, a product of sphingomyelin degradation, can induce cell programmed death [21] and can also be converted to other non-cytotoxic metabolites, such as glucosylceramide, which has the effect of promptly eliminating ceramide level and consequently promoting cell survival [17][18][19].In examining [ 3 H]sphingomyelin degradation, we found similar degradation of [ 3 H] sphingomyelin in flu-treated MEC-2 cells and flu-resistant clonal cells (Figure 3B) although the accumulation of [ 3 H] ceramide was not observed in flu-resistant clonal cells (Figure 3A).The results indicate that the formation of ceramide in flu-resistant clonal cells is likely converted to other metabolites.To identify possible metabolites, we analyzed the same samples and found a significant increase of [ 3 H]glucosylceramide in flu-resistant clonal cells (Figure 3C).To determine whether the accumulation of glucosylceramide is associated with GCS overexpression or activation, we further performed immunoblotting to determine expression of GCS in MEC-2 cells and flu-resistant clonal cells.Figure 3D clearly shows that GCS expression is up-regulated in flu-resistant clonal cells.Next, we treated MEC-2 cells with different concentrations of glucosylceramide for 24 hrs and then determined the effect of glucosylceramide on GCS expression and cell proliferation.The results showed that glucosylceramide enhanced expression of GCS and CD34 (Figure 3E) and promoted cell proliferation (Figure 3F). ",
"section_name": "Accumulation of glucosylceramide and overexpression of glucosylceramide synthase in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Our results indicate that the conversion of ceramide to glucosylceramide is clearly increased in flu-resistant clonal cells.To further confirm whether this conversion is associated with CLL cell flu-resistance, we use PDMP to block the conversion of ceramide to glucosylceramide.PDMP is a ceramide analog and can block the glycosylation of ceramide by inhibiting GCS [25].The cells were prelabeled with [ 3 H]palmitic acid for 24 hrs, and then incubated with different concentration of PDMP in 100 µM flu-containing medium for 24 hrs.Total cellular lipids were extracted and analyzed for the ",
"section_name": "PDMP inhibits the formation of glucosylceramide and restores chemo-sensitivity in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Cancer stem cells are a small subpopulation of cancer-initiating cells that tend to be drug resistance and have the capabilities of self-renewal, proliferation, differentiation and tumorigenicity [26,27] doubling times of ~30 ± 1.4 hours that were significantly longer than the 22 ± 2.1 hour doubling time measured for MEC-2 cells.It is clear that flu-resistant clonal cells have slow-growing and self-renewal capacity.We further analyzed expression of CD34, a marker antigen expressed on the surface of LSCs [26,27], in MEC-2 cells and fluresistant clonal cells.Figure 5B shows that expression of CD34 is significantly up-regulated in flu-resistant clonal cells (Figure 5B).To confirm that flu-resistant clonal cells are LSC-like cells, we used methylcellulose-based medium for colony formation.Figure 5C-5H showed the colonies of MEC-2 cells and flu-resistant clonal cells in the presence or absence of flu.More colonies were found in flu-resistant clonal cells compared to parental MEC-2 cells, in particular with flu treatment (Figure 5I).Based on slow-growing, self-renewal capacity, up-regulation of CD34 and colony formation, flu-resistant clonal cells compared to parental cells tend to be more LSC-like cells. ",
"section_name": "Flu-resistant clonal cells are LSC-like cells",
"section_num": null
},
{
"section_content": "As shown in Table 1, the PBMCs from four CLL patients are flu-insensitivity.We lyzed multiple CLL patient's PBMCs from chemo naïve, treated with either the combination of bendamustine and rituximab or FCR, and then the samples were processed for immunoblotting to determine the expression of GCS and CD34.We found that expression of GCS or CD34 was significantly upregulated in flu-insensitive samples compared to flusensitive samples (Figure 6).These results are similar to flu-resistant clonal cells, and indicate that flu-insensitivity in PBMCs is also associated with the alteration of ceramide metabolism and the development of LSC-like cells. ",
"section_name": "Overexpression of GCS and CD34 in fluinsensitive PBMCs",
"section_num": null
},
{
"section_content": "The resistance to flu-based therapies is one of the predominant reasons for treatment failure and is a major challenge for CLL treatment.In analyzing CLL patients resistant to flu, Moussay et al. found various genomic abnormalities (deletion or gain) in more than twenty genes that are involved in p53, DNA damage and repair, cell cycle and apoptosis signaling [28].Using piggyBac transposon-mediated mutagenesis combined with nextgeneration sequencing, one recent report also found that some new candidate genes such as deoxycytidine kinase and BMP-2-inducible protein kinase could be associated with flu-resistance in HG3 cells, a human modified CLL cell line [29].Identifying the genes that are involved in fluresistance is important because these cytogenetic mutations may be prognostic markers.However, genomic alterations are not enough because the proteins coded by these genes are involved in multiple different signaling pathways that can play opposite roles in the regulation of cellular functions.Understanding the signaling pathways is a key to develop new strategies for overcoming flu-resistance. There are few CLL cell lines available for research.Current several flu-resistant cell lines, such as malignant B-1 cell line (a mouse model of CLL) [30], K562 cells (a cell lines from chronic myelogenous leukemia patient) [31] and HG3 cells (a human modified CLL cell line) [29], are not ideal cell model for defining flu-resistant signaling pathways because these cell lines are either a modified cell line or not human CLL cell lines.Here, we used MEC-2 cells, a cell line established from the peripheral blood of a patient with B-chronic lymphocytic leukemia [22] to establish flu-resistant CLL clonal cells and used the clonal cells as a platform to study molecular mechanism of flu-resistance.Our recent study shows that MEC-2 cells respond to flu treatment similarly to the PBMCs from CLL patients [32].By comparing parental cells to flu-resistant clonal cells, we found that flu-resistant clonal cells like their parent cells express very high CD 20, a B-cell CD marker, but the flu-resistant clonal cells exist the significant alteration of ceramide metabolism that is associated with overexpression of GCS and the development of LSC-like cells that up-regulates CD34 expression.Importantly, up-regulation of GCS and CD34 expression was also found in flu-resistant PBMCs from CLL patients (Figure 6).This could be proved by the fact that the conversion of ceramide to glucosylceramide in CLL cells plays a key role in flu-resistance. Ceramide induced by numerous apoptotic stimuli (e.g.cytokines, anticancer drugs or cytotoxic agents, irradiation and environmental stresses) is recognized as a proapoptotic signaling molecule.Increasing the levels of cellular ceramide can enhance many proapoptotic molecules such as NH 2 -terminal Jun kinase, caspase-3, and reactive oxygen species [20] and suppress antiapoptotic molecules such as phosphatidylinositol 3-kinase, AKT and mTOR [33,34].Comparing to parental cells, our data clearly showed that flu-resistant clonal cells altered ceramide metabolism and up-regulated GCS expression (Figure 3 and Figure 6).Schwamb et al.. identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide in drug-resistance of primary CLL cells [35].Earlier reports indicate that glucosylceramide can stimulate DNA synthesis and cell growth (Figure 3F) [36,37].More and more evidence supports the accumulation of glucosylceramide in multidrug resistant cancer cell lines isolated from different solid tumors [38,39].Overexpression of GCS was also reported in adriamycin-resistant K562 cells, vincristineresistant HL-60 cells and clinical multidrug resistant samples of acute myeloid leukemia, acute lymphocytic leukemia and chronic myeloid leukemia [40][41][42][43].Our results and the data from many others [38][39][40][41][42][43] indicate the biochemical significance of accumulation of glucosylceramide and overexpression of GCS in drug-resistant cancer cells, and the inhibition of GCS has therapeutic potential for restoration of chemo-sensitivity and reversal of drug-resistance. Our data demonstrate that overexpression of GCS alters ceramide metabolism and promotes cancer cell survival.P-gp is the first described and most extensively studied multidrug resistant efflux protein that results in resistance to many structurally unrelated drugs [44]. Earlier studies showed that glucosylceramide is a substrate for P-gp and that both ceramide and glucosylceramide regulate P-gp expression and function [41,[45][46][47].Using a dithionite fluorescence quenching technique, Eckford et al. [45] showed that P-gp is a broad-specificity outwardlydirected flippase which enhances glycosphingolipid translocation.On the other hand, both cyclosporin A and GF120918 (p-gp inhibitors) can increase C8-ceramide mediated apoptosis [46].Using siRNA to silence GCS, knockdown of GCS expression affects P-gp expression and function [42].All these data clearly shows that either ceramide or glucosylceramide plays an important role in the regulation of P-gp expression and function [47].We found up-regulation of P-gp expression in flu-resistant clonal cells (Figure 1E), but the modulation of glucosylceramide levels by adding or depleting glucosylceramide does not significantly regulate P-gp expression in MEC-2 cells and flu-resistant clonal cells (Figures 3E and4D).Whether P-gp expression is regulated by ceramide metabolites and whether P-gp interacts with GCS need to be further studied. Cancer stem cells were first identified in myeloid leukemia with the cell surface marker combination of CD34 + and CD38- [48].These cells exhibit a slowing growth and pronounced self-renewal and differentiation capacity.Recently, accumulating evidence supports that cancer stem cells are considered as a major source of cancer recurrence and therapeutic resistance [49][50][51].Based on overexpression of CD34, slow-growth and self-renewal capacity and colony formation (Figure 5), we conclude that flu-resistant clonal cells are LSC-like cells.One recent study shows that glucosylceramide synthase is enhanced in breast cancer stem cells but not in normal mammary epithelial stem cells [52].With the accumulation of glucosylceramide and up-regulation of GCS and CD34 expression in fluresistant clonal cells, it indicates that ceramide metabolism is likely associated with the development of LSC. In conclusion, our data present signaling pathways that are involved in flu-resistance (Figure 7) and show consistent evidence that flu-resistant clonal cells are associated with ceramide metabolism (decreasing ceramide level and increasing glucosylceramide level) and that reducing GCS expression and activity can reverse flu-resistance and restore drug-sensitivity.Moreover, fluresistance is also associated with the up-regulation of CD34 expression which links to the development of LSClike cells. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "All chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Chemicals (St. Louis, MO) unless specified otherwise.Cell culture reagents were provided by HyClone.(Logan, UT).Methylcellulose-base medium (MethoCult H4434 classic) was purchased from Stem Cell Technologies (Cambridge, MA).Flu and PDMP were obtained from Cayman Chemical (Ann Arbor, MI).Glucosylceramide was supplied by Matreya, LLC (College Station, PA).Ceramide was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama).[ 3 H]palmitic acid (30-60 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St Louis, MO).Ficoll-Paque Plus was obtained from Amersham Biosciences (Piscataway, NJ, USA).The monoclonal anti-CD20 (L26), anti-GAPDH (0411), and anti-cytochrome c (7H8) antibodies, and the polyclonal anti-UGCG (glucosylceramide synthase, H-300) and anti-CD34 (H-140) antibodies were provided by Santa Cruz Biotechnology, Inc. (Dallas, TX).The monoclonal anti-P-glycoprotein (F4) antibody was supplied by Sigma-Aldrich (Saint Louis, Mo).CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT) was purchased from Promega (Madison, WI, USA).Halt protease and phosphatase single-use inhibitor cocktail, SuperSignal West Pico chemiluminescent substrate and BCA protein assay reagent were obtained from Thermo Scientific (Rockford, IL). ",
"section_name": "Materials",
"section_num": null
},
{
"section_content": "Blood was obtained from CLL patients as defined by NCI96 criteria 28 [53] following a receipt of written informed consent under an IRB protocol approved by Saint Louis University.PBMCs were isolated from whole blood immediately following donation using Ficoll density gradient centrifugation.Isolated cells were plated in 96-well assay plates at a concentration of 10-50,000 cells (depend on patient cell numbers) per well in 100 µl of RPMI 1640 media with 10% FBS with or without 10 µM flu.The cells were cultured for 72 hrs, and then cell viability was determined using Promega's CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT) according to the manufacturer's instructions [32].Absorbance at 570 nm was recorded using a BioTek Epoch Reader (Winooski, VT).The rest of PBMCs from CLL patients were harvested and lysed for immunoblotting. ",
"section_name": "PBMC isolation and treatment",
"section_num": null
},
{
"section_content": "",
"section_name": "Cell culture and establishment of flu-resistant clones",
"section_num": null
},
{
"section_content": "MEC-2 cells were treated with or without 100 µM flu for 3 hrs and flu-resistant clonal cells were maintained in the regular medium containing 100 µM flu.After treatment, cells were harvested and washed once with 1 × PBS.The cells were homogenized in a buffer containing 20 mM Hepes, 2 mM MgCl 2 , 1 mM EDTA and 1 mM DTT with protease and phosphatase inhibitor cocktails and centrifuged at 14,500 rpm to yield a pellet and supernatant.Cell lysates and cellular fractions were measured for protein concentration using the BCA protein assay reagent with BSA as a standard, and then adjusted to equal amounts of cellular protein in 1 × loading buffer.The samples were boiled for 10 min and 15 µg/lane were subjected to SDS-PAGE, and processed for immunoblotting with the appropriate antibodies [54]. In the experiments for analyzing cell viability, MEC-2 cells and flu-resistant clonal cells were plated in 96-well assay plates at a concentration of 50,000 cells per well in 100 µl of culture medium with different concentrations of flu.The cells were treated for 72 hrs, and then cell viability was determined.MEC-2 cells were incubated with different concentrations of glucosylceramide for 24 hrs, and the samples were further processed for immunoblotting analysis.For PDMP-treated experiments, MEC-2 cells and flu-resistant clonal cells (in \"maintaining\" medium containing 100 µM flu) were cultured in 96-well assay plates with different concentrations of PDMP for 72 hrs and then analyzed for cell viability.In some experiments, flu-resistant clonal cells were cultured in 6-well plates and treated with 50 µM PDMP for 24 hrs, and the samples were used for immunoblotting analysis. ",
"section_name": "Cell treatment, immunoblotting and cell viability assay",
"section_num": null
},
{
"section_content": "MEC-2 cells and flu-resistant clonal cells were cultured in 6-well plates containing 0.5 ml medium with 2 μCi/ml of [ 3 H]palmitic acid and 0.5 ml medium with or without 100 µM flu.After 24 hr treatment, the cells in the medium were collected and centrifuged at 1,500 rpm for 5 mins.Total cellular lipids in the cells were extracted by chloroform: methanol: water (5.5:5.5:5,v/v).In some experiments, the cells were prelabeled with [ 3 H]palmitic acid for 24 hrs in 100 µM flu-containing medium and then treated with different concentrations of PDMP for another 24 hrs.The individual radiolabeled lipid was resolved from the total cellular lipids by thin layer chromatography and identified by co-migration with commercial standards in different solvent systems: I) chloroform: acetic acid (90:10, v/v) for ceramide, II) chloroform: methanol: ammonium hydroxide (40:10:10, v/v) for glucosylceramide, and III) chloroform: methanol: acetic acid: and water (50:25:8:4, v/v) for sphingomyelin.The standards were visualized with iodine vapor, and the areas corresponding to ceramide, glucosylceramide or sphingomyelin were scraped into scintillation vials and quantitated by liquid scintillation spectrometry. ",
"section_name": "Cell radiolabeling and lipid metabolite analysis",
"section_num": null
},
{
"section_content": "MEC-2 cells were treated with or without 100 µM flu for 24 hrs and flu-resistant clonal cells were cultured in \"maintaining\" medium containing 100 µM flu.After treatment, cells (dead and alive) were harvested by centrifugation at 1,500 rpm for 2 mins, and the pellets were re-suspended in 0.5 ml of lysis buffer containing 5 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 0.5% Triton X-100 and placed on ice for >60 mins.The samples were then centrifuged at 14,500 rpm for 20 mins, and the supernatant containing DNA cleavage products with the same amount of cellular proteins was precipitated by isopropyl alcohol for 15 hrs.The samples were centrifuged at 14,500 rpm for 20 mins, and the pellets were re-suspended in Tris-EDTA buffer with proteinase K and RNase A for 2-3 hrs at 37° C. DNA fragments were separated on a 1.2% agarose gel, visualized with ethidium bromide, and photographed using the Bio-Rad Image System. ",
"section_name": "Measurement of DNA fragmentation",
"section_num": null
},
{
"section_content": "MEC-2 Cells and flu-resistant clonal cells were harvested, counted and adjusted to 2 × 10 4 cell/ml.To set up colony forming assay, we removed 5 ml MethoCult medium to a set of tubes and then added 0.5 ml cell suspense to each tube.The cells were mixed with MethoCult medium by vortex, dispensed into 48-well plates and cultured in the 37° C. The 25 μl regular medium or 100 µM flu-containing medium was carefully added to the designed wells in day 1 and day 8.The colonies were photographed by AMG EVOS Core Cell Imaging System.To quantify the colonies, the plates were determined using Promega's CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT). ",
"section_name": "Colony forming unit setting and analysis",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by the grants from the Elsa U. Pardee Foundation and Saint Louis University. ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The data were analyzed for significance using oneway repeated measures of ANOVA followed by Tukey's test for comparisons between the experimental groups shown in the figures. The materials and all data generated or analyzed during this study are available from the corresponding author on reasonable request. ",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "The data were analyzed for significance using oneway repeated measures of ANOVA followed by Tukey's test for comparisons between the experimental groups shown in the figures. ",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "The materials and all data generated or analyzed during this study are available from the corresponding author on reasonable request. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The authors are responsible for the reported research, and have participated in the concept and design, analysis and interpretation of data, drafting or revising of the manuscript, and have approved the manuscript as submitted. Blood was obtained from CLL patients following a receipt of written informed consent under an IRB protocol (00005304) approved by Saint Louis University. CLL: Chronic lymphocytic leukemia; FCR: fludarabine, cyclophosphamide and rituximab; flu, fludarabine; GCS: glucosylceramide synthase; LSC: leukemia stem cell; PBMCs: peripheral blood mononuclear cells; P-gp: P-glycoprotein. CH conducted the experiments; YT collected the patient blood samples; CH wrote the paper; and all authors participated in the experimental design, result discussion and interpretation, and edited and approved the final draft of the paper. The authors declare that they have no competing interests. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors are responsible for the reported research, and have participated in the concept and design, analysis and interpretation of data, drafting or revising of the manuscript, and have approved the manuscript as submitted. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "Blood was obtained from CLL patients following a receipt of written informed consent under an IRB protocol (00005304) approved by Saint Louis University. ",
"section_name": "Ethics approval",
"section_num": null
},
{
"section_content": "CLL: Chronic lymphocytic leukemia; FCR: fludarabine, cyclophosphamide and rituximab; flu, fludarabine; GCS: glucosylceramide synthase; LSC: leukemia stem cell; PBMCs: peripheral blood mononuclear cells; P-gp: P-glycoprotein. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "CH conducted the experiments; YT collected the patient blood samples; CH wrote the paper; and all authors participated in the experimental design, result discussion and interpretation, and edited and approved the final draft of the paper. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.1158/0008-5472.22395708
|
Supplementary Methods from miRNA-130a Targets <i>ATG2B</i> and <i>DICER1</i> to Inhibit Autophagy and Trigger Killing of Chronic Lymphocytic Leukemia Cells
|
<jats:p><p>PDF file - 69K</p></jats:p>
|
[
{
"section_content": "(TO-PRO-3) and 520 nm (GFP) for each sample.The acquired images were analyzed with IDEAS software (Amnis).First, a scatter plot of aspect ratio/area was used to gate for single cells.Second, a gradient RMS (root mean square for image sharpness) histogram was used to gate for cells in focus.Third, the intensity of TO-PRO-3 staining was used to exclude dead cells.On the remaining living, single cells in focus, image analysis was performed to determine the formation of autophagosomes by the extent of GFP-LC3 clustering.To do that, a spot mask was generated to automatically recognize the cellular regions containing GFP clusters.The level of fluorescence clustering was extracted from the individual cellular images and was represented by the bright detail intensity R3 feature.This feature computes the intensity of localized bright spots within the masked area in the image.The bright detail intensity was quantified at the cell population level for each sample and the data was analyzed using MATLAB (Mathworks, Inc., Massachusetts, USA).First, a lillietest for normality distribution was performed on the cellular bright detail intensities, showing that the data are non-normally distributed.Therefore, the data were analyzed using a non-parametric description derived from the Kolmogorov-Smirnov (K-S) statistics.The bright detail intensity distributions were shown as cumulative distribution functions.We defined that the autophagic flux can be described at the population level by the difference between the cumulative distribution functions between bafilomycin-treated cells and untreated cells.The difference between 2 cumulative distributions was calculated according to K-S statistics, by subtracting the values for each feature channel (histogram bin value).The resulting autophagic flux distributions were normalized to the maximum channel of the sample with the highest autophagic flux (set as 100%).Normalized autophagic flux distributions of 4 independent experiments were averaged and the data shown as mean ± SEM for each channel.In addition, a Student's t-test was performed for each channel in order to determine the statistical significance of the population frequency changes in function of the autophagic flux. ",
"section_name": "",
"section_num": ""
}
] |
[] |
10.3390/cancers16183182
|
The Economic Impact of Treatment Sequencing in Chronic Lymphocytic Leukemia in Canada Using Venetoclax plus Obinutuzumab
|
<jats:p>Background: Bruton tyrosine kinase inhibitors (BTKis) represent an advancement in chronic lymphocytic leukemia; however, these agents are administered continuously until disease progression or unacceptable toxicity, raising concerns about their affordability. Venetoclax in combination with obinutuzumab (VO) is a fixed-duration (12-month) treatment, approved in Canada in 2020. This study estimated the total cumulative cost of different treatment sequences and evaluated the economic impact of introducing treatment sequences with/without VO, from a Canadian health care system perspective. Methods: A 10-year partitioned survival model was developed, considering key clinical parameters and direct medical costs. Results were stratified by TP53 aberration. Results: Treatment sequences starting with first-line (1L) VO resulted in lower 10-year cumulative costs compared to sequences starting with BTKis administered until disease progression, across both TP53 aberration subgroups. With a maximum of three lines of treatment over a 10-year period, cumulative costs were largely determined by the first two lines of treatment. When comparing sequences with the same 1L treatment, sequences with BTKis in second-line incurred greater costs compared to fixed-duration regimens. Conclusions: Overall, the economic impact of treating all patients with VO led to 10-year cumulative savings of CAD 169,341 and CAD 293,731 per patient, without and with TP53 aberration, respectively. These savings are mainly due to reductions in treatment costs associated with fixed treatment duration.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Canada, representing almost half of all leukemia cases [1][2][3][4].Although there is no cure for CLL, the development and availability of highly effective therapies, including Bruton tyrosine kinase inhibitors (BTKis), have resulted in prolonged survival for these patients [3,5,6].A substantial proportion of health care spending on CLL is attributed to drug treatment, which poses an economic burden on patients, payers, and society [2,[7][8][9][10].Higher costs are also a consequence of therapy that results in adverse events (AEs), infections, and drug resistance [9,10].Given that CLL treatments were traditionally agnostic to molecular profiles, patients with adverse molecular features have short remissions, resulting in increased costs of first-line treatment (1L), especially with chemoimmunotherapy [11].The use of additional lines of therapy is also associated with further decline in quality of life (QoL) and greater economic burden [9].Additionally, with the evolution of molecular testing, the presence of TP53 aberration is associated with chemoimmunotherapy resistance, while patients with unmutated immunoglobulin heavy-chain variable (IGHV) have shorter remissions [12][13][14][15].Newer agents, including continuous BTKis (e.g., ibrutinib [IBRU] and acalabrutinib [ACAL]), have demonstrated improved outcomes among CLL patients with these mutations, which has resulted in greater utilization of these therapies in 1L and, hence, greater drug spending on these patients [6,14,16]. Though BTKis represent a significant advancement in the management of CLL, their high costs raise concerns about their financial impact and affordability [8,16].A part of this burden is associated with their use on a continuous basis until disease progression or unacceptable toxicity [8,17].In a previous study, a model was developed to predict the future prevalence and economic burden of CLL in the era of BTKis in Canada (from 2011 to 2025), which found that the prevalence of CLL is projected to increase almost two-fold and, accordingly, the total annual cost of CLL management is expected to be approximately 16 times greater (rising from approximately CAD 61 to CAD 958 million) by 2025 [8].The introduction of oral targeted therapies administered for a fixed duration (e.g., venetoclax [V]-based combinations) offers new options for CLL patients with the potential to achieve long-term remission with manageable side effects for a limited period and, most importantly, time off therapy, due the depth of response and marrow clearance [17,18].From a health care system perspective, a fixed treatment duration has the potential to offer better budget predictability and reduced costs [8,16]. Considering the rapidly evolving treatment landscape in CLL, ongoing evaluation of CLL treatment sequencing is vital to the optimal management of these patients while ensuring fiscal sustainability [16].Venetoclax (V) in combination with obinutuzumab (VO) is a fixed-duration treatment (12 months) approved in Canada.VO demonstrated significant improvement in progression-free survival (PFS) compared to chemoimmunotherapy (CIT), even four years following treatment cessation [19].Given its fixed duration of treatment, VO also has the potential to reduce the cost burden of treating CLL.Evaluating the cost implications of VO in various CLL treatment sequences would be beneficial to capture the potential cost savings for the Canadian healthcare system. The primary objective of this study was to estimate the total cumulative cost per patient of different treatment sequences for adults with CLL, considering 1L and subsequent lines of treatment, over a 10-year time horizon, from a Canadian health care system perspective.A secondary objective was to evaluate the economic impact of introducing treatment sequences with VO as 1L for all CLL patients, compared to sequences without VO in 1L to assess the impact of novel agent fixed-duration therapy.The aim of the study is not to recommend or evaluate clinical protocols.Healthcare professionals should consult Canadian evidence-based guidelines for clinical guidance on CLL treatment [20,21]. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "The population of interest included all CLL patients requiring frontline therapy.A subgroup analysis was conducted according to TP53 aberration to assess the economic impact within these subgroups of the CLL population. ",
"section_name": "Patient Population",
"section_num": "2.1."
},
{
"section_content": "A 10-year partitioned survival model was developed which took into consideration the clinical course of the disease and included the following health states: 1L, second-line treatment (2L), third-line treatment (3L), supportive care, and death (Figure 1) [22]. The aim of the study is not to recommend or evaluate clinical protocols.Healthcare professionals should consult Canadian evidence-based guidelines for clinical guidance on CLL treatment [20,21]. ",
"section_name": "Model Structure",
"section_num": "2.2."
},
{
"section_content": "A 10-year partitioned survival model was developed which took into consideration the clinical course of the disease and included the following health states: 1L, second-line treatment (2L), third-line treatment (3L), supportive care, and death (Figure 1) [22]. ",
"section_name": "Model Structure",
"section_num": "2.2."
},
{
"section_content": "Patients enter the model in 1L.If patients fail to respond to 1L, they proceed to 2L.After failure to a 2L, patients proceed to the 3L treatment.If disease progression continues, patients are placed in supportive care (palliative state).Patients in each of the health states could transition to death.Patients could not revert to previous health states. The probabilities of health-state transitions were estimated based on PFS and overall survival (OS) from pivotal clinical trials (Table 1) [15,19,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38].Trials were selected based on the Canada's Drug Agency (CDA), formerly known as the Canadian Agency for Drug and Technologies in Health (CADTH), funding algorithm published in May 2021 and supported with more abstracts/publications when available [39].A PubMed search was performed on June 30, 2022, including pivotal clinical trials (i.e., phase III trials) of key treatments, excluding real-world data.Many new data were also extracted from conference abstracts, which are often not indexed in bibliographical databases such as PubMed.PFS was used to estimate the transition from 1L to 2L, 2L to 3L, and 3L to supportive care.OS was used to determine the transition to death, from all health states.PFS and OS probabilities were converted into 28-day model cycle probabilities using the conversion equations published by Fleurence (2007) [40].Note that PFS and OS data for 2L and 3L were assumed to be similar, due to the paucity of clinical trial data distinguishing the line of treatment in relapsed/refractory CLL. ",
"section_name": "Simulated Clinical Pathway",
"section_num": "2.3."
},
{
"section_content": "Patients enter the model in 1L.If patients fail to respond to 1L, they proceed to 2L.After failure to a 2L, patients proceed to the 3L treatment.If disease progression continues, patients are placed in supportive care (palliative state).Patients in each of the health states could transition to death.Patients could not revert to previous health states. The probabilities of health-state transitions were estimated based on PFS and overall survival (OS) from pivotal clinical trials (Table 1) [15,19,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38].Trials were selected based on the Canada's Drug Agency (CDA), formerly known as the Canadian Agency for Drug and Technologies in Health (CADTH), funding algorithm published in May 2021 and supported with more abstracts/publications when available [39].A PubMed search was performed on June 30, 2022, including pivotal clinical trials (i.e., phase III trials) of key treatments, excluding real-world data.Many new data were also extracted from conference abstracts, which are often not indexed in bibliographical databases such as PubMed.PFS was used to estimate the transition from 1L to 2L, 2L to 3L, and 3L to supportive care.OS was used to determine the transition to death, from all health states.PFS and OS probabilities were converted into 28-day model cycle probabilities using the conversion equations published by Fleurence (2007) [40].Note that PFS and OS data for 2L and 3L were assumed to be similar, due to the paucity of clinical trial data distinguishing the line of treatment in relapsed/refractory CLL.Descriptions: 1L: first-line, 2L: second-line, 3L: third-line, ACAL: acalabrutinib, ACAL + O: acalabrutinib in combination with obinutuzumab, AEs: adverse events, BR: bendamustine in combination with rituximab, C: cycle, CAD: Canadian dollars, CLB + O: chlorambucil in combination with obinutuzumab, CLB + R: chlorambucil in combination with rituximab, FCR: fludarabine, cyclophosphamide, rituximab, FR: fludarabine in combination with rituximab, IBRU: ibrutinib, OS: overall survival, PFS: progression-free survival, V: venetoclax, VO: venetoclax in combination with obinutuzumab, VR: venetoclax in combination with rituximab.* PFS and OS probabilities extracted from clinical trials were converted to 28-day model cycle probabilities using the conversion equations published by Fleurence (2007) [40].** Note that PFS and OS data for 2L and 3L are assumed to be similar.a all costs are shown in 2022 Canadian dollars. ",
"section_name": "Simulated Clinical Pathway",
"section_num": "2.3."
},
{
"section_content": "Treatment sequences (Tables S1 andS2) were defined according to the CDA provisional funding algorithms for CLL published in May 2021 as well as the Alberta clinical guidelines and adapted by CLL clinical experts [35,39,48].Sequences are also in line with the Canadian evidence-based guidelines for the frontline treatment of CLL [20].The included treatments are approved and funded therapies in at least one jurisdiction in Canada.Compassionate use was not considered within this model since the objective was to capture the economic impact of various CLL treatment sequences from the public health care system perspective.Note that this research was conducted in 2021/2022 and further changes to the treatment landscape following the study is not captured. The identification process of treatment sequences was performed by two CLL clinical experts, over two consecutive meetings.The selection process was facilitated by an Excel worksheet (Microsoft Office 365, version 2408), for the selection of 1L, 2L, and 3L treatments.The selected treatment sequences were reviewed and approved by both CLL clinical experts.Note that the total number of treatment sequences was arbitrarily suggested to be limited to 100, of which only 88 were identified. ",
"section_name": "Treatment Sequencing",
"section_num": "2.4."
},
{
"section_content": "This economic evaluation was conducted from a Canadian publicly funded health care system perspective.Only direct medical costs relevant to a provincial health care payer were considered, including treatment acquisition costs, administration costs, follow-up and monitoring costs (e.g., laboratory and professional fee costs), cost of tumor lysis syndrome (TLS) prophylaxis, cost of AEs, and cost of supportive care (Tables 2 andS3-S12).Cost and resource use data were obtained from various Canadian sources, including published literature, public databases, and clinical experts, to the extent feasible [44,45,[49][50][51][52][53][54][55][56][57][58].All costs were inflated to 2022 Canadian dollars.Discounting was not applied considering that this study is a cost burden analysis, which is modelled similarly to budget impact analyses. ",
"section_name": "Cost Data",
"section_num": "2.5."
},
{
"section_content": "The unit cost of treatments was obtained from IQVIA Delta PA, as of December 10, 2021.Treatment regimens were obtained from Cancer Care Ontario (CCO) as well as clinical trials [49].For IV treatments, a body surface area of 1.89 m 2 and a weight of 76 kg were used to calculate total treatment costs.Drug wastage was not considered.The total treatment costs per 28-day cycle are detailed in Table S3. ",
"section_name": "Treatment Acquisition Costs",
"section_num": "2.5.1."
},
{
"section_content": "No administration costs were considered for oral therapies, including IBRU, ACAL, and V.For intravenous (IV) regimens, administration costs were estimated based on pharmacist and nurse workloads using information retrieved from CCO, calculated per 28-day treatment cycle [49].The mean time per occupation was multiplied by their respective median wage, obtained from the Job Bank of Canada (Table 2) [51].For the pharmacist, only the median annual salary was available; the hourly wage was obtained by assuming 52 weeks of work per year, with an average of 38.5 hours of work per week, as determined by Statistics Canada [52].The physician supervision cost was also considered and retrieved from the Ontario Schedule of Benefits for Physician Services (Table 2) [50].The average administration costs per 28-day cycle, per treatment, are detailed in Table S4. ",
"section_name": "Administration Costs",
"section_num": "2.5.2."
},
{
"section_content": "Routine follow-up and monitoring frequencies per treatment were derived from CCO regimens and included a physician consultation fee, laboratory tests, as well as costs of TLS prophylaxis [49]. The physician consultation fee was obtained from the Schedule of Benefits for Physician Services (Table 2) [50].The total professional fee costs per treatment per 28-day cycle are detailed in Table S5.The unit cost of each laboratory test was retrieved from the Ontario Schedule of Benefits for laboratory services (Table 2) [53].The total routine costs of laboratory test for each treatment regimen, per 28-day cycle, are detailed in Table S6.In clinical trials, TLS prophylaxis was given to patients who were administered V [6][7][8]; therefore, the cost of TLS prophylaxis was only applied to regimens including V (e.g., VO, V, and V in combination with rituximab [VR]).Costs were assumed to be different for low-risk, moderate-risk, and high-risk patients.The proportions of risk category reported in V clinical trials were used (Table S7).The unit cost of resources for TLS prophylaxis are detailed in Table S8, while the algorithms used to determine the resource use for VO, as well as V/VR, are shown in Tables S9 andS10, respectively.The algorithms were reviewed and approved by clinical experts. ",
"section_name": "Follow-Up and Monitoring Costs",
"section_num": "2.5.3."
},
{
"section_content": "The main AEs considered in the model were anemia, neutropenia, febrile neutropenia, thrombocytopenia, bleeding, infection, and atrial fibrillation (Table 2).Only grade 3 or 4 AEs were considered.The rate of AEs for each treatment regimen was obtained from their respective clinical trials.The weighted average cost of AEs per treatment regimen for 1L and 2L/3L are presented in Tables S11 andS12, respectively. ",
"section_name": "Cost of Adverse Events",
"section_num": "2.5.4."
},
{
"section_content": "The total 10-year cumulative costs per patient, of each treatment sequence, was calculated.Analyses included the stratification of treatment sequences by 1L as well as the first two lines of treatment.In addition, an analysis of the economic impact of treating all CLL patients with treatment sequences starting with VO versus treatment sequences without VO was also performed.The proportion of use of each treatment sequence, for both scenarios, was established by clinical experts in the field of CLL in Canada (Tables S1 andS2).Experts guided their assessment of use according to the funding status and availability of treatments across Canada as well as the Canadian CLL epidemiology and patient stratification (i.e., age, fitness level, IGHV mutation status).All results were stratified by TP53 aberration. ",
"section_name": "Model Outcomes",
"section_num": "2.6."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "",
"section_name": "10-Year Cumulative Costs",
"section_num": "3.1."
},
{
"section_content": "Total 10-year cumulative costs of treatment sequences by 1L treatment are summarized in Table 3. Treatment sequences starting with VO had lower 10-year cumulative costs compared to sequences starting with other novel agents (i.e., IBRU, ACAL, acalabrutinib in combination with obinutuzumab [ACAL + O]).For patients without TP53 aberration, the lowest cost sequence starting with VO was VO → VR → VR, provided they had a PFS of at least 12 months after completing the prior therapy.Among patients with TP53 aberration, the lowest cost sequence starting with VO was VO → IBRU → V. Among patients without TP53 aberration, cumulative costs per patient of sequences starting with VO ranged from CAD 327,574 to CAD 418,213, whereas sequences starting with IBRU monotherapy or ACAL monotherapy ranged from CAD 772,127 to CAD 930,700.Similarly, in patients with TP53 aberration, cumulative costs per patient of sequences starting with VO ranged from CAD 500,639 to CAD 536,507, while sequences starting with IBRU monotherapy or ACAL monotherapy ranged from CAD 770,737 to CAD 862,554. Note that patients with TP53 aberration have slightly higher costs for sequences starting with VO compared to patients without TP53, since these patients experience earlier relapses on VO.Therefore, more patients with TP53 aberration require additional lines of treatment, which mostly include continuous BTKis, which are more expensive.This explanation is also applicable for patients with TP53 receiving CLB + O. ",
"section_name": "First-Line Treatment",
"section_num": "3.1.1."
},
{
"section_content": "In our model, with a maximum of three lines of treatments over 10 years, cumulative costs for treatment sequences were largely determined by the first two lines of treatment.When comparing treatment sequences starting with the same 1L (e.g., any sequence starting with bendamustine in combination with rituximab [BR]), sequences with continuous BTKis in 2L (e.g., IBRU or ACAL monotherapy) resulted in higher costs compared to sequences with fixed-duration therapies (e.g., VR) in 2L (Figures 2 and3). Additionally, among sequences starting with VO, retreatment with a V-based regimen in 2L often resulted in relatively low cumulative costs (from CAD 339,370 to CAD 396,054 for patients without TP53 aberration and CAD 511,641 for patients with TP53 aberration). ",
"section_name": "First Two Lines of Treatment",
"section_num": "3.1.2."
},
{
"section_content": "In a scenario assuming that all patients are treated with sequences starting with VO (i.e., VO in 1L for all patients in the model), 10-year cumulative costs were estimated at CAD 359,001 and CAD 518,726 for patients without and with TP53 aberration, respectively.In a scenario assuming that all patients are not treated with sequences with VO (i.e., without VO for all patients), 10-year cumulative costs were estimated at CAD 528,343 and CAD 812,457 for patients without and with TP53 aberration, respectively. For patients without TP53 aberration, the economic impact of treating all patients with sequences of treatment starting with VO led to 10-year cumulative savings of CAD 169,341 per patient for the Canadian healthcare system.10-year cumulative savings were higher for patients with TP53 aberration at CAD 293,731 per patient. ",
"section_name": "VO vs. Non-VO Treatment Sequences",
"section_num": "3.1.3."
},
{
"section_content": "The results of this study provide 10-year cumulative cost estimates of various CLL treatment sequences for patients with and without TP53 aberration, in Canada.Overall, sequences starting with VO had lower costs compared to those starting with other novel agents.It was also found that, when comparing treatment sequences starting with the same 1L, sequences with continuous BTKis in 2L incurred greater costs compared to sequences with fixed-duration treatment regimens in 2L.This is an important finding as CLL treatment costs are mostly driven by the first two lines of therapy within the model, considering the inclusion of a maximum of three lines of treatment over a 10-year period.For CLL patients without TP53 aberration and those with TP53 aberration, initiating treatment with VO as 1L was estimated to reduce drug costs by CAD 169,341 and CAD 293,731 per patient, respectively, compared to treatment sequences without VO over 10 years.Given the expected increase in spending on CLL in Canada [8], this study indicates a possible strategy to mitigate these rising costs in a publicly funded health care system.It is also important to note that the projected savings calculated in this study were for the Canadian healthcare system in Canadian dollars; potential savings would be even greater when replicated in a more expensive healthcare system, such as the United States (US). Prior studies have established the health economic value of VO.In terms of budget impact analysis (BIA), a US study found that the implementation of VO as 1L, including ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The results of this study provide 10-year cumulative cost estimates of various CLL treatment sequences for patients with and without TP53 aberration, in Canada.Overall, sequences starting with VO had lower costs compared to those starting with other novel agents.It was also found that, when comparing treatment sequences starting with the same 1L, sequences with continuous BTKis in 2L incurred greater costs compared to sequences with fixed-duration treatment regimens in 2L.This is an important finding as CLL treatment costs are mostly driven by the first two lines of therapy within the model, considering the inclusion of a maximum of three lines of treatment over a 10-year period.For CLL patients without TP53 aberration and those with TP53 aberration, initiating treatment with VO as 1L was estimated to reduce drug costs by CAD 169,341 and CAD 293,731 per patient, respectively, compared to treatment sequences without VO over 10 years.Given the expected increase in spending on CLL in Canada [8], this study indicates a possible strategy to mitigate these rising costs in a publicly funded health care system.It is also important to note that the projected savings calculated in this study were for the Canadian healthcare system in Canadian dollars; potential savings would be even greater when replicated in a more expensive healthcare system, such as the United States (US). Prior studies have established the health economic value of VO.In terms of budget impact analysis (BIA), a US study found that the implementation of VO as 1L, including costs associated with AEs, routine care, and monitoring, resulted in cost savings of USD 1.6 million per 1 million members under the US health plan over a 3-year time horizon [59].Another BIA conducted in France demonstrated that although there is an increase in costs with treatment sequences including VO during the first year, it is followed by cost savings in subsequent years compared to other CLL treatment sequences, resulting in total budget savings of EUR 860 million over 10 years [60].A US study on CLL treatment sequencing also demonstrated budget savings with VO, with estimated cost reductions of approximately USD 13 million and USD 56 million over 5 and 10 years, respectively, in a hypothetical one-million-member health plan [61].In terms of cost-effectiveness, a US study by Alrawashdh et al. estimated the 10-year cost-effectiveness of 1L treatments for CLL and determined that CITs yielded less health benefits (i.e., life-years (LYs) and quality-adjusted LYs [QALYs]) at higher costs compared to VO; other targeted therapies were also found to be more costly but resulted in greater health benefits than VO [62].In another cost-effectiveness study conducted from the Canadian public funding perspective, Chatterjee et al. found that VO is the most cost-effective option in CLL treatment under most willingness-to-pay thresholds.They concluded that VO was a dominant treatment option for unfit 1L CLL patients, meaning that VO accrues higher QALYs and lower total costs, relative to the majority of comparators.Although ACAL accrued higher QALYs than VO, it is also more expensive than VO, and was not found to be cost-effective [63]. Note that this study is unique as it is the first to assess the costs of all possible sequences of treatment in CLL, from a Canadian healthcare system perspective.Although the costeffectiveness study by Chatterjee et al. considered subsequent treatments in their cost data, the cost for each treatment sequence as well as sub-analyses by 1L and 2L treatment were not analyzed.Our study presents data on a granular level, demonstrating the impact of different subsequent treatments on total cumulative costs.Furthermore, this study also assesses the potential savings from treating all Canadian CLL patients with VO in 1L, which has not been previously reported. Bruton tyrosine kinase inhibitors have become a treatment of choice for most CLL patients, providing salvage therapy and an alternative treatment option to chemoimmunotherapy.However, now that additional treatment options are available to treat CLL, it is important to assess the impact of treatments on the patients as well as the payers.Consequently, a strength of this study was that it estimated the long-term (i.e., 10-year) budget impact of a number of possible CLL treatment sequences, as opposed to just individual regimens.It also considered a relevant CLL subgroup (i.e., TP53 aberration).This is important as higher-risk patients are expected to incur more costs, which can affect the overall budget impact.Note that at as the time of this analysis, IGHV mutation status was not considered since IGHV testing was not uniform across all Canadian provinces and does not play a major role in the efficacy of novel targeted therapies.The model inputs were retrieved from the most suitable trial data and Canadian resources to ensure that the results would be representative and applicable to the Canadian public health care system.Clinical expert input was also acquired to confirm the validity and accuracy of the model and its input parameters.Additionally, the model included numerous cost items (i.e., treatment acquisition, administrative, monitoring, TLS prophylaxis, AE-related, and support care) to ensure that all costs relevant to manage patients with CLL were considered. This study also had some limitations.Allogeneic stem cell transplantation (allo-SCT) is another treatment option for certain patients with CLL [64,65], but it was not included as a possible treatment option in this analysis (only drug therapies were considered).According to key opinion leaders in Canada, allo-SCT is part of the standard of care; however, with access to novel treatments, the number of patients eligible for this treatment is diminishing.Furthermore, although the TP53 aberration subgroup was captured within this study, clinical data were not stratified by TP53 mutation type.It has been reported that patients may have single or multiple TP53 abnormalities, with increased risk for shorter time to therapy as well as increased risk of death with increased number of abnormalities [66,67].Single clinical data were retrieved and no distinction was made with regards to different TP53 aberration patient profiles, which might not provide accurate costing results for these patients.Another limitation is the exclusion of costs associated with COVID-19 infections.Although not reported in the pivotal clinical trials included in this study, COVID-19 infections represent a significant burden on healthcare costs and clinical admissions related to CLL.Additionally, this model considered that patients treated with oral targeted therapies are seen every three months.However, in clinical practice this frequency varies based on duration of time on therapies and toxicities and will be the subject of future studies as systems adapt to these regimens.Another limitation is that patients could only receive up to three lines of treatment followed by supportive care in this model; however, this may not be reflective of a real-world clinical setting, as patients may also receive further lines of treatment.In addition, the model also disregarded patients who transition to the next line of treatment due to intolerance; only PFS data were used to assess the transition to the following line of treatment, which may underestimate the proportion of patients requiring subsequent lines of treatment.With regards to PFS and OS data, a direct treatment comparison was not available, while an indirect treatment comparison of all treatment comparators would not be feasible and not be justified considering the extent of treatments and lines of treatments included in this analysis.Therefore, a cross-comparison of trials without adjustment was performed, which has its limitations pertaining to the differences within the trials and the populations studied.It was also assumed that drug prices and dosing remained constant over time.Drug prices can change (e.g., generic drug entry) and dose modification may occur throughout a patient's follow-up, which can, ultimately, impact their treatment costs.Lastly, since data on the proportion of use of treatment sequences are not publicly available, clinical experts provided a hypothetical proportion of use of each treatment sequence, based on funding status across Canada and CLL patient subpopulations.These numbers are hypothetical and may not reflect accurately the entirety of the CLL treatment paradigm in Canada.Health Canada approved the combination of fixed-duration ibrutinib in combination with V in March 2023 based on the GLOW and CAPTIVATE studies [68][69][70].Although CDA provided a positive recommendation for the reimbursement of this treatment combination in November 2023, it has not yet received public funding in any Canadian jurisdiction [71].This study does not compare fixed-duration VO to a fixed-duration oral therapy, and it remains to be determined if ibrutinib in combination with V will be cost-effective.Some of these limitations are factors to consider in future research in this area. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In conclusion, treatment sequences starting with time-limited therapy VO in 1L resulted in lower costs compared to sequences without VO.These savings were mainly due to reductions in the treatment costs associated with a fixed treatment duration. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers16183182/s1,Table S1.Treatment Sequence Utilization for Patients without TP53 Aberration; Table S2.Treatment Sequence Utilization for Patients with TP53 Aberration; Table S3.Treatment Acquisition Costs; Table S4.Administration Costs per Treatment; Table S5.Professional Fee Cost per Treatment; Table S6.Laboratory Monitoring Frequencies and Cost per Treatment; Table S7.TLS Risk Distribution; Table S8.Resources used for TLS Prophylaxis Unit Cost; Table S9.TLS Prophylaxis Algorithm Applied to VO Regimen; Table S10.TLS Prophylaxis Algorithm Applied to V/VR Regimens; Table S11.Severe AE Frequencies per Treatment-1L; Table S12.Severe AE Frequencies per Treatment-2L/3L Author Contributions: K.G. and K.M. were involved in the study conception and design, data collection, modeling, analysis and interpretation of the data, and drafting the manuscript.J.L., N.P.R., S.-J.B., D.T., B.S.M. and S.B. were involved in the study conception and design, participated in data collection, revision of the manuscript, and study supervision.C.H. and V.B. were involved in the study conception and design, data interpretation and revision of the manuscript.All authors have read and agreed to the published version of the manuscript. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Christopher Vannabouathong and Ariane Gosselin for revising the manuscript.Christopher Vannabouathong received funding from PeriPharm Inc. Ariane Gosselin is an employee of AbbVie Corporation. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research received external funding from AbbVie Corporation.No honoraria or payments were made for authorship.No grant number applicable. Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "Funding: This research received external funding from AbbVie Corporation.No honoraria or payments were made for authorship.No grant number applicable. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Informed Consent Statement: Not applicable. ",
"section_name": "Institutional Review Board Statement: Not applicable.",
"section_num": null
},
{
"section_content": "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
}
] |
10.1186/1476-4598-10-138
|
Targeting notch pathway enhances rapamycin antitumor activity in pancreas cancers through PTEN phosphorylation
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Pancreas cancer is one of most aggressive human cancers with the survival rate for patients with metastatic pancreas cancer at 5-6 months. The poor survival demonstrates a clear need for better target identification, drug development and new therapeutic strategies. Recent discoveries have shown that the role for Notch pathway is important in both development and cancer. Its contribution to oncogenesis also involves crosstalks with other growth factor pathways, such as Akt and its modulator, PTEN. The mounting evidence supporting a role for Notch in cancer promotion and survival suggests that targeting this pathway alone or in combination with other therapeutics represents a promising therapeutic strategy.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Using a pancreas cancer tissue microarray, we noted that Jagged1, Notch3 and Notch4 are overexpressed in pancreas tumors (26%, 84% and 31% respectively), whereas Notch1 is expressed in blood vessels. While there was no correlation between Notch receptor expression and survival, stage or tumor grade, Notch3 was associated with Jagged1 and EGFR expression, suggesting a unique relationship between Notch3 and Jagged1. Inhibition of the Notch pathway genetically and with gamma-secretase inhibitor (GSI) resulted in tumor suppression and enhanced cell death. The observed anti-tumor activity appeared to be through Akt and modulation of PTEN phosphorylation. We discovered that transcriptional regulation of RhoA by Notch is important for PTEN phosphorylation. Finally, the mTOR inhibitor Rapamycin enhanced the effect of GSI on RhoA expression, resulting in down regulation of phospho-Akt and increased <jats:italic>in vitro</jats:italic> tumor cytotoxity.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Notch pathway plays an important role in maintaining pancreas tumor phenotype. Targeting this pathway represents a reasonable strategy for the treatment of pancreas cancers. Notch modulates the Akt pathway through regulation of PTEN phosphorylation, an observation that has not been made previously. Furthermore, we discovered that this regulation is dependent on RhoA/Rock1 activation. Enhanced phospho-Akt suppression when GSI is combined with rapamycin suggests that targeting both pathways will lead to a greater efficacy in the treatment of patients with pancreas cancer.</jats:p> </jats:sec>
|
[
{
"section_content": "The Notch pathway is an evolutionarily conserved pathway important for cell fate determination in development as well as in cancer.In development, Notch is involved in tissue patterning and morphogenesis through cell differentiation, proliferation and apoptosis.The Notch family in mammals consists of four receptors (Notch1-4) and five ligands (Jagged1, 2 and Delta-like 1, -3, -4).In the canonical pathway, Notch receptors are activated by membrane-bound ligands, resulting in several intramembrane proteolytic cleavages that untether the cytoplasmic domain (NICD) from the cytoplasmic membrane.The NICD translocates to the nucleus and activates the transcription of target genes, such as those belonging to the Hairy/enhancer of split and Hairy/enhancer of splitrelated with YRPW motif families [1].In cancer, Notch crosstalks with numerous oncogenic pathways, such as Akt, TGF-β and src signaling [2][3][4].In certain context, the interaction between Notch and other oncogenic pathway is independent of the canonical HEY and HES activation [5]. While accounting for only 4% of estimated new cases of cancer in both men and women, pancreas cancer is the fourth leading cause of cancer-related death in the United States [6].The median survival for patients with advanced pancreas cancer remains at 5-6 months, a rate that has not changed significantly over the last decade [7].Thus, identification of new targets is needed to improve clinical outcome.Current literature suggests that Notch pathway plays an instrumental role in pancreas cancer.In the developing pancreas, Notch regulates the ratio between the exocrine and endocrine cell mass, supporting its role in controlling cell-fate determination [8].RT-PCR showed that Notch pathway components were overexpressed in a small set of pancreas tumors.Furthermore, activated Notch cooperates with TGF-β in the expansion of undifferentiated precursor cells and in the promotion of PanIN progression to anaplastic pancreas cancer [9,10]. In this study, we examined the prevalence of Notch receptors and ligands in a large number of patients with pancreas cancers.Using immunohistochemistry (IHC) on a tissue array, we discovered that Notch3 was most often overexpressed in pancreas cancer, followed by Notch4.Conversely, Notch1 was expressed in the vasculature within the tumor mass but not in malignant cells.Furthermore, inhibiting Notch activation reduced tumor phenotypes and Akt phosphorylation in pancreas cancer.While previous studies have shown that Notch-dependent activation of Akt is a result of transcriptional downregulation of PTEN, we noted that in our system, Notch regulated PTEN phosphorylation but not PTEN expression.Our results show that this regulation is dependent on RhoA and Rock1, an observation that has not been previously described.Finally, rapamycin, an inhibitor of the mTOR pathway, greatly enhanced Notch-dependent inhibition of Akt and tumor cytoxicity in vitro.This effect appears to be dependent of RhoA.Taken together, our observations further support a role for Notch in pancreas cancer and suggest a new strategy in targeting pancreas cancer. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "The prevalence in expression of a potential oncogene helps determine the significance of its role in cancer.To better understand the role of Notch pathway in pancreas cancer, we developed a pancreas tissue microarray with associated clinical data from 86 patients (Table 1).We also examined the expression of Notch1-4 and their ligands, Jagged1 and DLL4.Notch3 was most prevalent with greater expression in 84% of resected cancers, followed by Notch4 at 31% (Table 2 Figure 1).Interestingly, none of the tumor cells expressed Notch1, and only one of the 86 tumors surveyed expressed Notch2.Notch1 and DLL4 were expressed predominantly in endothelial cells, suggesting that, while not significantly expressed in tumor cells, they are important in tumor angiogenesis.We also tested the dataset for correlation between different Notch family members and clinical characteristics, such as overall survival, stage and tumor grade.No association between Notch receptors and clinical characteristics was observed.However, we noted that Notch3 expression correlated with Jagged1, but not for Delta-like 4, suggesting that Jagged1 is the ligand for Notch3 (Figure 1C, Table 3) [11]. Of note, eighty-five percent of the tumors surveyed with IHC exhibited high expression of EGFR (Figure 1B).Notch3 also correlates with EGFR expression (Figure 1D, Table 4), consistent with our previous finding in lung cancer that Notch3 and EGFR pathways cooperate in maintaining the oncogenic phenotype [12].Notch receptors are activated by proteolytic cleavages after ligand binding, resulting in the release of the cytoplasmic domain (NICD). We were able to demonstrate that several human pancreas cancer cell lines expressed the activated forms or NICD of Notch receptors (Figure 2A).In addition, pancreas cancer cell lines developed from overexpressing K-rasG12D and TGF-β knockout mice showed Notch1 ICD and Notch3 ICD expression (Figure 2B), further supporting the role of Notch pathway in pancreas cancers [13].Similar to our previous observation, Jagged1 is also highly expressed in nearly all of cell lines tested [14].We found no difference in Notch expression between cell lines with K-ras mutation alone (K162, K512, K518) and those with both K-rasG12D and TGF-β knockout (K375, K389, K399).When K162 and K399 were treated with MRK003, γsecretase inhibitor, dose-dependent down regulation of activated Notch3 was observed (Figure 2C).Interestingly, while we observed suppression of the activated form of Notch, we observed a rise in HES1 and HEY1 transcripts, suggesting that Notch modulates cancer phenotype in pancreas through non-canonical pathways (Figure 2D,E). ",
"section_name": "Notch Receptors and Ligands Are Expressed in Resected Pancreas Cancer",
"section_num": null
},
{
"section_content": "To determine whether inhibiting Notch activation reduces tumor phenotype, we utilized both dominant-negative Notch3 receptor and a γ-secretase inhibitor (GSI).When BxPc3 was transfected with dominant-negative Notch3 or treated with 25 μM of MRK003, colonies were significantly reduced in number, as compared to vector controls (VC) or DMSO control (C) (Figure 3A).A significant body of literature has supported a role for Notch signaling in apoptosis.Similar to our previous observation in lung cancer, inhibiting Notch in serum-free condition resulted in enhanced cancer cell death measured with PI staining (Figure 3B).The Bcl-2 family plays an important role in apoptosis through the activation of the mitochrondria-dependent caspase pathway.Using Notch3 siRNA, we showed that Notch regulates Bcl-xL expression and Bcl-2 (Figure 3C).When MRK003 was used, a similar effect on Bcl-xL could be found, accompanied by an increase in cleaved PARP, a marker of caspases activation (Figure 3D).To determine whether γ-secretase inhibitors possess activity in vivo, we inoculated xenografts with K162 and K399 cell lines developed from a mouse model of pancreas cancer.The γ-secretase inhibitors DAPT and MRK003 suppressed tumor growth by 25% to 50%, suggesting that the Notch pathway plays a role in the survival of cancer cells in both in vitro and in vivo models (Figure 3E). ",
"section_name": "Inhibiting Notch Activation Reduces Malignant Phenotype and Induces Apoptosis",
"section_num": null
},
{
"section_content": "The Notch pathway is known to crosstalk with other oncogenic pathways such as the EGFR and the Akt pathway [12,15].Interestingly, unlike observations in lung cancer, inhibition of the Notch pathway in pancreas cancer had no appreciable effect on ERK activation (Figure 4A).On the other hand, Akt phosphorylation was inhibited by MRK003 in pancreas cancer cell line K399.PTEN (phosphatase and tensin homolog) is a well-known negative regulator of Akt.In hypoxia, Notch1 has been shown to suppress PTEN transcription, leading to Akt activation [15].However, while Notch is known to regulate Akt through the transcriptional regulation of PTEN, we did not detect a difference in total PTEN levels.Rather the phosphorylation of PTEN at Ser380 was altered, when GSI was used (Figure 4A).While not much is known about the phosphorylation of PTEN, recent evidence suggests that it regulates protein stability [16].While some findings indicate that phosphorylation of PTEN improves stability but reduces PTEN function, others have shown that the loss of phospho-PTEN in migrating cells leads to the activation of Akt [17].Cdc42, a member of the Rho GTPase family, is important in Akt-mediated cell survival and motility, and its activation is inhibited by PTEN [18,19].We noted a decrease in Cdc42 when treated with GSI, suggesting that Notch regulates Akt-dependent cell survival through PTEN and Cdc42.How PTEN is regulated through phosphorylation is intensely investigated.In a recent model of chemotaxis proposed by Li et al., Rock1, a member of the Rho-associated, coiled-coil containing protein kinases, is activated by Rho-GEF (guanine nucleotide-exchange factor) and RhoA, another Rho GTPase family member.Activated Rock1 then binds and phosphorylates PTEN [17,20].Rho proteins and Rock proteins are important regulators of cell migration, proliferation and apoptosis [21].To examine the role of the Rho GTPase pathway in Notch-induced PTEN phosphorylation in pancreas cancer, we examined the effect of GSI on Rock1 and RhoA.Interestingly, we noted an increase in the expression of RhoA with increasing dose of GSI, whereas the expression of Rock1 remained essentially unchanged (Figure 4B).The effect of Notch signaling on RhoA appears to be transcriptionally mediated (4C).To determine whether Notch modulation of PTEN phosphorylation is dependent on RhoA/Rock1, we examined the effect of GSI in the presence of Rock1 inhibitor Y27632 (4D).Whether the observations in the chemotaxis model can be translated into a cancer model requires further validation.The loss of PTEN phosphorylation by GSI in the presence of Y27632 suggests, however, that the Notch effect on PTEN depends on the RhoA/Rock1 pathway. ",
"section_name": "GSI Inhibits Akt Activation and PTEN Phosphorylation",
"section_num": null
},
{
"section_content": "The observed redundancy in oncogenic pathways may require that multiple pathways are inhibited in order to enhance tumor cytotoxicity.The PI3K/Akt/mTOR pathway is activated in the majority of pancreas cancers.Because of the crosstalk between Notch and Akt, we examined whether the combination of the mTOR inhibitor Rapamycin and MRK003 will result in improved tumor cytotoxicity.While some studies suggest that Rapamycin induces Akt activation, we noted that in K399 rapamycin inhibits Akt phosphorylation, and that this inhibition was enhanced, when Rapamycin was combined with MRK003 [22] (Figure 5A).Again, we observed a change in phospho-PTEN, but not total PTEN, when Notch pathway is inhibited.Furthermore, the level of phospho-PTEN was increased when MRK003 was combined with rapamycin.Foxo3a is a member of the forkhead family which acts as tumor suppressor by promoting cell cycle arrest and apoptosis.It is inactivated by Akt.The combination of Rapamycin and MRK003 led to a slight increase in the tumor suppressor Foxo3a and pro-apoptotic Bim, a member of the BH-3 only Bcl-2 family.Moreover, we noted an increased expression of RhoA, when cancer cells were treated with MRK003, and the change was enhanced when Rapamycin was added (Figure 5B).No change in Rock1 level was detected.Taken together, these observations support the hypothesis that Notch and mTOR cooperate in regulating Akt through PTEN phosphorylation and RhoA. ",
"section_name": "Rapamycin Enhances GSI Antitumor Activity Through the Regulation of Akt",
"section_num": null
},
{
"section_content": "While results from preclinical studies using mTOR inhibitors in pancreas cancers have been promising, their low efficacy in early clinical studies indicate that these agents possess minimal clinical activity when administered as single agents [23].Redundancy in the biological system and results from clinical trials suggest that targeting multiple targets will result in augmented tumor suppression.Because we observed Akt suppression when GSI was added to Rapamycin, we tested whether inhibiting the Notch pathway will enhance tumor suppression with mTOR inhibitor in vitro.In both human and murine pancreas cell lines, K399 and Panc-1, respectively, the combination of MRK003 and rapamycin inhibited proliferation to a greater degree than Rapamycin or MRK003 alone (Figure 5C,D).These findings suggest that Notch can enhance Rapamycin in inhibiting pancreas cancer growth through the modulation of Akt. ",
"section_name": "Notch Inhibition Enhanced Rapamycin-dependent Growth Suppression in pancreas Cancer Cells",
"section_num": null
},
{
"section_content": "Overexpression of Notch receptors and ligands in pancreas cancer supports the hypothesis that this developmental pathway plays an important role in this type of cancer.However, the lack of correlation between Notch pathway compounds, clinical characteristics and outcome does not support their use as biomarkers.We observed that Notch3 is expressed in cancer cells, whereas Notch1 is mainly expressed in blood vessels.Differences in expression pattern among the various Notch pathway components suggest a non-redundancy in functions.We hypothesize that in cancer Notch3 is important for tumor survival, whereas Notch1 mediates the response to hypoxia through the regulation of angiogenesis.This hypothesis is supported by previous observations from other investigators [12,15,24].Furthermore, our observations suggest that a less specific Notch inhibitor will be more effective for targeting cancer cells and the tumor microenvironment, albeit with higher toxicity profile.However, only further clinical testing can ascertain this supposition. While none of the Notch receptors have been shown to be useful as biomarkers, our in vitro and in vivo data provide evidence that the Notch pathway is oncogenic.Targeting this pathway genetically or with small molecules such as γ-secretase inhibitors may reduce tumor phenotype and represent a viable option for the treatment of patients with pancreas cancer.Because of the redundancy in oncogenic signals, targeting multiple Notch pathways will likely improve clinical outcomes.Similar to Notch, the PI3K/AKT/mTOR signaling pathway mediates key cellular processes, including cell growth, proliferation, and survival [25].Furthermore, Akt is found to be activated in 59% of tumors.Our findings demonstrate that Notch modulates Akt, supporting a crosstalk between the pathways.While the mechanisms for this crosstalk needs further elucidation, our data suggest that one mechanism involves the modulation of PTEN phosphorylation. PTEN is a tumor suppressor and functions as a phosphatidylinositol phosphate (PIP) phosphatase.Dephosphorylation of PI (3,4,5)P3 by PTEN prevents the phosphorylation and activation of Akt kinase [26].Earlier studies suggest that, while phosphorylation of PTEN at the C2 domain enhances PTEN stabilization, it also promotes a closed conformation, inhibiting PTEN activity [16,27].Conversely, in inflammatory cells, Rock1 was found to bind to PTEN and is essential for PTEN phosphorylation and activation [20].Bone marrow cells from mice lacking functional Rock1 showed loss of PTEN activity and increased Akt activation [17].Thus, similar to many complex biological systems, the phenotypic outcome of PTEN and RhoA/Rock pathways activation is highly context dependent. In our system, we observed no difference in Rock1 expression with GSI, but RhoA expression was enhanced.RhoA is a member of the Rho family of small GTPases.It is required for Rock1 activation [20].The Notch-dependent increase in PTEN phosphorylation is inhibited by Rock1 inhibitor, suggesting that Notch regulates PTEN through the RhoA/Rock1 pathway.Our study is the first to show that Notch regulates the phosphorylation of PTEN through the RhoA pathway in pancreas cancer. We have demonstrated that the Notch pathway plays an important role in pancreas cancer.Furthermore, our findings suggest thst a cooperative relationship between the Notch pathway and the Akt/mTOR pathway may exist and this interaction is mediated by the Rho GTPase pathway.Similar to Notch, other studies have indicated a contradictory role of Rho proteins in cancer, suggesting that its role is highly context dependent.However, from the treatment perspective, Notch can be considered a target for intervention, since the inhibition of this pathway mitigates the malignant phenotype.Moreover, due to the crosstalk with the mTOR pathway, combination treatment may improve therapeutic outcome. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Human pancreas cancer cell lines Panc-1, HRAF-II and BxPC3 were obtained from American Type Culture Collection (ATCC).Murine pancreas cancer cell lines K399, K389, K375, K162, K152, and K518 were developed ex vivo from tumors of mice overexpressing K-rasG12D and TGF-β knockout, and were obtained from Dr. H. Moses [13].The formulation and the in vivo dosing schedule of γ-secretase inhibitor MRK003 were provided by Merck Co., Inc, and were described previously [28].The mTOR inhibitor rapamycin and the Rock1 inhibitor Y27632 were obtained from Sigma-Aldrich and CalBiochem, respectively.The γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), was also obtained from Sigma-Aldrich.The dominant-negative Notch3 (DN) and VC (control) constructs were transfected into BxPC3 and selected with G418, as previously described [12].Notch3 siRNA3 sequences were also described previously [14]. ",
"section_name": "Cell lines, Constructs and Inhibitors",
"section_num": null
},
{
"section_content": "De-identified tumor and adjacent normal tissues were obtained under an IRB-approved protocol at Vanderbilt University Medical Center.Before constructing a TMA block, serial 5-μm sections were cut from each donor block.One of these sections was stained with H&E for marking morphologically representative areas of the tumor.Using a Beecher Instruments Tissue Arrayer (Silver Springs, MD), tissue cylinders with a diameter of 0.6 mm were punched from the four targeted areas in each donor block and deposited into a 9 × 14 (~126 cores) TMA block, which contained 76 cores of adenoma tissue and 50 cores of adjacent, non-malignant tissue as controls.The TMA blocks were warmed to 36°C for 30 minutes, and multiple serial 5 μm sections were cut and placed on charged slides. ",
"section_name": "TMA Construction, TMA Slide Preparation",
"section_num": null
},
{
"section_content": "The Notch3 antibody 1E4 (a gift from Dr. Anne Joutel) was used for immunohistochemistry, and the method was described previously [29].Jagged1 (C-20) and Notch4 (H-225) were purchased from Santa Cruz, whereas Notch1 (3608), DLL4 (HPA023392) and Notch2 (C651.6D6HN)antibodies were obtained from Cell Signaling Technology, Sigma-Aldrich, and the Developmental Studies Hybridoma Bank (DSHB), respectively.Human EGFR antibody (31G7) was purchased from Zymed.The IHC staining was scored on a composite scale of 0 to 3 by two independent observers, including one pathologist.In case of disagreement, the decision was deferred to the pathologist.The tumors that scored 2 or better were considered positive.For immunoblotting, Notch1 (3447), Notch3 (2889), phospho-Akt, total Akt, PTEN, pPTEN, RhoA, Rock1, cdc42, Bcl-xL, Bcl-2 and PARP were obtained from Cell Signaling Technology.For specific use in murine cell lines, Jagged1 (H114), Notch1 (C-20), and Notch3 (M-134) were obtained from Santa Cruz, and Notch2 (C651.6D6HN)and Notch4 were purchased from DSHB and Orbigen, respectively. ",
"section_name": "Antibodies",
"section_num": null
},
{
"section_content": "Total RNA was isolated from K399 cells using Sure Prep RNA Purification Kit (Fisher Scientific).cDNA synthesis was carried out using iScript cDNA Synthesis Kit, according to manufacturer's recommendation (Bio Rad).Primers for murine GAPDH were AATGGGGTGAGGCCGGTG (sense) and CAGAAGGGGCGGAGATGATG (antisense).Murine RhoA primers were CCATGTACCCAAAAGC GCC (sense) and CAAATGTGCCCATCGTCCTG (antisense).Experiments were performed at annealing temperature of 55°C for 39 cycles. ",
"section_name": "Real-time RT-PCR",
"section_num": null
},
{
"section_content": "Cells were plated into 96-well microtitre plates at 10% FCS and at 50% confluency in 200 μl DMEM.After 48 hours of treatment with inhibitors, 50 μl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solution (2 mg/ml) was added to each well, and the plates were incubated for 4 hours.MTT formazan crystals were then resolubilized by adding 150 μl 100% dimethylsulfoxide (DMSO) to each well.Plates were agitated on a plate shaker for 5 min, and the absorbance at 540 nm was determined using a scanning multi-well spectrophotometer (BioRad).For soft agar assays, transfected cells were plated at a density of 5000 cells/plate using 35 mm Petri dishes and suspended in 0.4% agar containing 10% FCS RPMI and 50 μg/ml of G418 selective antibiotic over 0.8% base agar.The plates were incubated at 37°C and 5% CO2 in a humidified chamber for 14 days.Cell death was determined as follows: Cells were stably transfected with Notch3 DN or treated with MRK003 for 24 hours and were maintained in 10% FCS-RPMI or serum-free medium.Then, they were stained with propidium iodide (Calbiochem, La Jolla, CA).The percentage of dead cells was determined with a Beckman Coulter FACS Calibur Flow Cytometer. ",
"section_name": "Proliferation Assays, Soft Agar and Cell Death Analysis",
"section_num": null
},
{
"section_content": "Animal experiments were performed according to the protocol approved by Vanderbilt University IACUC.Athymic 4-to 6-week-old female nude mice (nu+/nu+) were used for the tumor xenograft models.Panc1 or K399 (1 × 106 cells in the volume of 200 ml of PBS) was inoculated subcutaneously (s.c.) into the right posterior legs of nude mice.Treatment was initiated when tumors were palpable.MRK-003 (150 mg/kg) was administered orally for three consecutive days per week for 2 weeks.MRK-003 was diluted in 0.5% methylcellulose.The tumors were measured every 2 days with a caliper.Tumor Volume (TV) was calculated with the formula: TV = (Length) × (Width)2/2.Percentage tumor volume (% TV) on day X was calculated as: %TV = (tumor volume on day ×/tumor volume on day 1) × 100. ",
"section_name": "In Vivo Tumorigenicity",
"section_num": null
},
{
"section_content": "The size of implanted tumors at precise time points after treatment was compared with that of control groups.Unless specifically stated, statistical inference in all comparative experiments both in vivo and in vitro was obtained using unpaired, two-sided Student's t-tests.For TMA, protein expression was correlated using Pearson's correlation coefficients.For all determinations, differences were considered significant at P < 0.05. ",
"section_name": "Statistical Analyses",
"section_num": null
}
] |
[
{
"section_content": "We like to thank Sandy Olson from Vanderbilt University Medical Center and the University of Virginia Biorepository and Tissue Research Facility for their assistance in performing IHC. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Authors' contributions AG and KW are pathologists.KW was responsible for production of pancreas cancer TMA, collection of clinical data, and tumor grading.AG was responsible for IHC evaluations.JB was instrumental in reviewing patients' clinical data and manuscript editing.KV and BA performed the experiments.The corresponding author is responsible for the manuscript preparation and oversaw the experiments.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 AG and KW are pathologists.KW was responsible for production of pancreas cancer TMA, collection of clinical data, and tumor grading.AG was responsible for IHC evaluations.JB was instrumental in reviewing patients' clinical data and manuscript editing.KV and BA performed the experiments.The corresponding author is responsible for the manuscript preparation and oversaw the experiments.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
}
] |
10.21926/obm.genet.1804054
|
Distinct Mechanisms of Alterations in DNA Methylation/Demethylation Leading to Myelodysplastic Syndromes/Acute Myeloid Leukemia and Chronic Lymphocytic Leukemia
|
Myelodysplastic syndromes (MDS) represent malignant myeloid disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and an increased risk of progression to acute myeloid leukemia (AML). AML is a highly heterogeneous disease characterized by the presence of large chromosomal translocations, gene fusions as well as mutations in the genes involved in hematopoietic proliferation and differentiation resulting in the accumulation of poorly differentiated myeloid cells. Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia, and is characterized by the clonal proliferation and accumulation of mature, long-lived CD5+ B cells. Epigenetic dysregulations are present in both myeloid disorders and in CLL, but, in contrast to MDS and AML that present gene fusions (TET1/LCX) and somatic mutations in epigenetic regulators (DNMT3A, TET2, IDH1/2), in CLL, epigenetic modifications are not qualitative but mostly quantitative (DNMT3A, TET2) and associated with disease progression. As a consequence, in MDS, AML and CLL, the focal or global DNA methylation/demethylation process is altered. In conclusion, a better qualitative and quantitative understanding of epigenetic regulators during myeloid/lymphoid differentiation, their localization and the co-recruitment of other proteins at specific DNA target sites, could offer us the possibility to modulate hematopoiesis, restore the initial balance, and control disease progression.
|
[
{
"section_content": "Myelodysplastic syndromes (MDS) comprise a diverse group of clonal and malignant myeloid disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and an increased risk of progression to acute myeloid leukemia (AML), either gradually or rapidly [1].AML is the most frequently occurring acute leukemia in adults, with malignant transformation occurring in primitive hematopoietic stem and progenitor cells, leading to increased proliferation and impaired differentiation [2,3].A wide heterogeneity of genetic mutations has been identified in both MDS and AML, the most frequent involving epigenetic processes, including the control of DNA methylation, RNA splicing, histone modifications, transcription factor function, DNA repair, and kinase signaling. Another hematologic pathology presenting important epigenetic modifications, is chronic lymphocytic leukemia (CLL), which is the most common type of leukemia, with a median age at diagnosis ranging from 67 to 72 years [4,5].CLL is characterized by the clonal proliferation and accumulation of mature, long-lived CD5+ B cells in peripheral blood, bone marrow, lymph nodes and secondary lymphoid organs [6].Several hypotheses have been formulated regarding the cell of origin for CLL B cells.Initially, based on the subdivision of CLL patients into two main subgroups according to their immunoglobulin heavy chain variable region (IGHV) mutational status, it was suspected that CLL cells were derived either from naïve B cells, in the case of the IGHV unmutated (UM) CLL B cells, or from memory B cells, in the case of mutated (M) CLL B cells.Subsequently, this dichotomy lost support with the development of gene expression microarray analysis, which is considered to be the gold standard technique for classifying the different lymphoid malignancies based on their cell of origin.Indeed, according to Klein et al. and validated since, transcriptome analysis revealed only a small number of differences between IGHV-UM and IGHV-M CLL B cells, supporting the possibility that both subsets are related to memory B cells [7].A recent study has confirmed these findings by integrating DNA methylation data and comparing normal B cell subsets to malignant B cells from 268 patients with CLL.This analysis showed that CLLs are derived largely from a continuum of memory B cell maturation states ranging from low-to high-maturity [8] In addition, such analysis verified the observation that the CLL methylation status resembles the that of memory B cells, accounting for 70%-100% of the programming for high-maturity memory B cells.However, the debate is not closed regarding the origin of CLL cells, and there is mounting evidence supporting the occurrence of the primary oncogenic event not at the memory B cell stage, but earlier in the HSC counterpart, and that the leukemogenic transformation is a step-wise process involving the accumulation of oncogenic events during B cell differentiation to the memory B cell stage [9].In MDS/AML, somatic mutations are presented in epigenetic regulators.In contrast, in CLL, epigenetic modifications are not qualitative, but mainly quantitative and affect the expression of epigenetic sensitive genes, such as CD5 and CD20, with differences reported during CLL disease progression [10][11][12].Such a dichotomy may explain the absence of the simultaneous occurrence of CLL and MDS/AML, except in CLL patients receiving chemotherapeutic and mutagenic agents. Accordingly, the aim of this review is to discuss the parallels between MDS/AML and CLL, diseases that represent two different mechanisms by which the epigenetic landscape is modified: by somatic mutations (qualitative) in MDS/AML, and at the expression level (quantitative) in CLL.Concerning MDS/AML, epigenetic dysregulation is an early event, conferring a competitive advantage on the myeloid lineage and leading to self-renewal.On the other hand, in CLL, there is a step-wise process in which methylation dysregulation represents a secondary event and contributes to disease progression. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "DNA methylation occurs predominantly at position 5 of the cytosine and represents the most studied epigenetic marker.Research on this cytosine methylation in cancer began in the 1970s when analysis of tumors revealed significant aberrations in 5-methylcytosine .Furthermore, in 2009, rapid breakthroughs came with the characterization of an active DNA demethylation enzymatic process that starts with the conversion of 5-mCyt to 5-hydroxy(h)mCyt (Figure 1) [13,14]. Currently, due to important advances in DNA methylation analysis technologies, whole-genome scale methylation maps are available for several human cell types and show that the methylation is mainly restricted to cytosine-phospho-guanosine (CpG) sites.The human genome contains approximately 29 million CpG sites, 60% to 80% of which are generally methylated and with important differences among cell types [15].Compartment-specific analysis of the methylome showed that DNA methylation has an important role in maintaining the latent transcriptional repression of pericentromeric repeats and transposable repetitive elements [16][17][18][19].This is essential for genomic stability and normal mitosis, by controlling chromosome alignment, segregation and integrity.The human genome is globally methylated with the exception of a small fraction (1%-2%) of the total genome which possesses CpG rich spots, referred to as CpG islands.These regions are predominantly located in transcriptionally active gene areas and are usually close to the first exon of a gene.The asymmetric distribution of DNA methylation between cell types has been shown to have functional implications in the epigenetic guidance of development, cellular differentiation and more recently, in nuclear organization; however, our understanding of this distribution and its effects is still incomplete [20][21][22][23].Approximately 70% of gene promoters overlap with CpG islands and it is generally accepted that the state of CpG methylation in these DNA regions, regulates gene expression through modification of chromatin structure and accessibility to the transcription machinery [24,25].The methylation process is catalyzed by the DNA methyltransferase (DNMT) enzymes, including DNMT1, DNMT3A, and DNMT3B, with the help of accessory proteins, such as DNMT3L and UHRF1.(Note: In MDS/AML, the main DNA methylation/demethylation regulators are DNMT1 (overexpressed), DNMT3A and TET2, presenting loss of function due to mutations, and IDH1/2 that repress TET2 through α-KG inhibition when mutated.In CLL, DNMT3A and TET2 expression is altered during disease progression.Cyt: cytosine; 5-mCyt: 5methylcytosine; 5-hmCyt: 5-hydroxymethylcytosine; α-KG: α-ketoglutarate; 2-HG: 2hydroxy-glutarate; IDH1/2: isocitrate dehydrogenase.MDS: myelodysplastic syndrome; AML: acute myeloid leukemia; CLL: chronic lymphocytic leukemia.)DNMTs catalyze the transfer of a methyl group from the methyl donor S-adenosyl-L-methionine (SAM), to position 5 of cytosine residues in DNA [26].SAM, in turn, is consumed by increased activation of the polyamine synthesis and recycling pathways [27].The first DNMT to be purified was shown to have a preferential affinity for hemi-methylated DNA substrates, and therefore is considered responsible for maintaining the methylation pattern during DNA replication [28].The gene encoding this enzyme was subsequently cloned and sequenced by the same group and named DNMT1 [29]. The observation that DNA methylation is not completely lost after DNMT1 inactivation suggested that cells must contain a de novo methylation activity.Later, Li and colleagues identified and cloned two novel methyltransferases [30].These genes are expressed as two proteins, DNMT3A and DNMT3B, with similar structure and domain organization.These enzymes do not differentiate between hemi-methylated and unmethylated CpG sites and are responsible for de novo DNA methylation, in association with DNMT3L, which has a regulatory function and no methylation capacity per se [31]. Regarding the active DNA demethylation process, 5-hmCyt was identified shortly after the discovery of 5-mCyt [32].After this discovery, no remarkable research was published on the subject, until the ten-eleven translocation (TET)1 protein was discovered and shown to be capable of erasing methylation marks via oxidation to 5-hmCyt [14].By definition, active DNA demethylation refers to the enzymatic process by which 5-mCyt and demethylation intermediates are replaced by an unmodified cytosine in a replication-independent manner [33].In contrast, the failure to maintain DNA methylation marks across cell divisions, resulting in the dilution of 5-mCyt during replication, is referred to as passive DNA demethylation.TET1 belongs to a family of enzymes which also includes TET2 and TET3 and catalyze the successive transformation of 5-mCyt to 5-hmCyt, 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-CaCyt) in a Fe(II)-and 2oxoglutarate-dependent manner [34,35]. Methylation genome mapping provided important insights into the role of 5-mCyt.Similarly, genome mapping of the demethylation process intermediates, and in particular 5-hmCyt, have been shown to be associated with important epigenetic regulatory functions.Indeed, 5-hmCyt content is highly variable in normal human tissues and higher levels of 5-hmCyt content are found in areas of transcriptional control, including promoters, transcription factor binding sites and gene bodies [36].However, as highlighted in a recently published study, the relationship between 5-hmCyt peaks and gene transcription is complex, which raises important questions and challenges based on some of the data provided so far by DNA methylome analysis using microarrays or the reduced representation bisulfite sequencing (RRBS) techniques [37].In this study, Zhang et al. demonstrated that the 5-mCyt distribution is only poorly correlated with gene expression, whereas the 5-hmCyt displays a \"bi-modal\" pattern in HSC with a high 5-hmCyt distribution in the promoter and gene body associated with highly expressed genes, while high 5-hmCyt levels in the transcription start sites (TSS) reveal lower relative gene expression [37].The positive association between high 5-hmCyt content in the gene bodies with gene expression in embryonic HSC was also demonstrated in a study by Huang et al. which, additionally, demonstrated a correlation with TET2 activity [38]. Another interesting observation concerning the mechanisms by which the fragile balance between methylation and hydroxymethylation is adjusted is that DNMT3A and TET1 function in a complementary and competitive manner, as demonstrated by Gu et al [39].This study showed that normal epigenetic landscapes and gene expression are maintained by TET1, which limits DNA methylation partially by protecting the cytosine target from DNMT3A and establishing boundaries for DNA methylation. Methylation and demethylation enzymes have also been shown to contribute to the adjustment of the epigenetic landscape and transcriptional regulation in an enzyme-independent manner, by simply recruiting other enzymes and co-factors.For example, there is a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT) [40].TET2/3-OGT co-localizes on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitantly decreased transcription.Independent of its catalytic activity, TET1 also has a role in transcriptional repression through its interaction with the SIN3A complex [41].These interactions complicate the interpretation of the link between the localization on the genome of methylation/hydroxymethylation enzymes, methylated/ hydroxymethylated CpGs and transcriptional regulation. ",
"section_name": "DNA Methylation and Demethylation",
"section_num": "2."
},
{
"section_content": "The Link between DNA Methylation, Myeloid or the Lymphoid Lineage Commitment, Self-Renewal and Differentiation The hematopoietic system and its hierarchical cascade of differentiation provides comprehensive information on the DNA methylation/demethylation process during differentiation (Figure 2).Indeed, important DNA methylation/demethylation maps have been shown to reflect cell types and differentiation stages [42]. Figure 2 The link between DNA methylation, myeloid or the lymphoid lineage commitment, self-renewal and differentiation -knowledge gained from mouse models.(Note: Lymphoid/myeloid differentiation in the bone marrow compartment is tightly regulated at the epigenetic level, with an observed increase in DNA methylation levels following lymphoid commitment, predominating at myeloid transcription factor binding sites, and a decrease as myeloid differentiation progresses.DNMT1 inhibition in hematopoietic progenitors skews progenitors toward the myeloid state, causing enhanced cell cycling of myeloid progenitor cells and reduced lymphoid differentiation potential.Homozygous DNMT3A and DNMT3B deficiency at the hematopoietic stem cell (HSC) stage of differentiation generates global DNA demethylation associated with HSC multipotency, self-renewal gene upregulation and downregulation of differentiation factors, which results in a severe block in myeloid/lymphoid differentiation and enhanced HSC self-renewal.Heterozygous Dnmt3a null (Dnmt3a +/-) mice develop myeloid skewing over time and myeloid malignancies after a long latent period.TET1-deficient mice develop a lymphoproliferative disease at an advanced age (approximately 2 years), with a mature germinal center B cell phenotype.TET2deficient mice present an altered cell differentiation with skewing toward monocytic/granulocytic lineages observed and development of chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).The B cell-specific TET2-knockout, on the other hand, leads to abnormalities in the B1 cell subset and a development of B cell malignancies.The combined loss of TET1/TET2 in HSC generates the development of lethal B cell neoplasms whereas the dual loss of TET2/TET3 results in aggressive myeloid leukemia, with an almost complete loss of 5-hmCyt.IDH1-R132H knock-in mice shared similar phenotypes with the Tet2-knockout mice, including global 5-hmCyt reduction, altered DNA methylation, impaired hematopoietic differentiation, myeloid skewing and the development of myeloid disorders.IDH2-R140Q transgenic mice showed normal HSC numbers and lineage differentiation, but exhibited increased extramedullary hematopoiesis and, when combined with HoxA9/Meis1 or FLT3 mutations in hematopoietic cells, produced acute myeloid leukemia.)Lymphoid/myeloid differentiation in the bone marrow compartment is tightly regulated at the epigenetic level, with an observed increase in DNA methylation levels following lymphoid commitment, and a decrease as myeloid differentiation progresses [43].DNA methylation mapping at the CpG level in the primitive hematopoietic compartment has revealed a progressive and specific methylation accumulation during lymphoid differentiation that predominates at myeloid transcription factor binding sites [42].This suggests that DNA methylation is important for cell fate determination and the balanced commitment to the myeloid or the lymphoid lineages. Multiple transgenic mouse models have been established to study the role of epigenetics in the hematopoietic lymphoid/myeloid differentiation process.The fact that DNA methylation profiling of murine hematopoietic progenitors involves more global methylation enrichment during early lymphopoiesis than during myelopoiesis could explain why DNMT1 inhibition skews progenitors toward the myeloid state [43].In other words, these data suggest that DNMT1 differentially controls the myeloid and lymphoid lineages, causing enhanced cell cycling of myeloid progenitor cells and reduced lymphoid differentiation potential [43]. Next, to determine whether constitutive methylation is continuously required throughout lymphoid differentiation, DNMT1 was also inhibited in lymphoid progenitors.In contrast to the previous mouse model, normal numbers and undisturbed homeostasis of B lymphoid cells were detected.Therefore, once the B cell program is established, a lower sustained constitutive DNA methylation is sufficient to maintain B cell identity and maturation [43]. Although it has been clearly shown that lymphoid commitment of the HSC is protected by DNMT1 and involves a global increase in DNA methylation, the roles of DNMT3A and DNMT3B at this early stage of B cell differentiation are less clear.DNMT3A-null HSCs exhibit global DNA demethylation but show both increased and decreased DNA methylation at different loci [44].These modifications are associated with upregulation of HSC multipotency and self-renewal genes as well as downregulation of differentiation factors.Serial transplantation of DNMT3A-null HSC progressively impairs HSC differentiation [44].Conditional loss of DNMT3B in HSC, another de novo methyltransferase, results in minimal phenotypic effects; however, combined loss of DNMT3B and DNMT3A results in a more severe block in myeloid/lymphoid differentiation and an enhanced HSC self-renewal similar to that observed in DNMT3A-null HSCs [45] Together, these data suggest a crucial role of de novo methyltransferases, particularly DNMT3A, in determining HSC differentiation and self-renewal, their expression being required to allow HSC to differentiate into the myeloid/lymphoid lineage. Hematopoiesis was also evaluated in mice heterozygous for a constitutive Dnmt3a null mutation.With no other manipulations, Dnmt3a +/-mice developed myeloid skewing over time and their HSCs exhibited a long-term competitive transplantation advantage.Furthermore, Dnmt3a +/-mice also spontaneously developed transplantable myeloid malignancies after a long latent period [46]. Interestingly, in a different mouse model bearing a defective DNMT3a gene under control of the immunoglobulin heavy chain enhancer (Eμ-DNMT3a-/-), DNMT3A inactivation occurred in both embryonic and adult stem and progenitor hematopoietic cells.Moreover, mice did not develop a myeloid disease but, in contrast, a lymphoproliferative disease resembling CLL or a peripheral T cell lymphoma phenotype [47]. DNA hydroxymethylation enzymes also play important roles in HSC differentiation.In transgenic TET1 knockout mice, HSC analysis showed global loss of 5-hmCyt with greater losses in 5-hmCyt occurring in gene bodies (exons and introns), important gains in 5-mCyt at promoters, and increased self-renewal potential [48].TET1-deficient mice further developed a lymphoproliferative disease at an advanced age (approximately 2 years), with a mature germinal center (GC) B cell phenotype [48].In Tet2-deficient mice, an altered cell differentiation skewed toward monocytic/granulocytic lineages was observed and Tet2-knockout mice develop chronic myelomonocytic leukemia (CMML), MDS and AML with increased proliferation of the HSC compartment [49].It is important to note that the B cell-specific Tet2-knockout (CD19-TET2-/-), on the other hand, leads to abnormalities in the B1 cell subset and a development of B cell malignancies showing similarities to human CLL, including CD5 expression and sensitivity to ibrutinib-mediated B cell receptor (BCR) signaling inhibition [50].Interestingly, the combined loss of Tet1/Tet2 in HSC is associated with the development of lethal B cell neoplasms, while the dual loss of Tet2/Tet3 results in aggressive myeloid leukemia with an almost complete loss of 5-hmCyt [51,52]. Heterozygous mutations in the catalytic arginine residues of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are common in MDS and LAM and the mutant enzymes acquire a neomorphic activity by converting the TET substrate α-ketoglutarate (α-KG) to D-2-hydroxyglutarate (2-HG).The IDH1-R132H knock-in mice shared similar phenotypes with the Tet2 knockout mice, including global 5-hmCyt reduction, altered DNA methylation, impaired hematopoietic differentiation, myeloid skewing and the development of myeloid disorders [53].The IDH2-R140Q transgenic mice showed normal HSC numbers and lineage differentiation, but exhibited increased extramedullary hematopoiesis and, when combined with HoxA9/Meis1 or FLT3 mutations in hematopoietic cells, produced acute leukemia [54]. ",
"section_name": "Knowledge Gained from Mouse Models:",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Alterations within Epigenetic Regulators in MDS/AML and CLL",
"section_num": "4."
},
{
"section_content": "DNMT1 is significantly overexpressed in AML [55,56].However, mutations of DNMT1 are rarely observed in contrast to the frequent appearance of DNMT3A mutations in AML and MDS [57,58] (Table 1).DNMT1 forms complexes with important transcription factors with established roles in AML, highlighting a possible pathoepigenetic interconnection in the disease biology.For example, recent studies suggest that DNMT1 transcription is modulated by the Sp1 transcription factor [59].The regulatory functions of Sp1 are mediated through its physical interaction with other transcription factors, such as those of the NF-κB family that are constitutively activated in AML [59,60].Such interactions were shown to lead to increased DNMT1 expression and consequently, gene silencing both in vivo and in vitro [59].RUNX1, which is mutated in 20% of AML and MDS cases, is a weak transcription activator although, its transcriptional potential is enhanced by acting in synergy with lineage specific transcription factors.DNMT1 inhibition in RUNX1 AML restores terminal differentiation through the switch of the RUNX1-Pu.1 coactivator/corepressor activity complex via the interaction of Pu.1 with DNMT1 [61].Minimally decreased expression of Pu.1 in mice may induce a pre-leukemic state with dysplastic features and initiate AML [62]. An important feature of AML is the existence of characteristic fusion genes, which might confer a worse (mixed lineage leukemia (MLL)-TET1/LCX fusions) or better (PML-RARα) prognosis.The PML-RARα onco-fusion induces hypermethylation by recruiting DNMT1 and DNMT3A in target promoters, such as RARb2, and is also required for the normal localization of DNMT1, which is otherwise dispersed in the nucleus [63].Similarly, through its physical association with DNMT1, the AML-ETO fusion binds and hypermethylates AML target gene promoters [64].DNMT1 is indirectly regulated via the miR-29b-Sp1 complex, which binds physically to the DNMT1 promoter to positively regulate its expression.Hence, there is a miR-29b-Sp1-DNMT1 axis in AML that is deregulated beginning with the underexpression of miR-29b.Forced miR-29b expression leads to restored expression of DNMT1 and its target genes p15 and ESR1, inducing partial differentiation of blast cells [65,66]. However, the role of the miR-29 family in epigenetic regulation is much more complex, because not only DNMT3A, DNMT3B, but also TET1 and thymine DNA glycosidase (TDG) are direct targets of miR-29.Furthermore, miR-29 overexpression has been shown to upregulate the global DNA methylation level in some cancer cells and downregulate DNA methylation in others [67,68]. In comparisons of DNMT1 expression in CLL with normal CD19+ B cells and memory B cells, no differences were observed [12,69].In addition, we have previously published data demonstrating an absence of significant differences between the IGHV-UM and IGHV-M CLLs and stable expression of DNMT1 during disease progression, which further supports a minor role for DNMT1 in disease progression [12]. ",
"section_name": "DNMT1",
"section_num": "4.1."
},
{
"section_content": "DNMT3A can be mutated in both MDS and AML.DNMT3A mutations, which occur in less than 10% of MDS cases and 12%-35% of AML, are most commonly heterozygous.DNMT3A R882(C/H) represents the most frequent mutation and is associated with poorer survival [65,78,79].DNMT3A R882H mutants show both decreased methyltransferase activity and dominant negative functions as its overexpression results in hypomethylation [80,81].Therefore, a partial, rather than complete inactivation of DNMT3A is likely more relevant in the pathogenesis of AML/MDS.Although they occur less frequently in MDS, DNMT3A mutations may represent a risk factor for progression to AML [82].Furthermore, several studies have provided evidence that DNMT3A mutations represent early events in leukemogenesis, and may also be present in T lymphocytes derived from AML patients.This observation shows that DNMT3A mutations are harbored within founding clones that retain the capacity to give rise to both myeloid and lymphoid lineages [83][84][85]; DNMT3A mutations are also thought to occur early in MDS [78]. However, acquired DNMT3A mutations are highly prevalent in age-associated clonal hematopoiesis in normal individuals with low or no biological impact and an, as yet, undefined oncogenic risk [86].This suggests that mutations in epigenetic modifiers, such as DNMT3A, are permissive for leukemogenesis but not sufficient to initiate transformation alone. The effect of DNMT3A mutations on DNA methylation in AML is controversial, possibly due to the different technical platforms used by various investigators.By using liquid chromatography tandem mass spectrometry or ELISA-based DNA methylation measurements to identify global changes in 5-mCyt, no statistical difference was detected in bulk 5-mCyt [72,87].However, by using HM-450K BeadChip arrays and separating DNMT3A wild-type, DNMT3A R882-mutant, and DNMT3A non-R882-mutant AMLs, the R882 mutant AMLs were found to have lower mean methylation values than were detected in both the non-R882 and DNMT3A wild-type patients [81].Hypomethylation is rather more focal than global and mainly localized in CpG islands and shores. In CLL, several studies, including our own recently published report, have shown DNMT3A downregulation during disease progression, compared to normal CD19+ B cells or memory B cells [12,69,88].Indeed, DNMT3A transcript levels decrease in CLL patients who progress from the indolent Binet A stage to the more aggressive Binet B/C stages [12].On the other hand, in patients with stable disease (Binet A) for more than 5 years, there was no significant difference in the DNMT3A levels between the two time points [12].Thus, it is unsurprising that low DNMT3A levels are associated with negative prognostic impact cytogenetic anomalies, such as 17p deletion, trisomy 12 or a complex karyotype and with a shorter treatment-free survival (TFS) [12,89].Our group also evaluated the association of DNMT3A downregulation in the progressive CLL disease group with changes in 5-mCyt levels by using the base resolution sequencing technique, RRBS, which revealed a significant decrease in DNA 5-mCyt in all regions of the genome (unpublished data).These findings support the view that loss in DNMT3A activity represents a driver for MDS/AML progression, and a risk factor for disease activity in CLL as reported previously in the DNMT3a+/-and Eμ-DNMT3a-/-mice, respectively. ",
"section_name": "DNMT3A",
"section_num": "4.2."
},
{
"section_content": "TET1 was first identified as a fusion partner, TET1/LCX, of the MLL gene in AML carrying t (10,11)(q22;q23).Furthermore, TET1 represents a direct target of MLL-fusion proteins, such as HOXA9, MEIS1, and PBX3, and is significantly upregulated in MLL-rearranged leukemia, leading to a global increase in 5-hmCyt levels [90].In normal cytogenetic AML, TET1 overexpression is associated with poor survival [91]. In CLL, somatic mutations in the TET1 gene represent rare events (2.8%) and CLL B cells have similar transcriptional levels of TET1 to those observed in control memory B cells [12,74].However, low levels of TET1 are associated with shorter TFS [12]. ",
"section_name": "TET1",
"section_num": "4.3."
},
{
"section_content": "TET2 is mutated in approximately 12%-20% of AMLs, 23%-35% of MDS and 50% of CMML [92,93].These loss-of-function mutations are diverse and impair TET2 activity [93,94].In primary MDS and AML patients harboring TET2 mutations, significantly reduced global 5-hmCyt levels have been revealed [87,95]. However, TET2 mutations, like those present in DNMT3A, are frequent in age-associated clonal hematopoiesis in normal individuals with low or no biological impact, which shows these mutations are not sufficient to initiate leukemogenesis alone [86]. In CLL, only 3.2% of patients present somatic mutations in TET2.In the early stage of the disease TET2 transcript levels in CLL are similar to those of control memory B cells, while important variations in TET2 levels are reported between CLL patients [12,73].In these two reports, a reduction in TET2 is associated with a lower median TFS, supporting an unexpected role for TET2 as an oncogenic repressor. ",
"section_name": "TET2",
"section_num": "4.4."
},
{
"section_content": "The isocitrate dehydrogenase-1 (IDH1) and -2 (IDH2) genes encode NADP+ dependent enzymes that convert isocitrate to -KT in the Krebs cycle.IDH1 functions in the cytoplasm whereas IDH2 is localized to the mitochondria.Mutations in the genes encoding IDH1 and IDH2 tend not to occur together in the same clone and have been described in 15%-30% of AMLs [96].Mutations result in a new enzymatic function resulting in the aberrantly accelerated conversion of -KG to 2-HG [97].The accumulation of the metabolite 2-HG has been shown to be responsible for the inhibition of TET2 function, thus ultimately inhibiting hydroxymethylation of DNA.Mutations in IDH1/2 and TET2 mutations are mutually exclusive in AML and, as expected, patients show similar DNA methylation profiles [98].While the prognostic impact of IDH mutations in AML is controversial and remains unclear, clinical trials using inhibitors that are relatively specific for IDH1 and IDH2 are ongoing [99][100][101]. In CLL, IDH1/2 mutations are extremely rare (0.9%) and decreased expression of IDH2 in addition to IDH1 overexpression were revealed in CLL B cells compared to the levels detected in normal CD19+ B cells [74]. ",
"section_name": "Isocitrate Dehydrogenase-1 (IDH1) and -2 (IDH2)",
"section_num": "4.5."
},
{
"section_content": "Advances in gene expression profiling and the development of new and improved highthroughput techniques for exploring methylation and hydroxymethylation of DNA have provided a more accurate and detailed understanding of the genetic and epigenetic basis for the development of hematopoietic diseases.Since their discovery, many important advances have also been made in our mechanistic understanding of the enzymes implicated in DNA methylation and demethylation processes. However, although these enzymatic processes have been thoroughly characterized and highresolution co-crystal structures of human DNMTs and TETs and their substrates have provided structural insights into their activities, several fundamental aspects of these processes and their implications in hematologic myeloid and lymphoid malignancies remain to be fully elucidated.First, concurrent recruitment of auxiliary factors by DNMT and TET proteins to genomic target sites is far from clear at the moment.Although several interactions have been demonstrated, studying these interactions in association with specific oncogenes or tumor suppressor genes implicated in hematologic malignancies could provide important information concerning their roles in these pathologies. Second, the mechanisms that govern recruitment and localization of DNMTs and TETs to DNA are not fully understood.Addressing these questions will be an important part of future investigations. We chose to discuss epigenetic modifications from two perspectives -the myeloid lineage pathologies, MDS/AML, in which epigenetic regulators are dysregulated because they present somatic mutations, and the lymphoid lineage pathology, CLL, in which epigenetic regulators are rarely mutated, but are highly dysregulated (Figure 3).With regard to MDS/AML, epigenetic dysregulation is an early event in the development of HSC.In CLL, on the other hand, there is a step-wise process of leukemogenesis and disease progression, and epigenetic regulators and methylation have an important dynamic correlation with disease progression and prognosis. Irrespective of the underlying molecular events, DNMT3A and TET2 in particular are dysregulated in MDS/AML and CLL, leading to altered DNA methylation and hydroxymethylation pattern.Advances in gene expression profiling and the development of new and improved highthroughput techniques for exploring methylation and hydroxymethylation of DNA have provided a more accurate and detailed understanding of the genetic and epigenetic basis for the development of hematopoietic diseases.Since their discovery, many important advances have also been made in our mechanistic understanding of the enzymes implicated in DNA methylation and demethylation processes. However, although these enzymatic processes have been thoroughly characterized and highresolution co-crystal structures of human DNMTs and TETs and their substrates have provided structural insights into their activities, several fundamental aspects of these processes and their implications in hematologic myeloid and lymphoid malignancies remain to be fully elucidated.First, concurrent recruitment of auxiliary factors by DNMT and TET proteins to genomic target sites is far from clear at the moment.Although several interactions have been demonstrated, studying these interactions in association with specific oncogenes or tumor suppressor genes implicated in hematologic malignancies could provide important information concerning their roles in these pathologies. Second, the mechanisms that govern recruitment and localization of DNMTs and TETs to DNA are not fully understood.Addressing these questions will be an important part of future investigations. We chose to discuss epigenetic modifications from two perspectives -the myeloid lineage pathologies, MDS/AML, in which epigenetic regulators are dysregulated because they present somatic mutations, and the lymphoid lineage pathology, CLL, in which epigenetic regulators are rarely mutated, but are highly dysregulated (Figure 3).With regard to MDS/AML, epigenetic dysregulation is an early event in the development of HSC.In CLL, on the other hand, there is a step-wise process of leukemogenesis and disease progression, and epigenetic regulators and methylation have an important dynamic correlation with disease progression and prognosis. Irrespective of the underlying molecular events, DNMT3A and TET2 in particular are dysregulated in MDS/AML and CLL, leading to altered DNA methylation and hydroxymethylation patterns that confer a competitive advantage to the myeloid or lymphoid lineage and an imbalance between self-renewal and differentiation.A better understanding of their localization and their co-recruitment of other proteins at specific DNA target sites could provide us with the possibility to modulate hematopoiesis, restore the initial balance, and control disease progressions that confer a competitive advantage to the myeloid or lymphoid lineage and an imbalance between self-renewal and differentiation.A better understanding of their localization and their co-recruitment of other proteins at specific DNA target sites could provide us with the possibility to modulate hematopoiesis, restore the initial balance, and control disease progression.(Note: In MDS and AML, epigenetic dysregulation is an early event caused by somatic mutations in hematopoietic stem cells, which generate a loss of function at the protein level and a poor prognosis.In CLL, epigenetic regulators are rarely mutated, but highly dysregulated, with their expression diminishing in association with disease progression.) ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Authors express their gratitude to Simone Forest and Geneviève Michel for their help in editing the paper.This study was supported by research funding from the \"Association Laurette Fugain\" (ALF 2015/03), the \"ligue against the cancer sections 29/35/49, the \"Region Bretagne\" and from the REpiCGO an Epigenetic network of the \"Cancéropole Grand Ouest\". ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The original manuscript was written by CBa and YR (main expertise in epigenetics/CLL) and the other authors (main expertise in MDS/AML and/or epigenetics) modified and approved the final manuscript. The authors have declared that no competing interests exist. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "The original manuscript was written by CBa and YR (main expertise in epigenetics/CLL) and the other authors (main expertise in MDS/AML and/or epigenetics) modified and approved the final manuscript. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interests exist. ",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.1007/s40120-013-0009-y
|
Listeria monocytogenes Meningoencephalitis Mimicking Stroke in a Patient with Chronic Lymphocytic Leukemia
|
Listeria monocytogenes is an important opportunistic pathogen affecting patients with immunosuppression and shows a high tropism for the central nervous system. The clinical manifestations of central nervous system listerial infections are variable and represent a diagnostic challenge.The authors report the case of a 59-year-old woman who was admitted for confusion, agitation, and right-lower extremity weakness. The patient was treated for 3 months with fludarabine and 2 months with corticosteroids for chronic lymphocytic leukemia and hemolytic anemia, respectively. At the time of admission, the neurological examination revealed grade 4 right-lower extremity weakness with reflex asymmetry and right-sided Babinski sign; no signs of meningeal irritation were detectable. Physical examination was notable for grade 1 obesity and subfebrility. The cerebral computed tomography scan demonstrated a hypodense lesion in the left frontal lobe. Cerebral magnetic resonance imaging revealed a hyperintense lesion in the left frontal lobe with extension toward the basal ganglia (T2 and Fluid-Attenuated Inversion Recovery [FLAIR] sequences), and small nodular enhancing lesions after gadolinium infusion in the affected territory. Blood analyses revealed pancytopenia and elevated liver enzymes. During the second day after admission, the patient developed fever and neurological examination revealed signs of meningeal irritation. The cerebrospinal fluid (CSF) analyses revealed: red blood cells 24 cells/mm(3); white blood cells 829 cells/mm(3) (76% lymphocytes, 22% neutrophils, 2% monocytes); protein level 111.2 mg/dL; glucose level 10.2 mg/dL. Empiric anti-infection treatment was started with intravenous ceftriaxone, ciprofloxacine, aciclovir, and fluconasole. Both blood cultures and CSF cultures were positive for L. monocytogenes. The antimicrobial regimen was changed to ampicillin. The clinical and imaging outcome was excellent.The supratentorial focal lesions secondary to Listeria meningoencephalitis are rare. The cases with focal neurological signs without fever at onset can resemble stroke.
|
[
{
"section_content": "admission, the patient developed fever and neurological examination revealed signs of meningeal irritation.The cerebrospinal fluid (CSF) analyses revealed: red blood cells 24 cells/mm 3 ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Listeria monocytogenes is a foodborne pathogen affecting mostly newborns, pregnant women, and non-pregnant adults with either primary or secondary immunosuppression conditions, especially cellular immunity disturbances. Listeria monocytogenes shows a high tropism for the central nervous system (CNS).The most frequent neurological manifestation is meningitis. Meningoencephalitis and encephalitis (cerebritis), which can progress to brain abscess and rhombencephalitis (brainstem encephalitis), are less common [1][2][3]. Chronic lymphocytic leukemia (CLL) is the most common leukemia that occurs in adulthood and frequently is accompanied by a multitude of immune abnormalities.Defects in both the cell-mediated and humoral-mediated immunity are responsible for the disease specific complications of CLL, including the infectious complications [4][5][6].Beside the disease-related immune abnormalities, further immunosuppression related to therapy with cytotoxic drugs, steroids, and monoclonal antibodies predisposes patients with CLL to infections [7]. Cases Cerebral computed tomography (CT) scan revealed a hypodense lesion in the left frontal lobe suggestive of an ischemic stroke (Fig. 1). During the second day after admission, the patient developed fever (101.12 8F; 38.4 8C) and neurological examination revealed signs of meningeal irritation (positive Kernig's sign). Cerebral magnetic resonance imaging (MRI) revealed a hyperintense lesion in the left frontal lobe with extension toward the basal ganglia (T2 and Fluid-Attenuated Inversion Recovery [FLAIR] sequences) (Fig. 2), and small nodular enhancing lesions after gadolinium infusion in the affected territory (Fig. 3). Lumbar puncture was performed on the second day and the CSF analysis revealed the following abnormalities: red blood cells (RBCs) 24 cells/mm 3 ; WBCs 829 cells/mm 3 (76% lymphocytes, 22% neutrophils, 2% monocytes); protein level 111.2 mg/dL; glucose level 10.2 mg/dL (serum glucose 86 mg/dL).CSF and blood samples were sent to laboratory for culture.Empiric anti-infection treatment was started with intravenous (i.v.) ceftriaxone, ciprofloxacine, aciclovir, and fluconasole.The neurological status of the patient was unchanged 2 days after the initiation of anti-infection therapy and the patient presented septic fever.CSF In CLL, both the humoral and cellular immunity are affected.There are cellular qualitative and quantitative defects in B-cells, T-cells, NK cells, neutrophils, and the monocyte and/or macrophage lineage that are inherent to the disease process and to its progression.These immunological defects are further complicated by the immunosuppressive properties of the drugs used in the treatment of CLL [4]. It is estimated that up to 80% of the patients with CLL will develop infectious complications during their disease course, and these infections are responsible for up to 60% of deaths [4].Patients undergoing chemotherapy are not only at risk of acquiring infections but also at risk of reactivation of latent infections.Patients treated with potent drugs, such as purin analogues (e.g., fludarabine) and monoclonal antibodies, are predisposed to infections with opportunistic pathogens, such as Pneumocystis jiroveci, L. monocytogenes, herpes viruses, JC virus, fungus (Candida, Cryptococcus, Aspergillus), and Toxoplasma gondii [4,12]. Glucocorticoid therapy is an important predisposing factor to listeria infection.In a large case series of CLL patients, listeria infection occurred in seven of 248 patients treated with prednisone and fludarabine, and no listerial infection occurred in 160 patients treated with fludarabine alone or in 387 patients treated with conventional chemotherapy [13]. The most common type of CNS damage in listerial infection is meningitis.In more rare cases the damage can be meningoencephalitis or encephalitis (mainly rhombencephalitis) [14].The clinical presentation of Listeria meningoencephalitis ranges from mild illness with fever and mental state changes to fulminant disease with coma.The meningeal signs are not present in all cases.In a large case series of 820 patients, 42 % presented without signs of meningeal irritation.The presence of focal neurological signs (cranial nerve palsies, ataxia, tremor, hemiparesis) indicates an encephalitic component [15]. Cases without fever at onset and focal neurological signs can resemble stroke.Elzie ´re et al. [16] [17].The gram stain has a low sensitivity and may resemble pneumococci or diphtheroids [15].RBCs can be present in the CSF of patients with listeria CNS infection in the absence of a CNS bleed or traumatic tap.In this case, the differential diagnosis must include the herpes simplex virus encephalitis and tuberculous meningitis [17]. The positivity of CSF and blood cultures is variable.Negative CSF culture results occur in [11-30% of patients with L. monocytogenes meningitis [18].The blood cultures are positive in approximately 50% of cases with CNS infection.Positive blood cultures rarely occur in the presence of negative CSF cultures [18].infectious diseases [19][20][21][22]. For the detection of the cerebral lesions secondary to listerial infection, MRI is more sensitive than CT.MRI reveals a high signal intensity in T2 and FLAIR sequences, and enhancing lesions in T1-weighted images after administration of gadolinium [16,23]. In the treatment of listerial CNS infection, ampicillin is the first-line agent.There is an important synergistic effect between aminoglycoside and ampicillin or penicillin. Many clinicians recommend the addition of an aminoglycoside to ampicillin for at least the first week of treatment.Bacteremic patients with normal CSF may be treated for 2 weeks.Patients with brain abscess, encephalitis, or rhombencephalitis should be treated for at least 6 weeks.In cases of penicillin hypersensitivity, trimetoprim-sulfametoxazol is the treatment of choice.Cephalosporins have limited activity against listeria.Vancomycin, imipenem, and meropenem also have been used successfully to treat cases of listeriosis.Some newer quinolones and linezolid show good in vitro activity [24].The value of adjunctive corticosteroids in listerial infection is not known [24][25][26][27]. The prognosis of listerial infections is highly variable.CNS infection is an important risk factor for morbidity and mortality.Neurological sequelae are common among the survivors of CNS infections [28][29][30]. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "The supratentorial focal lesions secondary to Listeria meningoencephalitis are rare.The cases with focal neurological signs without fever at onset can resemble stroke. ",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "No funding or sponsorship was received for this study or publication of this article.Dr. Bajko ´is the guarantor for this article, and takes responsibility for the integrity of the work as a whole. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
}
] |
10.1038/s41598-018-28420-y
|
Rbpj direct regulation of Atoh7 transcription in the embryonic mouse retina
|
<jats:title>Abstract</jats:title><jats:p>In vertebrate retinal progenitor cells, the proneural factor <jats:italic>Atoh7</jats:italic> exhibits a dynamic tissue and cellular expression pattern. Although the resulting <jats:italic>Atoh7</jats:italic> retinal lineage contains all seven major cell types, only retinal ganglion cells require <jats:italic>Atoh7</jats:italic> for proper differentiation. Such specificity necessitates complex regulation of <jats:italic>Atoh7</jats:italic> transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous <jats:italic>in vivo</jats:italic> mouse genetic studies established the cell autonomous suppression of <jats:italic>Atoh7</jats:italic> transcription by <jats:italic>Notch1</jats:italic>, <jats:italic>Rbpj</jats:italic> and <jats:italic>Hes1</jats:italic>. Here we identify four CSL binding sites within the <jats:italic>Atoh7</jats:italic> proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by <jats:italic>in vitro</jats:italic> electromobility shift, calorimetry and luciferase assays and, <jats:italic>in vivo</jats:italic> via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses <jats:italic>Atoh7 t</jats:italic>ranscription using both Notch-dependent and –independent pathways.</jats:p>
|
[
{
"section_content": "Recombination Signal Binding Protein for immunoglobulin kappa J region) in human/mouse, Su(H) (Suppressor of Hairless) in Drosophila, Lag-1 (lin12 and glp-1 phenotype) in C. elegans) and the Maml (Mastermind-like) co-activating factor.This protein complex activates downstream target gene transcription by binding to a variety of CSL sites within noncoding DNA [13][14][15][16] .Well-characterized effector genes of the Notch pathway include the Hes (Hairy/Enhancer of Split (E(Spl)) and Hey gene families, which encode transcriptional repressor proteins [17][18][19] .Intriguingly, during Drosophila retinal development, Notch signaling reiteratively regulates Atonal transcription 20,21 .In undifferentiated cells anterior to the eye disc morphogenetic furrow (a moving differentiation boundary), Notch signaling activates Atonal in a continuous stripe of cells.But more posteriorly, the Notch complex, via E(Spl) activity, suppresses Atonal expression in cells adopting non-R8 photoreceptor fates.This latter activity embodies the classic neurogenic role of Notch signaling.Importantly, these phases of Atonal regulation are separated in both time and space, and utilize distinct Atonal enhancers, as reviewed in 22 . Predicted CSL binding sites are interspersed throughout metazoan genomes, but it is challenging to know which are in vivo targets.However, the probability of any gene being a direct target is enhanced when its expression is affected by Notch pathway loss-and gain-of-function mutants.Previous examinations of mouse retinal mutants showed that loss of Notch1, Rbpj or Hes1 derepresses Atoh7 mRNA expression and RGC neurogenesis [23][24][25][26][27][28][29] .By contrast, overexpression of activated Notch1 (NICD1) stimulates proliferation, thereby blocking Atoh7 LacZ expression and RGC formation 26,30 .Yet, the mechanism(s) underlying Notch pathway regulation of Atoh7 remain unresolved. A classical view of Notch regulation of bHLH factors holds that an activated Notch protein complex transcriptionally activates Hes1, which in turn would repress Atoh7 transcription.But, other models are possible, including Rbpj direct regulation of Atoh7, either in a corepressor complex (repression), or in a Notch complex (activation) 21,31,32 .However, all genetic data consistently support Rbpj suppression of Atoh7 transcription.In this study, we characterize four CSL binding sites situated in Atoh7 5′ regulatory DNA.We found that at the peak of Atoh7 retinal expression, Rbpj occupies one CSL site, R3 within the distal primary enhancer, and this binding site mediates transcriptional repression.We also discovered that simultaneous loss of all four binding sites reduced Atoh7 transcription, suggesting a distinct mode of Rbpj regulation, consistent with locus priming for efficient transcriptional regulation by other factors 33 .Importantly, both Rbpj-mediated repression at site R3, and activation via multiple sites are Notch-independent, although Notch1/3 signaling has a measurable impact on Atoh7 mRNA levels.Overall our data are suggestive of three separate, yet simultaneous modes of Rbpj regulation: locus priming, direct repression and canonical Notch pathway suppression.We propose that integrated regulatory inputs are important for precise control of Atoh7 pulsatile expression in the developing retina. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Predicted Rbpj binding sites in Atoh7 regulatory DNA.The goal of this study was to identify and test putative Rbpj consensus (CSL) binding sites relative to Atoh7 transcriptional activity.Because the Atoh7 primary enhancer recapitulates endogenous mRNA expression in frog, chick, zebrafish and mouse transgenic studies, and contains two CNEs, it was the strongest candidate to contain CSL binding sites 8,10,11,34 .However, we used the CSL consensus TRANSFAC MATCH algorithm (5′-[C/T]GTG[G/T]GAA-3′) to perform an unbiased search of 6 Kb of noncoding DNA surrounding the Atoh7 coding exon [13][14][15] .Four putative CSL sites (termed R1-R4) were identified; all positioned 5′ to the ATG codon (Fig. 1A).Three of these sites (R2-R4) reside in the primary enhancer CNEs, while the other site (R1) is 300 bp upstream of the primary enhancer (Fig. 1A,B).Of the four sites, R1, R3, and R4 are highly conserved between mouse and human (Fig. 1B), with only R3 and R4 having analogous sequences in the chick and frog Atoh7/Ath5 locus (not shown).Interestingly, site R3 is situated in between previously characterized Pax6 and Neurog2 (E4/E3) binding sites (Fig. 1A,B) 8,10,11 .Site R4 lies within the proximal CNE, very close to the TATAA box and two highly conserved Ebox binding sites (Fig. 1A,B).In the adult frog retina, this CNE is critical for maintaining Atoh7/Ath5 expression in the ciliary marginal zone 8 . ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Both commercial and academic laboratory-made antibodies have been generated for detecting mammalian Rbpj or Atoh7 proteins.In a separate project, we rigorously tested a polyclonal antibody with high specificity for human and mouse Atoh7 proteins 35 .Previously, a rat anti-Rbpj antibody was used in our exploration of Notch signaling during mouse lens development 36,37 .Here we further confirmed the specificity of this monoclonal antibody, during mouse retinogenesis, and evaluated its usefulness in biochemical assays (Fig. 2).We immunolabeled E13.5 retinal cryosections from α-Cre;Z/EG;Rbpj CK °/+ control and α-Cre;Z/EG;Rbpj CKO/CKO mutant embryos (Fig. 2A,B).The α-Cre transgene induces Cre-mediated recombination in the distal retina 38 and Cre-mediated recombination is permanently marked by GFP expression from the Z/EG transgene 39 .In α-Cre;Z/EG;Rbpj CKO/+ controls, Rbpj protein is ubiquitous, including within the Cre lineage (GFP+ cells) (Fig. 2A).However, the same α-Cre cells lacking Rbpj showed a cell autonomous loss of protein expression (Fig. 2B).We also used this antibody for Western blotting, using denatured protein extracts from genotyped and pooled embryonic lenses, including from Rbpj conditional mutants 40,41 .A single immunoreactive band of 56 kDa was recognized in the wild type and heterozygous western lanes, but completely missing from homozygous mutant lens extracts (Fig. 2C). Next, we compared Atoh7 and Rbpj protein expression during in vivo embryonic retinal development.The spatiotemporal expression pattern of Atoh7 protein is highly dynamic, restricted to the central optic cup at E11.5 (Fig. 3A), but expressed by a broader group of E13.5 RPCs (Fig. 3B), and then confined to the periphery by E16.5 (Fig. 3C).Because Rbpj expression is ubiquitous, complete coexpression was expected.However, we noted that at every age almost all Atoh7+ cells exhibit brighter Rbpj expression than the Atoh7-negative cells.This is suggestive of differential Rbpj protein expression, although immunohistochemistry is a nonquantitative technique.To rule out non-specific secondary antibody cross-reactivity, we performed control experiments on retinal sections that were incubated with only one primary antibody (rabbit anti-Atoh7 or rat anti-Rbpj), prior to simultaneous application of both secondary antibodies (Fig. S1). ",
"section_name": "Rbpj and Atoh7 antibody validation and protein colocalization.",
"section_num": null
},
{
"section_content": "For initial assessment of Rbpj binding to the four putative CSL sites, we conducted in vitro electromobility shift assays (EMSA), using bacterially expressed and purified mouse Rbpj protein (residues 53-474), previously shown to bind DNA 42 .Rbpj protein was incubated with biotin-labeled double stranded oligonucleotides, in which the putative binding site is centrally located (Fig. 4A, Supplemental Table S4).We noted that all four sites shifted upon incubation with Rbpj protein (Fig. 4A).When key nucleotides within each CSL site were mutated, based on previous CSL-DNA structural studies, binding was abolished.We conclude that Rbpj protein specifically binds to each CSL binding site in vitro. We also performed isothermal titration calorimetry (ITC) to quantitate binding, using purified Rbpj with the oligomeric DNA duplexes that correspond to the four putative CSL binding sites (Fig. 4B-F and Supplemental Table S5).As positive and negative controls, we tested Rbpj with the CSL binding site from the Hes1 proximal promoter element (GTTACTGTGGGAAAGAAAG) and the non-specific sequence (GCTACTCATACCTAGAACG), respectively, and detected binding from the Hes1 site (~1 μM K d ), but did not detect binding from the non-specific site (data not shown).The results showed Rbpj bound to three of the four putative CSL sites with comparable affinity to the well-characterized Hes1 site (Fig. 4B) 43 .However, we found that one consensus site, R3, displayed a lower binding affinity (Fig. 4E).While we were unable to separate any potential effects of nucleotide variation and flanking sequence, interestingly, R3 lies in between validated Pax6 and Neurog2 binding sites 10,11 . Next, we wished to determine which CSL consensus sites are occupied by Rbpj during in vivo retinal development.Previously we demonstrated that Pax6 occupies consensus site J in the human ATOH7 gene, using chromatin from Ad12Her10 retinal cell line 10 .As a positive control, we performed Pax6 ChIP in parallel here, using mouse E14.5 retinal chromatin.This age was selected because it is the peak of Atoh7 expression 44 .Dissected retinas from individual embryonic litters were pooled and frozen en masse (n = 3 litters).After lysis, cross-linking, quantification and sheering, chromatin was immunoprecipitated with rat anti-Rbpj, rabbit anti-Pax6 or relevant IgG controls (see Methods).The antibody-bound chromatin complexes were purified, crosslinks reversed, and the isolated genomic DNA used as a template for real-time PCR.Each primer set amplified an amplicon specific for each putative binding site (Fig. 5A, Supplemental Table S4).As expected, Pax6 occupies site J in the Atoh7 primary enhancer (Fig. 5C).Although there was measurable occupancy of Rbpj at all four sites (R1-R4) relative to 3′UTR, only site R3 shows statistically significant enrichment (Fig. 5B).We conclude that Rbpj directly regulates Atoh7 transcription, via site R3.Although occupancy of the other three sites at E14.5 was not statistically significant, we hypothesized there could be more robust enrichment at other developmental ages, or that Rbpj simultaneous occupancy of multiple sites might contribute to a local Atoh7 chromatin configuration 33 . ",
"section_name": "Rbpj binding to Atoh7 5′ regulatory DNA in vitro and in vivo.",
"section_num": null
},
{
"section_content": "and at least site R3 in vivo, it is unclear if it does so via the Notch complex (activation), or a corepressor complex (repression).To address these possibilities formally we used luciferase reporter assays to measure the activity and requirement for each site individually, versus simultaneous mutation of all 4 sites, within a previously identified primary gene Atoh7 enhancer 7,10 .However, first we compared Atoh7 primary gene enhancer expression to that of the endogenous protein, which had not been previously reported 8,10,11,34,45,46 .For this we created a transgenic construct with 2.4Kb of mouse Atoh7 5′ DNA joined to a minimal human β-globin promoter (BG) and red fluorescent monomeric Cherry reporter (mCherry) 47 .We then used antibody colabeling to evaluate mCherry expression in retinal cryosections from E13.5 transient transgenic embryos.We found that endogenous Atoh7 significantly overlaps with Cherry+ cells (Fig. 6A) in the proliferative neuroblast layer, but not in the differentiated ganglion cell layer (GCL).This difference can be attributed to the greater stability of the fluorophore protein compared to Atoh7.Coexpression here was roughly equivalent to what was previously reported for another mouse transgenic line in which the human ATOH7 shadow enhancer drives BG-mCherry expression 7,35 . Next, we performed luciferase assays in both HEK293T kidney-derived and AD12Her10 retinal-derived human cell lines, under identical conditions 48,49 .This strategy was chosen because our previous study of Pax6 regulation of Atoh7 transcription suggested that retinal-specific context influences assay output 10 .Yet we found no differences between these cell lines, although HEK293T cells endogenously express RBPJ, but not ATOH7 50,51 , whereas AD12Her10 cells express both genes (Supplemental Fig. S2).Individual CSL site mutations (ΔR1, ΔR2, or ΔR4) did not affect transcriptional output, relative to wild type (Fig. 6A).By contrast, our mutation of CSL site R3 (ΔR3) derepressed luciferase activity over wild type levels, in both HEK293T and AD12Her10 cells, in the absence of exogenous Notch intracellular domains (Fig. 6A, Supplemental Fig. S2).We concluded that this particular binding site normally represses Atoh7 transcription.By contrast, when all four sites (ΔR1-4) were simultaneously mutated, there was a significant decrease in Atoh7 transcriptional levels (Fig. 6B, Supplemental Fig. 2).This is suggestive of coordinated, Rbpj-mediated transcriptional activation, among multiple (possibly all four) binding sites. Rbpj occupancy of site R3 represses Atoh7, presumably reflecting corepressor complex activity.However, the coordinated enhancement via multiple sites might be attributable to direct regulation of Atoh7 transcription, via a Notch complex.Therefore, we tested for Notch-dependence by coexpressing the intracellular domains of Notch1 (NICD1) or Notch3 (NICD3) in our luciferase assays.Each receptor alone, as well as in combination, was previously shown to be required in vivo for particular aspects of Atoh7 expression 26 .Plasmids containing the NICD1, NICD3, or a mixture of the two, were cotransfected with either the Atoh7 wild type or ΔR1-R4 mutant luciferase constructs 52,53 .Increasing NICD1 levels stimulated Atoh7 transcription but had no impact on the ΔR1-R4 mutant (Fig. 6B).NICD3 or NICD1 + NICD3 coexpression did not affect wild type Atoh7 activity, but further suppressed transcription in the ΔR1-R4 mutant (Fig. 6B).We interpret these outcomes to mean that multiple CSL binding sites help maintain Atoh7 basal level transcription.The elevated luciferase activity seen after NICD1 overexpression might represent Notch-dependent regulation of sites R1, R2, and R4, or an ectopic effect of NICD1 acting as a Rbpj sink, de-repressing R3 similar to R3 mutagenesis.In addition, Notch-mediated regulation of Atoh7 must utilize other noncoding sequences based on the decrease of transcriptional activity with NICD3 overexpression, potentially through Hes consensus N-boxes 16,43,54,55 . Multiple mammalian \"co-repressor\" genes encode proteins that interact with Rbpj via CSL binding sites 56 .These genes are unrelated at the primary sequence level, but predicted to link Rbpj to HDAC machinery, and include Smrt/Ncor2, Spen/Mint/Sharp, Fhl1b//KyoT2, Rita, Skip, L3mbtl3 and Kdm1a/Lsd1 [57][58][59] .Interestingly, Kdm1a/Lsd1 is expressed in the retina from E17-P15, and its pharmacologic inhibition in retinal explants induced an upregulation of bHLH factor expression 60 .Because nothing further is known about the retinal expression other Rbpj co-repressor genes, we used RT-PCR to test for transcription of four genes (Spen; Fhl1b; L3mbtl3; Kdm1a) in the E13.5 mouse retina (Fig. 6C).The Fhll/KyoT gene encodes multiple splice products, so we assayed for one exon common to all splice variants, as well as specific Fhl1b and Fhl1c exons that uniquely contain Rbpj-interaction domains [61][62][63] .We found that all of these co-repressor mRNAs are expressed during embryonic retinal neurogenesis (Fig. 6C).Given that at least four of seven putative co-repressors are present in the mammalian retina, elucidation of their specific mechanisms of action will require in-depth, future studies. ",
"section_name": "Rbpj differentially regulates Atoh7 transcription. Although Rbpj binds to all four CSL sites in vitro",
"section_num": null
},
{
"section_content": "Previous studies of Notch signaling in the vertebrate retina genetically linked Rbpj activity to Atoh7 expression and retinal ganglion cell differentiation [24][25][26][27][28][29] .Notch activity normally suppresses both Atoh7 and RGC neurogenesis, but the molecular mechanisms for this remain unresolved.Here, we explored the possibility that Rbpj can directly regulate Atoh7 transcription. Bioinformatic analysis of noncoding sequences surrounding the mouse Atoh7 gene identified four putative Rbpj-CSL consensus binding sites.While a CSL consensus sequence is useful for predicting Rbpj target genes [13][14][15] , it is not the only nucleotide motif this protein can bind 16,43 .One particular binding site arrangement, called SPS (Su(H)-paired site), consists of two CSL binding sites in a head to head configuration separated by ~16 nucleotides 64 .These SPS elements are occupied by dimeric NICD/Rbpj complexes, to regulate transcription [65][66][67][68] .Although the Atoh7 upstream CSL binding sites lack a canonical SPS arrangement, there remains some possibility for such a mechanism, since a cryptic CSL element in the Hes5 promoter acts in a dimeric SPS complex 65 . Among the CSL sites analyzed here for the embryonic retina, R3 stands out as unique.Counterintuitively, CSL has the weakest affinity for R3 in vitro, but was the only site that ChIPped CSL in vivo with statistical significance.Similar phenomenon has been observed for Su(H), the fly CSL ortholog, binding of the sparkled (spa) enhancer in Drosophila, whereby the low affinity Su(H) sites are critical for proper gene expression and patterning driven by spa 69 .The location of R3 within the distal CNE is flanked by a Neurog2-dependent Ebox (30 bp upstream) and a Pax6 binding site (20 bp downstream).Such spacing allows one or two helical turns to separate each of these sites.In the developing pancreas, this same distance is permissive for Rbpj physical interaction with the bHLH transcription factor Ptf1a 70 .Interestingly, Ptf1a is also expressed in the developing retina and influences neurogenesis of several retinal cell types [71][72][73] .In Drosophila and Xenopus there are other examples of CSL/Rbpj protein interactions with bHLH factors that activate transcription 66,74,75 .Hence, we cannot discount the possibility that Neurog2 (or another bHLH factor) may physically interact with Rbpj.Alternatively, Rbpj occupancy of site R3 might affect local chromatin architecture, thereby displacing activating factors.Because we detected both Rbpj and Pax6 occupancy in E14.5 retinal chromatin (within the same preparation), a mutually exclusive binding mechanism would seem implausible.One caveat was the use of whole retina chromatin, which could obscure distinct configurations of transcription factor binding at the Atoh7 primary enhancer, among a heterogenic population of retinal cells.For example, in mitotically-active RPCs that do not express Atoh7, Rbpj could act as a repressor at site R3.When these cells enter their terminal mitosis, they may activate Atoh7 expression, via Pax6 or Neurog2 binding, which would also displace nearby Rbpj-corepressor complexes.Conversely, during terminal differentiation, this relationship could be reversed, with Rbpj-corepressor binding at site R3 dislodging either Pax6 or Neurog2.Only the generation of single-cell genomic datasets can map the occupancy of particular enhancer binding site to the developmental status of cells, at distinct stages of retinogenesis. Here we also provided some insight into in vivo context for our biochemical data, via direct comparison of Rbpj and Atoh7 protein expression patterns.Rbpj is well established as ubiquitously expressed, so co-localization with Atoh7 protein was already predicted.But, transcription factors that are coexpressed with their target genes typically activate transcription, not repress it.Indeed, Hes1, a known repressor of neurogenesis, displays mutually exclusive expression with βgal in Atoh7 LacZ/+ eyes 26 .Yet, our transcriptional activity data clearly indicate that Rbpj repression of Atoh7 is the major mode of regulation, which correlates with all previous genetic findings 25,29 .However, we also note that Rbpj does not appear uniformly expressed, with brighter anti-Rbpj labeling coinciding with Atoh7 expression.The significance of this observation remains unclear.Better understanding will Distinct PCR primers for the Fhl1 (KyoT) gene were used that amplified an exon common to all splice products (pan Fhl1), and the specific splice variants Fhl1b (KyoT3) and Fhl1c (KyoT2) that uniquely contain the Rbpjinteraction domain 78 .All PCR reactions were run on a single gel, uncropped image provided in Supplemental require the determination of which Rbpj regulation activities at work during distinct stages of retinal cell development.Moreover, we must clarify whether Notch1/3 signaling invokes canonical pathway regulation, namely Notch-Rbpj-Maml binding to Hes gene promoters, which may in turn directly repress Atoh7 transcription. While highly speculative, we propose that lower levels of Rbpj protein expression are sufficient for binding to multiple CSL sites, which contributes to keeping the Atoh7 locus open and primed (yet transcriptionally silent) 33 , until its rapid, pulsatile expression is needed.Higher levels of Rbpj protein may subsequently be required to also engage in the activities of Notch and co-repressor complexes, which act at different regulatory sequences, and possibly at different rates to shut down Atoh7 transcription.Clearly additional transcription factors must simultaneously regulate Atoh7 (positively or negatively) since brighter-labeled Rbpj cells are capable of Atoh7 co-expression.The integration of these multiple modes of regulation allows for more precise modulation of target gene mRNA levels, particularly during highly dynamic developmental processes. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Ethics Statement.All mice were housed and cared for in accordance with the guidelines provided by the National Institutes of Health, Bethesda, Maryland, and the Association for Research in Vision and Ophthalmology, and conducted with approval and oversight from the Cincinnati Children's Hospital Research Foundation and UC Davis Institutional Animal Care and Use Committees. ",
"section_name": "Experimental Methods",
"section_num": null
},
{
"section_content": "A Rbpj tm1Hon conditional allele (termed Rbpj CKO ) were maintained on a 129/SvJ background and genotyped as described 41 .α-Cre transgenic mice were maintained on a CD-1 background and genotyped as described 38 .Le-Cre mice were maintained on an FVB/N background and genotyped as described 40 .Z/EG lineage tracing mice (Tg(CAG-Bgeo/GFP)21Lb3/J) use the CMV enhancer/chicken actin promoter to constitutively express lacZ, which is replaced with eGFP expression upon Cre activation 39 .These mice were acquired from Jackson Labs (Stock Number 003920), maintained on a CD-1 background and genotyped as in 39 .Embryonic gestational age was determined by timed matings, with the date of the vaginal plug as E0.5. The upstream, noncoding 2.4 Kb of mouse Atoh7 genomic DNA (nucleotides -3032 to -503 containing the primary enhancer but lacking the TATAA box) was PCR amplified using primers with engineered XbaI and BglII restriction sites, cloned into the Xba I-Bam HI sites of the pBGnCherry vector 47 in the normal transcriptional orientation, and verified by Sanger sequencing.The Atoh7 fragment was PCR amplified (EXPAND Hi-Fidelity polyermase) from a previously subcloned 6.5 Kb mouse Atoh7 genomic DNA template 3 , digested with BglII and XbaI and then purified.The pBGnCherry vector 47 contains a minimal human β-globin promoter and monomeric Cherry red fluorescent protein reporter cassette (mCherry), as well as a synthetic amino terminal nuclear localization signal (MAPKKKRKVEDV) downstream of the BamHI site.There is no intrinsic activity of this vector in transgenic mice 47 .Linearized DNA was microinjected into CD-1 mouse pronuclei by the CHRF Transgenic Core Facility.F 0 embryos were collected at E13.5 and screened for live mCherry fluorescence with a Leica MZ12 dissecting scope equipped with a Texas-Red filter.We harvested 4 Cherry +/44 embryos and each Cherry-positive embryonic head was cryoembedded, sectioned and analyzed using immunohistochemistry and confocal imaging (see below). Bioinformatics of Rbpj binding sites.Three kilobases of 5′ and three kilobases of 3′ noncoding genomic DNA from the mouse and human Atoh7 genes (Gene IDs 53404 and 220202) were aligned using the MacVector Clustal W algorithm (v.12).CNEs were identified in multiple vertebrate genomes using the UCSC genome browser MultiZ alignment and conservation features and mm10 genome assembly (http://genome.ucsc.edu).Putative CSL/Rbpj binding sites were identified using the TRANSFAC MATCH program with matrices M01111 (V$RBPJK_Q4) and M01112 (V$RBPJK_01).Previously defined Pax6 paired domain and E-box binding sites within the mouse Atoh7 primary distal CNE are included for reference 8,10,11 . Immunohistochemistry. Embryonic heads were fixed in 4% paraformaldehyde/PBS for 1 hour at 4 °C, processed through a sucrose/PBS series, cryoembedded and sectioned at 10 μm.Immunohistochemistry using our lab protocol 2 to label with chick anti-GFP (Abcam, 1:1000, AB13970), rat anti-Rbpj (CosmoBio, 1:100, SIM-2ZRBP-1), rabbit anti-Atoh7 (Novus Biologicals, 1:500, NBP1-88639), or goat anti-mCherry polyclonal (SICGEN, 1:500, AB0040-200) primary antibodies.Secondary antibodies were directly conjugated to Alexa Fluor 488 (Invitrogen, A21208), Alexa Fluor 594 (Jackson ImmunoResearch, 712-586-153), Alexa 594 (Invitrogen, A11058), Alexa Fluor 647 (Invitrogen, A21244), or Dylight 649 (Jackson ImmunoResearch, 711-495-152).Microscopic imaging used either a Zeiss Axioplan fluorescent microscope with a black and white camera, Apotome deconvolution device, and Axiovision (v.7.0) software, or a Leica DM5500 microscope equipped with a SPEII solid state confocal and Leica LASAF software.All digital micrographs were electronically adjusted equivalently for brightness, contrast and pseudocoloring using Adobe Photoshop CS5 software. Western blotting.Pairs of lenses were harvested from E14.5 Rbpj CKO/CKO , Le-Cre;Rbpj CKO/+ and Le-Cre;Rbpj CKO/CKO embryos and flash frozen.Twenty lenses (10 pairs) of the same genotype were pooled and lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40) containing Complete protease inhibitors (Sigma 11697498001).Total protein concentrations were determined by Bradford assay (Biorad, 500-0006).NuPAGE 4-12% Bis-Tris gels (Invitrogen, NP0322BOX) were loaded with 20ug of total lens protein per gel lane, electrophoresed and transferred onto nitrocellulose membranes (Invitrogen, LC2000).Standard western blotting was performed, using rat anti-Rbpj (Cosmo Bio Co, 1:500 SIM-2ZRBP-2) or mouse anti-β-actin (Sigma, 1:3000, A1978) primary antibodies, followed by HRP-conjugated anti-rat IgG or mouse IgG secondary antibodies (Jackson Immunoresearch, Rat 112-035-175 1:5000, Mouse 315-035-003, 1:10,000).Blots were developed using a Supersignal West Pico Chemiluminescent substrate kit (Thermo Scientific, 34078), Kodak standard x-ray film and film developer. ",
"section_name": "Animals.",
"section_num": null
},
{
"section_content": "Single-stranded complementary oligonucleotides containing predicted CSL binding sites were labeled using a Biotin 3′ end DNA labeling kit (Thermo Scientific, 89818).Double-stranded DNA probes were made by annealing biotin-labeled complementary oligonucleotide pairs at room temperature for one hour.0.5 μM purified mouse Rbpj protein (residues 53-474; Friedmann et al., 2008) and 1 nM of labeled oligonucleotide complexes, in the presence of 1.9 ng/μl poly[d(I-C)] (Sigma, 10108812001), were resolved on a 6% DNA retardation gels (Invitrogen, EC63652BOX) in 0.5 × TBE buffer and then transferred to nylon membranes.The LightShift Chemiluminescent EMSA kit assay (Thermo Scientific, 20148) was performed on the blots, which were developed using the Chemiluminescent Nucleic Acid Detection kit (Thermo Scientific, 89880), Kodak x-ray film and film developer. Isothermal titration calorimetry of CSL-DNA complexes.The production and purification of bacterially expressed Rbpj protein, residues 53-474, has been described 16,42 .Oligonucleotides from Integrated DNA Technologies (IDT) (Fig. 4A) were hydrated, purified, quantified and annealed as in 16 .All purified components were degassed, buffer matched and quantified as previously described 16 .A typical experiment was performed at 5 °C using a MicroCal VP-ITC microcalorimeter with the oligomeric duplex (~100 μM) in the syringe and Rbpj (~10 μM) in the cell and consisted of 40 injections of 7 μl each.Data analysis used the ORIGIN software and was fitted to a one-site binding model, with binding data representing the average of n = 3 experiments. Chromatin immunoprecipitation and Real-time PCR.ChIP was performed as described 10,76 with several modifications.30 E14.5 CD-1 pooled embryonic retinas were crosslinked with 1% formaldehyde and the reaction stopped by addition of 125 mM final concentration of glycine.Chromatin was sheered to 300-1000 bp size range with a Bioruptor UCD-200 sonicator + chiller (Diagenode), for 20 minutes at high power with 15 sec ON/30 sec OFF cycles.Either 3 μg rat anti-Rbpj antibody (Cosmo Bio Co, SIM-2ZRBP1) or rat IgG (Jackson ImmunoResearch, 012-000-003) were incubated with 40 μg sonicated chromatin overnight at 4 °C.Immune complexes were collected with Protein G agarose beads (Sigma, P7700), washed several times and eluted using 0.5 M NaHCO 3 , 1% SDS elution buffer.Pax6 ChIP was run in parallel from each retinal chromatin prep, by incubating 20 μg of sheared chromatin with 1 μg anti-Pax6 (Covance, PRB-278P), or rabbit IgG (Jackson ImmunoResearch, 011-000-003), coupled to Protein A sepharose beads (GE Healthcare, 17-0780-01).Input and immunoprecipitated chromatin samples were initially analyzed by performing 30 cycles of PCR amplification and agarose gel electrophoresis, then quantified by real time PCR, using Table S4 primers, fast SYBR Green master mixes and a StepOnePlus PCR system (Applied Biosystems, 4385612 and 4376600).A standard curve using serial dilutions of 1% input chromatin was used to calculate the percent input of each sample.The p-values were determined by ANOVA and a Bonferroni posthoc test (Rbpj) or a student's unpaired, 2-tailed t-test (Pax6) with GraphPad Prism software (v6). Luciferase Assay.The upstream 2.6 Kb noncoding DNA from the mouse Atoh7 locus was previously cloned into the pGL2 luciferase vector 10 .Rbpj binding site mutations were generated using PCR-based site directed mutagenesis 77 and verified by Sanger Sequencing.Either 3.5 × 10 5 HEK293T or 5 × 10 5 AD12HER10 cells were plated per well of a 6-well tissue culture plate.After 48 hours (~60% confluency) cultures were transfected according to the Fugene6 (Promega, E2692) protocol with a 5:1 Fugene6 to DNA ratio, with the DNA constituting 500 ng of luciferase plasmid and 50 ng of Renilla control plasmid (pRL).In Notch ICD overexpression experiments, 100 ng of NICD1/pBK-CMV, 100 ng of NICD3/p3XFLAG-CMV-7TM, or a mixture of 50 ng of each plasmid were cotransfected with the luciferase and Renilla plasmids.Cells were washed in PBS, harvested 48 hours after transfection in 500 μL 1 × PLB (Promega) and cell pellets stored at -80 °C.Cell extracts were assayed in technical triplicate using the Dual Luciferase Assay System (Promega, E1980) on a Perkin Elmer Victor X5 workstation.Luciferase activity levels were normalized to the control Renilla activity, and p-values determined with GraphPad Prism (v6) software, using a two-tailed, unpaired t-test with equal standard deviation and assuming a Gaussian distribution. ",
"section_name": "Electrophoretic Mobility Shift Assay (EMSA).",
"section_num": null
},
{
"section_content": "Total RNA was extracted using the RNeasy micro kit (Qiagen, Cat No 74004) from 1 pair of dissected E13.5 retinas, or using Trizol (Invitrogen Cat No 15596026) for HER-10 cells.For embryonic retinal RNA 100 ng was reverse transcribed into cDNA using the iScript Synthesis kit and product protocol (BioRad, Cat No. 178891).For HER-10 cells, 6.5 μg of total RNA was first treated with 1U of 10 U/μL of DNase (Roche Cat No. 04716728001) by incubating at 37 °C × 35 min, 80 °C × 5 min, 90 °C × 3 min.Then 2 μg of treated RNA was used for cDNA synthesis, with Superscript III and manufacturer protocol (Invitrogen/ThermoFisher, Cat No. 18080093).Both experiments included a mock synthesis (lacking total RNA) performed in parallel.For embryonic retinas, 1 μL of cDNA was combined with individual primer sets (Supplemental Table S4) and Go-Taq polymerase (Promega, cat # M7122) for 35 cycles of PCR at 95 °C × 30 sec, 55 °C × 30 sec, 72 °C × 30 sec.PCR products were electrophoresed on a 2% TAE agarose gel.Alternatively, 1 μL HER-10 cDNA was combined with individual primer sets (Supplemental Table S4), 1 × PCR Buffer and dNTPs, 1 × Masteramp (Epicentre/Illumina, ME81210) and 1U of Taq polymerase (5U/μL Roche/Sigma, Cat No. 11146173001) for 35 cycles of PCR at 95 °C × 30 sec, 60 °C × 30 sec, 72 °C × 30 sec.PCR products were electrophoresed on a 1% TBE agarose gel. ",
"section_name": "RT-PCR.",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Tasuku Honjo for Rbpj flox mice, Ruth Ashery Padan for α-Cre and Le-Cre transgenic lines, Shiming Chen and Guang-Hua Peng for guidance on ChIP assays, Brad VanderWielen for purified Rbpj protein, Jane Johnson for pBGnCherry vector, Malgorzata Quinn, Yuqi Cai and Tien Le for technical support, Joo-Seop Park and Tom Glaser for fruitful discussions and critical feedback.This work was supported by NIH R01 grants EY13612 to NLB, CA178974 to RAK, and NEI Training Grants T32EY015387 to JBM and T32ES007250 to ANC. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "All data generated or analyzed during this study are included within this published article and its Supplementary Information files. ",
"section_name": "Data availability.",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28420-y. 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": "",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28420-y. ",
"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
}
] |
10.3390/cells8090970
|
JNK1 Induces Notch1 Expression to Regulate Genes Governing Photoreceptor Production
|
<jats:p>c-Jun N-terminal kinases (JNKs) regulate cell proliferation and differentiation via phosphorylating such transcription factors as c-Jun. The function of JNKs in retinogenesis remains to be elucidated. Here, we report that knocking out Jnk1, but not Jnk2, increased the number of photoreceptors, thus enhancing the electroretinogram (ERG) responses. Intriguingly, Notch1, a well-established negative regulator of photoreceptor genesis, was significantly attenuated in Jnk1 knockout (KO) mice compared to wild-type mice. Mechanistically, light specifically activated JNK1 to phosphorylate c-Jun, which in turn induced Notch1 transcription. The identified JNK1–c-Jun–Notch1 axis strongly inhibited photoreceptor-related transcriptional factor expression and ultimately impaired photoreceptor opsin expression. Our study uncovered an essential function of JNK1 in retinogenesis, revealing JNK1 as a potential candidate for targeting ophthalmic diseases.</jats:p>
|
[
{
"section_content": "The retina is a thin sheet of neural tissue in the eye that senses light and transmits relevant information to the brain via the optic nerve.The vertebrate retina consists of six types of neurons and one type of glial cells (Müller glial cells), which form three cellular layers: photoreceptors in the outer nuclear layer (ONL); horizontal, bipolar, and amacrine interneurons and Müller glial cells in the inner nuclear layer (INL); and ganglion and displaced amacrine cells in the ganglion cell layer (GCL).These cell types play specific roles in the process of vision [1,2].The detection of light stimuli is mediated by photoreceptors, which contain two basic subtypes: rods and cones.Rod photoreceptors use rhodopsin to mediate night vision and can respond to single light quanta, whereas cone photoreceptors use opsins to respond to different wavelengths of light and mediate color perception [3]. Photoreceptors are generated from multipotent progenitors [4,5].Cones are early-born cells, with production peaking at E14.5 and E15.5 in the central and peripheral areas of the retina, respectively.The rods peaks are produced centrally, at P0, and between P0 and P2 in the peripheral parts of the retina [6].Crx [7][8][9], Otx2 [10], Rxrg [11], Neurod1 [12,13], Thrb2 [14,15], and Nrl [16,17] are all important transcriptional factors controlling photoreceptor formation and differentiation.Additionally, Notch signaling has been established as playing a pivotal role in photoreceptor genesis, which is essential for the transition from multipotent progenitor to photoreceptor precursor [18,19].Conditional deletion of Notch1 signaling components increases the production of both rod and cone photoreceptor precursors [20][21][22].Furthermore, the inhibition of Notch signaling induces the expression of several 2.4.Real-Time PCR Total cellular RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.The quantification of gene transcripts was performed by real-time PCR using SYBR Green PCR mix (Applied Biosystems).All values were normalized to the level of β-actin mRNA.The primers used are listed below: β-actin: sense (AAAGACCTGTACGCCAACAC), antisense (GTCATACTCCTGCTTGCTGAT); c-Jun: sense (GCAAGCCCTGAAGGAAGAG), antisense (GTCAT ACTCCTGCTTGCTGAT); Neurod1: sense (GACGGGGTCCCAAAAAGAAAA), antisense (GCCAAGCGCAGTGTCTCTATT); Crx: sense (GTTCAAGAATCGTAGGGCGAA), antisense (TGAGATGCCCAAAGGATCTGT); Rxrg: sense (CATGAGCCCTTCAGTAGCCTT), antisense (CGGAGAGCCAAGAGCATTGAG); Notch1: sense (CCGTGTAAGAATGCTGGAACG), antisense (AGCGACAGATGTATGAAGACTCA); Opn1sw: sense (CAGCCTTCATGGGATTTGTCT), antisense (CAAAGAGGAAGTATCCGTGACAG); Opn1mw: sense (ATGGCCCAAAGGCTTACAGG), antisense (AAGGGACCTTTGGTGCTGTT); Notch2: sense (GAGAAAAACCGCTGTCAGAATGG), antisense (GGTGGAGTATTGGCAGTCCTC); Notch3: sense (TGCCAGAGTTCAGTGGTGG), antisense (CACAGGCAAATCGGCCATC); Wnt2b: sense (CCGACGTGTCCCCATCTTC), antisense (GCCCCTATGTACCACCAGGA); Wnt5a: sense (CAACTGGCAGGACTTTCTCAA), antisense (CATCTCCGATGCCGGAACT); Rho: sense (CCCTTCTCCAACGTCACAGG), antisense (TGAGGAAGTTGATGGGGAAGC); Nrl: sense (TCCCAGTCCCTTGGCTATGG), antisense (CACCGAGCTGTATGGTGTG); Onecut2: sense (GGCTTCCGTCCATGAACAAC), antisense (CGAAATTGGGGCTGAGCATTTT); Chx10: sense (CTGAGCAAGCCCAAATCCGA), antisense (CGCAGCTAACAAATGCCCAG); Stx1a: sense (AGAGATCCGGGGCTTTATTGA), antisense (AATGCTCTTTAGCTTGGAGCG); Vim: sense (CGGCTGCGAGAGAAATTGC), antisense (CCACTTTCCGTTCAAGGTCAAG); Sox9: sense (CGGAACAGACTCACATCTCTCC), antisense (GCTTGCACGTCGGTTTTGG); Rbpms: sense (CCCGTAGGCTTTGTCAGTTTT), antisense (GTAAAGTGCAGGTACTGTGAGC); Prkca: sense (GTTTACCCGGCCAACGACT), antisense (GGGCGATGAATTTGTGGTCTT); ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Cell pellets were collected and resuspended in RIPA (Radio-Immunoprecipitation Assay) buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (Ethylene Diamine Tetraacetic Acid), 0.5% NP40, 0.25% Na-deoxycholate, 1 mM Na 3 VO 4 , 0.1% SDS (Sodium dodecyl sulfate), and 0.1 mM PMSF (Phenylmethanesulfonyl fluoride); Roche complete protease inhibitor set).The resuspended cell pellet was vortexed for 20 s and then incubated on ice for 20 min, followed by centrifugation at 12,000 rpm for 15 min.Afterward, supernatants were collected for subsequent western blot analysis.Retinas, which were isolated from mice, were homogenized with RIPA buffer and centrifuged, and then supernatants were collected for subsequent western blot analysis. ",
"section_name": "Western Blot",
"section_num": "2.5."
},
{
"section_content": "Harvested 661W cells were resuspended in 200 µL of buffer A (10 mM HEPES (N-2hydroxyethylpiperazine-N-ethane-sulphonicacid), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT (dl-1,4-Dithiothreitol), and 1 mM PMSF (PH 7.9)) containing 0.1% Nonidet P-40 and were incubated on ice for 15 min.The lysates were mixed and centrifuged immediately at 4000 rpm for 5 min to save supernatants containing the cytosolic fraction.The pellet was washed with 500 µL buffer A twice.Nuclear proteins were extracted by resuspending the nuclear pellets in 100 µL of buffer B (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, and 2 mM PMSF (PH 7.9)), holding them on ice for 0.5-2 h.They were vortexed every 10 min.The lysates were centrifuged at 12,000 rpm for 15 min to save supernatants containing the nuclear fraction.Both cytosolic and nuclear fractions were subjected to western blotting. ",
"section_name": "Subcellular Fractionation",
"section_num": "2.6."
},
{
"section_content": "For histology, eyes from wild-type, Jnk1 KO, and Jnk2 KO mice were enucleated, fixed in buffered mixed aldehydes (3% paraformaldehyde and 2% glutaraldehyde in PBS, pH 7.4), and embedded in paraffin.Sections of 5 µm were stained with H & E. For immunohistochemistry, eyes from wild-type, Jnk1 KO, and Jnk2 KO mice were enucleated, fixed in buffered 4% PFA (4% paraformaldehyde, in PBS, pH 7.4), and embedded in paraffin.Eyes were cut into 5-µm sections.After dewaxing and rehydration, the sections were soaked in sodium citrate buffer for heat-induced epitope retrieval and incubated with 10% goat serum for 1 h to block the nonspecific binding sites.Then, sections were incubated with anti-S-opsin antibody (ab229786, Abcam, 1:200), anti-M-opsin antibody (NB110-74730, Novus, 1:400), and anti-Rhodopsin antibody (NB120-3267, Novus, 1:300) overnight at 4 • C, followed by incubation with HRP (Horseradish Peroxidase) secondary antibodies for 1 h.The sections were developed by using a diaminobenzidine substrate kit (TIANGEN) and counterstained with hematoxylin.Images were obtained with an Olympus BX41 microscope. ",
"section_name": "Histology and Immunohistochemistry",
"section_num": "2.7."
},
{
"section_content": "Here, 661W cells were plated on coverslips in 2-cm dishes: 24 h later, cells were treated with or without light for 1 h.Coverslips with the cells were washed once with PBS and fixed in 3.7% formaldehyde in PBS for 15 min.After permeabilization with Triton X-100 (0.25%) in PBS for 15 min, cells were blocked with PBS containing BSA (5%) for 1 h and then incubated with primary antibodies overnight at 4 • C.After three separate washes, cells were incubated with secondary antibody for 1 h and then stained with DAPI for 2 min.The coverslips were washed extensively and fixed on slides.Eyes from wild-type, Jnk1 KO, and Jnk2 KO mice were enucleated, fixed in buffered mixed aldehydes (3% paraformaldehyde and 2% glutaraldehyde, in PBS, pH 7.4), and embedded in paraffin.For immunofluorescence, eyes were cut into 5-µm sections.After dewaxing and rehydration, the sections were soaked in sodium citrate buffer for heat-induced epitope retrieval and incubated with Cells 2019, 8, 970 5 of 19 10% goat serum for 1 h to block the nonspecific binding sites.Then, sections were incubated with anti-S-opsin antibody (ab229786, Abcam, 1:200), anti-M-opsin antibody (NB110-74730, Novus, 1:200), and anti-Rhodopsin antibody (NB120-3267, Novus, 1:200) overnight at 4 • C, followed by incubation with secondary antibody for 1 h and then staining with DAPI for 2 min.Images were captured using a Nikon Inverted Microscope Ts2/Ts2R (Nikon ECLIPSE Ts2/Ts2R). ",
"section_name": "Immunofluorescence",
"section_num": "2.8."
},
{
"section_content": "The chromatin immunoprecipitation (ChIP) assay was performed by essentially following the manufacturer's protocol (SimpleChIP ® Enzymatic Chromatin IP Kit, Agarose Beads, #9002, Cell Signaling).The following antibodies were used: 1 µg anti-c-Jun (sc-74753, Santa Cruz Biotechnology) and 1 µg normal mouse IgG (sc-2025, Santa Cruz Biotechnology).The genomic region contained a strong affinity binding site: 5 -CCCCACCCTTGCCAAAAGTGGGTTGGCGGCAGCTCCAAGCGCC TTAGATCACCGAGTTACCATCGCTAGAGCAGCAACAGAAAGGACTAGGGCTGTGCCTGCAGGC CCCACCCCTGCAGAGACATTGAGCACAGTGGGAAGAACACTGCAGAGGAAGTAATGAAAAAC GTGAGGCCCCTAAGGCCCTCAAGACCCTGTTATTCAATTACCATCCCCAGAGAAAGGGGAAAC CCCAGGGACTGCCCATGACAGGGTGCTCCCGTAACATACAGAGAGTCACATGTGGATTCCAAA CAGTTCATGAGCATCTGTAAAGGCAGGCTGGCTCCCTGCCCTAAGAGGCTCAGGGTATTGACAT GGTGGGTATTAGTTGGCTATGGGTCAGGGGAGGCCCAGCATGAAGAAAATACCAAGGGAAGGC ACTCTTATCAGCAGAAAGAAGGCTTGG-3 .For quantitative PCR assays, ChIP DNA was amplified using sense (5 -CCCCACCCTTGCCAAAAGTGGGTTG-3 ) and antisense (5 -CCAAGCCTTCTTTCTG CTGATAAGAGTGC-3 ) primers.The data were analyzed by the following formula: fold change enrichment = 2 -(Ct IP-Ct IgG) . ",
"section_name": "Chromatin Immunoprecipitation Assay",
"section_num": "2.9."
},
{
"section_content": "The full-length pr was amplified from mouse retinal genome DNA by standard PCR with sense primer 5 -AAAAAGCTTGGTTCGGAAGAAAGACGACT-3 and antisense primer 5 -AAACTCGAGGGGGTGGGAACGGTTTTCA-3 .The product was cloned into the luciferase reporter vector, pGL6-TA (Beyotime Biotechnology).The full-length pr, which contained 449 bp, was divided into 4 fragments (A, B, C, D): ∆A-pr was truncated of its A fragment, ∆B-pr was truncated of its B fragment, ∆C-pr was truncated of its C fragment, and ∆D-pr was truncated of its D fragment. ",
"section_name": "Plasmid Constructs",
"section_num": "2.10."
},
{
"section_content": "HEK293 cells were transfected with pGL6-TA reporter constructs and pSV-RL Renilla internal control plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.After 48 h, cells were treated with 100 ng/mL TPA for 30 min or 20 µmol/mL SP600125 for 2 h, and then cells were treated with 100 ng/mL TPA for 30 min, with DMSO treated as the control.Cells were lysed and luciferase activity was assayed with a dual-luciferase system by normalization to Renilla activity (Promega, Madison, WI, USA). ",
"section_name": "Transfection and Luciferase Assay",
"section_num": "2.11."
},
{
"section_content": "P30 retinas from wild-type and Jnk1 KO mice were dissected and incubated for 1 h at room temperature in dissociation solution (0.6% papain and 0.1% DNaseI in PBS), and then digested cell suspension was washed with PBS solution and filtered with a 45-µm cell strainer to a single-cell suspension.The cells were resuspended to approximately 1-5 × 10 6 cells/mL in ice cold PBS, which contained 5% FCS (fetal calf serum) and 1% sodium azide.Then, the primary antibodies, such as anti-S-opsin antibody (ab229786, Abcam, 1:200), anti-M-opsin antibody (NB110-74730, Novus, 1:200), and anti-Rhodopsin antibody (NB120-3267, Novus 1:200), were added and incubated for 30 min on ice in the dark.The cells were washed 3 times by centrifugation at 300× g for 5 min and were resuspended in ice cold PBS containing 5% BSA and 1% sodium azide.The fluorochrome-labeled secondary antibody Alexa Fluor ® R647 (FcMACS, 1:400) was added and incubated for 30 min on ice in the dark.Then the cells were washed 3 times by centrifugation at 300× g for 5 min and were resuspended again in ice cold PBS, which contained 5% BSA and 1% sodium azide.Cells were analyzed with a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany).Flow cytometry analysis was done with FlowJo software (7.2, Becton, Dickinson & Company, Franklin Lakes, NJ, USA). ",
"section_name": "Cell Isolation and Flow Cytometry",
"section_num": "2.12."
},
{
"section_content": "ERG responses were recorded in two groups, including 4 Jnk1 KO and 4 control 1-month-old littermates and 4 Jnk1 KO and 4 control 3-month-old littermates.All animals were recorded under the same settings and conditions.Mice were dark-adapted overnight before an ERG was performed.Under weak red light, mice were anesthetized with an intramuscular injection of ketamine (100 µg/g body weight).Pupils were dilated with tropicamide phenylephrine eye drops, and corneas were kept moist with a drop of hypromellose.During ERG recording, mice were tested under dark adaptation first.Then, they were exposed to full-field scotopic flashes of 1.3 ms duration, presented by the Espion E2 system and a Color Dome Ganzfeld stimulator (Diagnosys) at different intensities: 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10 cd.s/m 2 .Flash stimuli above 10 cd.s/m 2 were delivered by a Xenon lamp, and those below 10 cd.s/m 2 were delivered by a green (525 nm) LED (Light Emitting Diode).Mice were light-adapted with a saturating background (green, 20 cd.s/m 2 ) for 5 min, and five levels of stimuli were used for the photopic recordings (0.3, 1.0, 3.0, 10, and 30 cd.s/m 2 ).The a-wave amplitude was measured from baseline to the first negative peak, and the b-wave amplitude was measured from the a-wave trough to the next positive peak. ",
"section_name": "Electroretinogram (ERG) Recording",
"section_num": "2.13."
},
{
"section_content": "Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad software, San Diego CA, USA), Image pro plus 6.0 (Media Cybernetics, Rockville, MD, USA), and Microsoft Excel computer programs.The results are expressed as mean ± SEM for experiments conducted at least in triplicate.The Wilcoxon-Mann-Whitney test was used to assess the difference between two groups, and a value of p < 0.05 was considered to be statistically significant. ",
"section_name": "Statistical Analysis",
"section_num": "2.14."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "To explore the effect of JNKs on retinogenesis, hematoxylin and eosin (H & E) staining of wild-type, Jnk1 KO, and Jnk2 KO littermates was performed at P1 (postnatal day), P7, P14, and P30, respectively (Figure 1A-D).We confirmed that JNK1 and JNK2 were indeed depleted in the Jnk1 KO mice and Jnk2 KO mice, respectively (Figure S1A,B).Retinas from Jnk1 KO mice and Jnk2 KO mice exhibited normal cell layer lamination: the outer plexiform layer, inner plexiform layer, and GCL were of similar thickness to the corresponding layers of wild-type retinas and showed no obvious morphological abnormalities.The ONL, which consists of rod and cone photoreceptors, was subtly increased in Jnk1 KO mice.Furthermore, immunofluorescence of wild-type, Jnk1 KO, and Jnk2 KO littermates was carried out at P30 (Figure 1E).We determined the density and cell count of the ONL, INL, and GCL (Figure 1F).Interestingly, both the density and cell count of the ONL from Jnk1 KO mice were significantly higher than those of the wild-type mice and Jnk2 KO mice, whereas no apparent changes were observed in the INL or GCL layers.Retinas from Jnk2 KO mice showed no obvious changes compared to retinas from wild-type mice.These data suggest that JNK1, not JNK2, might regulate the production of photoreceptors. ",
"section_name": "Knocking Out JNK1 Promoted Retinogenesis",
"section_num": "3.1."
},
{
"section_content": "To confirm that JNK1 influenced the production of photoreceptors, immunofluorescence and immunohistochemistry were carried out on P30 wild-type and Jnk1 KO retinas using photoreceptor markers (M-opsin/S-opsin for cone photoreceptor cells and Rhodopsin for rod photoreceptor cells).As expected, the expressions of the three opsins were significantly higher in Jnk1 KO retinas than in wild-type retinas (Figure 2A-F).Western blot and real-time PCR analysis of the three markers substantiated the observations (Figure S2A,B).Consistently, the flow cytometry analysis revealed that rod photoreceptors in Jnk1 KO retinas displayed a 1.4-fold increase compared to wild-type retinas (Figure 2G,H).Notably, there was a 2.7-fold increase of S-opsin-positive cells and a 5-fold increase of M-opsin-positive cells in Jnk1 KO retinas compared to wild-type retinas.These data indicated that Jnk1 modulated the production of photoreceptors. During mouse retinogenesis, retinal ganglion cells (RGCs) are generated first, followed by cone photoreceptors and other cell types.To explore whether JNK1 alters the production of all early-born retinal cells, we used western blot and immunofluorescence to detect retinal ganglion cell marker (Nefl) expression in wild-type and Jnk1 KO retinas at P30 and observed no difference in these retinas (Figure 2I and Figure S2C).Furthermore, real-time PCR experiments were carried out to detect the expression level of horizontal cell markers (onecut2), bipolar cell markers (Chx10 and PKCα), amacrine cell marker (Syntaxin I), Müller cell markers (Sox9 and Vimentin), and ganglion cell markers (RBPMS).We observed no expression difference in these cell-type markers between wild-type and Jnk1 KO retinas (Figure S2D).Taken together, these data indicated that JNK1 specifically regulated the production of photoreceptors. ",
"section_name": "The Number of Photoreceptors Increased in Jnk1 KO Mice",
"section_num": "3.2."
},
{
"section_content": "To evaluate retinal function in vivo, we recorded ERG responses under various stimulus intensities in dark-and light-adapted conditions from 1-month-and 3-month-old wild-type and Jnk1 KO mice.At both ages, under the dark-adapted conditions, the amplitude of the ERG b-waves for Jnk1 KO mice was significantly higher than that for wild-type mice, indicating enhanced rod-mediated activity in Jnk1 KO mice (Figure 3A).As for light-adapted ERG responses, the results were the same, which suggested that cone-mediated activity was also enhanced (Figure 3B).These observations were consistent with the increased number of photoreceptors observed in Jnk1 KO mice. ",
"section_name": "Enhanced ERG Responses in the Jnk1 KO Mice",
"section_num": "3.3."
},
{
"section_content": "Light is indispensable for the vascular development of the eye [30].Photoreceptors are light-sensitive cells [31,32].By taking advantage of previously published RNA-seq (RNA-Sequencing) data (GSE106820) [33], we found that Jra, the orthologous gene of c-Jun in mice, was significantly induced after 3 h of blue light exposure in photoreceptors of both 1-and 6-day post-eclosion flies (Figure S3).We wondered if light could also influence the activation of JNK in photoreceptors in mice. To explore this, we used light to stimulate the photoreceptor cell line 661W (derived from murine retina photoreceptors) or control HEK293 cells and then checked the activation of JNK1.Interestingly, light stimulated the phosphorylation of JNK1 in a time-dependent manner.In addition, light triggered the phosphorylation of Ser-63 and Ser-73 on c-Jun, as expected [34,35].However, the light did not activate JNK1 in the HEK293 cells (Figure 4A).Notably, similar effects were not observed for JNK2 in 661W cells, indicating that light may specifically activate JNK1 (Figure 4A).Phorbol 12-myristate 13-acetate (TPA) has been reported to activate JNKs [36].As expected, TPA also stimulated the activation of JNK1 in 661W cells (Figure 4B).intensities in dark-and light-adapted conditions from 1-month-and 3-month-old wild-type and Jnk1 KO mice.At both ages, under the dark-adapted conditions, the amplitude of the ERG b-waves for Jnk1 KO mice was significantly higher than that for wild-type mice, indicating enhanced rodmediated activity in Jnk1 KO mice (Figure 3A).As for light-adapted ERG responses, the results were the same, which suggested that cone-mediated activity was also enhanced (Figure 3B).These observations were consistent with the increased number of photoreceptors observed in Jnk1 KO mice.Using the JNK-specific inhibitor SP600125 [37], it was observed that this inhibitor blocked the dual phosphorylation of c-Jun induced by light stimulation (Figure 4C).It is known that activated JNK translocates from the cytoplasm to phosphorylate c-Jun in the nucleus [35].Immunofluorescence and western blot analysis revealed that light enhanced the translocation of JNK1 into the nucleus, but did not enhance the translocation of JNK2 (Figure 4D,E).These observations established that light specifically induced the activation of JNK1, but not JNK2, in 661W cells. ",
"section_name": "Light Activated JNK1, but Not JNK2, in 661w Cells",
"section_num": "3.4."
},
{
"section_content": "We went on to explore how JNK1 impaired the photoreceptor opsin expression.Stimulating 661W cells with light significantly reduced the expression of transcription factors essential for photoreceptor fate (Figure 5A), which included Otx2, Crx, Neurod1, Rxrg, and Nrl [7][8][9][10][11][12][13]16].Additionally, we measured Opn1sw (S-opsin) and Opn1mw (M-opsin) mRNA levels in light-stimulated 661W cells (Figure 5B).As expected, a decrease of Opn1sw and Opn1mw mRNA levels was observed.In contrast, reducing the JNK1 activation using the SP600125 inhibitor boosted the expression of the above-mentioned transcription factors, and consequently, the mRNA levels of Opn1sw and Opn1mw also increased (Figure 5C,D).These observations suggest that the JNK1 activation triggered by light suppressed the expression of the transcription factors essential for photoreceptors, which in turn attenuated the expression of the opsins.stimulated the phosphorylation of JNK1 in a time-dependent manner.In addition, light triggered the phosphorylation of Ser-63 and Ser-73 on c-Jun, as expected [34,35].However, the light did not activate JNK1 in the HEK293 cells (Figure 4A).Notably, similar effects were not observed for JNK2 in 661W cells, indicating that light may specifically activate JNK1 (Figure 4A).Phorbol 12-myristate 13-acetate (TPA) has been reported to activate JNKs [36].As expected, TPA also stimulated the activation of JNK1 in 661W cells (Figure 4B). ",
"section_name": "JNK1 Attenuated the Transcription of Genes Important for Photoreceptors",
"section_num": "3.5."
},
{
"section_content": "We checked the possible alterations of the Wnt [38][39][40] and Notch [20][21][22] signaling pathways, which are important for photoreceptor fate.Notably, the mRNA levels and protein abundance of Notch1 were significantly decreased in the Jnk1 KO mice (Figure 6A,B).Consistently, light stimulation promoted the expression of Notch1 in 661W cells in a time-dependent manner (Figure 6C,D).In contrast, JNK-specific inhibitor SP600125 reversed the stimulatory effects on Notch1 expression (Figure 6E). ",
"section_name": "The Suppressive Effect of JNK1 Was Dependent on Notch1",
"section_num": "3.6."
},
{
"section_content": "We checked the possible alterations of the Wnt [38][39][40] and Notch [20][21][22] signaling pathways, which are important for photoreceptor fate.Notably, the mRNA levels and protein abundance of Notch1 were significantly decreased in the Jnk1 KO mice (Figure 6A,B).Consistently, light stimulation promoted the expression of Notch1 in 661W cells in a time-dependent manner (Figure 6C,D).In contrast, JNK-specific inhibitor SP600125 reversed the stimulatory effects on Notch1 expression (Figure 6E).Tangeretin is an inhibitor of Notch1 gene expression [41].Therefore, we used Tangeretin to test whether JNK1 attenuated photoreceptor-related transcription factors by influencing Notch1.It was observed that Tangeretin reversed the increase of the expression of the photoreceptor-related transcription factors, or opsins, when stimulating 661W cells with light (Figure 6F,G), indicating that JNK1 functioned upstream of Notch1. ",
"section_name": "The Suppressive Effect of JNK1 Was Dependent on Notch1",
"section_num": "3.6."
},
{
"section_content": "We speculated that JNK1 regulated Notch1 expression by activating c-Jun.To explore this possibility, we first tested whether c-Jun could directly modulate Notch1 transcription.Based on previously published chromatin immunoprecipitation (ChIP)-seq data [42][43][44], we focused on pr (the 449-bp fragment, position: chr2:26339064-26339512, mouse genome mm9) of the Notch1 transcription regulatory region, which possibly harbors the binding site for c-Jun (Figure 7A).ChIP assays demonstrated that the binding of c-Jun to the transcription regulatory region of Notch1 was increased in a time-dependent manner upon light stimulation in 661W cells (Figure 7B), which indicated that JNK1 activated c-Jun to bind to the transcription regulatory region of Notch1.Furthermore, we performed a luciferase reporter assay to confirm that c-Jun directly regulated the transcription of Notch1, and a pGL6-TA vector was used to construct a recombinant plasmid carrying the firefly luciferase reporter gene under the control of the Notch1 transcription regulatory region, which was responsive to c-Jun activation (Figure 7C).Compared to the DMSO control, TPA triggered the activation of c-Jun, which markedly increased luciferase activity in HEK293 cells transfected with the recombinant plasmid (Figure 7D).In contrast, the inhibition of JNK1 activity with the JNK-specific inhibitor SP600125 suppressed a TPA-induced increase in luciferase activity (Figure 7E).To pinpoint the binding site of c-Jun in the Notch1 transcription regulatory region, we created a series of Notch1 transcription regulatory region truncations, including ∆A-pr (lack of A fragment), ∆B-pr (lack of B fragment), ∆C-pr (lack of C fragment), and ∆D-pr (lack of D fragment) (Figure 7C).Interestingly, the luciferase reporter assay indicated that the constructs lacking an A domain or D domain remained responsive to a TPA-induced increase in luciferase activity.Notably, the elimination of either the B domain or C domain resulted in a significant decrease in luciferase activity (Figure 7F).In line with this result, all three c-Jun binding sites predicted using TRANSFAC (TRANScription FACtor database) were within the B domain and C domain regions (Figure S4).Collectively, our results revealed that JNK1 regulated Notch1 expression at the transcriptional level via activating c-Jun, which bound to the transcription regulatory region of Notch1. ",
"section_name": "JNK1 Regulated Notch1 Transcription via Activating c-Jun",
"section_num": "3.7."
},
{
"section_content": "JNK signaling is well established as regulating cell proliferation and differentiation.However, its potential function in modulating retinogenesis has remained largely unknown.In this study, we characterized JNK1 to specifically control the production of photoreceptor cells in retinogenesis.Unexpectedly, JNK2 apparently did not play any role in the retinogenesis.Several lines of evidence substantiated our claims.In vivo, JNK1 depletion significantly increased the production of photoreceptor cells and promoted photoreceptor-mediated ERG responses.In vitro, light specifically activated JNK1, but not JNK2.The activated JNK1 further phosphorylated the transcription factor c-Jun, which potentiated Notch1 transcription.The JNK1-c-Jun-Notch1 axis suppressed photoreceptor-related transcriptional factor expression and further impaired photoreceptor opsin expression (Figure 8). Notch1 is known to determine the progenitor multipotency in a developing retina [45][46][47][48][49][50].Notch signaling sustains the progenitor cells in an undifferentiated state.This action is mainly mediated by Notch1 downstream effectors, such as bHLH (Basic Helix-Loop-Helix) transcription factors [18,51,52].The inhibition of Notch1 channeled the progenitors toward differentiating into rod or cone photoreceptor cells [20][21][22].Our study filled a missing gap by demonstrating that light-activated JNK1 regulated Notch1 expression by phosphorylating the transcription factor c-Jun, which in turn bound to the cognate transcription regulatory region on Notch1. Several gain-and loss-of-function studies of the JNK pathway have revealed that JNK3 has functions distinct from JNK1 or JNK2 due to its relative tissue specificity.The functions of JNK1 or JNK2 could be inferred from the differences in the phenotypes of Jnk1 KO and Jnk2 KO mice in terms of their contributions to the cellular regulation of the nervous system.Specifically, the phenotype of Jnk1 KO mice is more dramatic than that of Jnk2 KO mice, with abnormal brain development [53].While our study revealed that JNK1 activated Notch1 to negatively regulate photoreceptor gene expression, a previous study found that JNK1 could be a positive regulator for rhodopsin expression through interacting with and regulating Nrl transcriptional activity [55].While the use of different cell culture models may have caused the result discrepancy, both studies revealed the crucial roles of JNK1 in regulating photoreceptor fate.Since both positive and negative signaling are commonly required for appropriate and smooth developmental transition and organ biogenesis, it is intriguing to speculate that JNK1 may play a dual role to fine-tune opsin expression during retinogenesis.Besides, it is also possible that JNK1 plays an opposite role in different developmental stages of retinogenesis.The role of JNK in retinogenesis and its detailed mechanisms still require further exploration. The gene regulatory network of photoreceptor genesis is a research frontier for biomedical investigations.Aberrations in photoreceptors result in such retinal diseases as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), which lead to visual impairment and blindness.Notch and Wnt signaling pathways have been proposed as potential targets to treat eye diseases, due to their fundamental importance in retinal development [56].In our study, the JNK1-c-Jun-Notch1 axis influenced some transcriptional factors and three opsins, such as Crx and Rhodopsin, which are drug targets of retinitis pigmentosa [57].It is possible that JNK1 becomes a potential target of eye diseases.It would be intriguing to explore the actions of the upstream regulators of JNK1 and integrate the network regulations of retinogenesis in future studies.The characterization of a new regulatory kinase or transcription factor will be instrumental for understanding the fine-tuning of photoreceptor genesis, ultimately providing novel therapeutic strategies to manipulate photoreceptor cells and improve vision accordingly. mice, Figure S3.The expression changes of Jra after 3 h blue light exposure in fly photoreceptor, Figure S4.The predicted c-Jun binding sites in the c-Jun ChIP-seq peak region. ",
"section_name": "Discussion",
"section_num": "4."
}
] |
[
{
"section_content": "Acknowledgment: We thank Dr. Lijian Hui (Shanghai Institute of Biochemistry and Cell Biology, CAS) for sharing Jnk1 and Jnk2 KO mice and Dr. Xin Zhang (the First School of Clinical Medicine, Nanjing Medical University) for sharing the 661W cell line. We thank Lijian Hui (Shanghai Institute of Biochemistry and Cell Biology, CAS) for sharing Jnk1 and Jnk2 KO mice and Xin Zhang (the First School of Clinical Medicine, Nanjing Medical University) for sharing the 661W cell line. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Acknowledgment: We thank Dr. Lijian Hui (Shanghai Institute of Biochemistry and Cell Biology, CAS) for sharing Jnk1 and Jnk2 KO mice and Dr. Xin Zhang (the First School of Clinical Medicine, Nanjing Medical University) for sharing the 661W cell line. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "We thank Lijian Hui (Shanghai Institute of Biochemistry and Cell Biology, CAS) for sharing Jnk1 and Jnk2 KO mice and Xin Zhang (the First School of Clinical Medicine, Nanjing Medical University) for sharing the 661W cell line. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The mice were maintained under specific pathogen-free (SPF) conditions at the Center for New Drug Safety Evaluation and Research, China Pharmaceutical University.Jnk1 KO and Jnk2 KO mice [28,29] were kindly provided by Dr. Lijian Hui.These strains were maintained on a C57BL/6 background.Age-matched C57BL/6 mice were used as a control.All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The protocol was approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University and the Institutional Ethics Committee of China Pharmaceutical University (Approval Number: 2019-08-001).Papain was purchased from Sigma Aldrich (St. Louis, MO, USA).DNase I was purchased from Roche.The following antibodies were used ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Funding: This research was funded by grants from the National Natural Science Foundation of China (31730018, 81672029), the National Key R&D Program of China (2016YFA0501800), the Open Project of the State Key Laboratory of Natural Medicines (SKLNMZZCX201802), the \"Double First-Class\" Project of China Pharmaceutical University (CPU2018GF10), and the Jiangsu Innovative and Entrepreneurial Talents Program. Funding: This research was funded by grants from the National Natural Science Foundation of China (31730018, 81672029), the National Key R&D Program of China (2016YFA0501800), the Open Project of the State Key Laboratory of Natural Medicines (SKLNMZZCX201802), the \"Double First-Class\" Project of China Pharmaceutical University (CPU2018GF10), and the Jiangsu Innovative and Entrepreneurial Talents Program. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1007/s00280-012-2033-5
|
Acadesine for patients with relapsed/refractory chronic lymphocytic leukemia (CLL): a multicenter phase I/II study
|
<jats:title>Abstract</jats:title> <jats:p>Abstract 4625</jats:p> <jats:p>CLL - Therapy, excluding Transplantation</jats:p> <jats:p>Acadesine induces cell death in B-cell chronic lymphocytic leukemia (CLL) cells in a dose-dependent manner. Acadesine enters B-cells where it is phosphorylated to ZMP, which induces apoptosis independently of ATM or p53. It is active in vitro against CLL cells from patients who have not responded to prior treatment with fludarabine and/or chlorambucil.</jats:p> <jats:p>A phase I/II open-labeled clinical study was designed to determine the safety and tolerability of acadesine given intravenously as a 4-hour infusion to patients with CLL. Part I is the dose escalation part of the study where patients receive a single dose of acadesine on Day 1 and are followed up to Day 22. In Part II, patients will receive up to 5 doses of acadesine at the maximum tolerated dose (MTD) identified in Part I over a period of up to 20 days. Patient population includes CLL patients with relapsed/refractory disease who have received one or more prior lines of treatment including either a fludarabine or an alkylator-based regimen. A patient is defined as having refractory disease if they fail to achieve less than a partial response (PR) according to the NCI working group guidelines, or relapse within the first 6 months after treatment after achieving at least partial response.</jats:p> <jats:p>Primary endpoints of the study evaluate the safety and tolerability of acadesine. Secondary endpoints evaluate the pharmacokinetics of acadesine and ZMP, and B and T-cell counts in peripheral blood as efficacy biomarkers.</jats:p> <jats:p>Twenty-one patients have been included to date, eighteen in Part I at doses of 50, 83.5, 139.5, 210 or 315 mg/kg, and three in Part II, with two doses at 210 mg/kg at days 1 and 4. Pharmacokinetic data showed acadesine is rapidly converted into ZMP inside blood cells.</jats:p> <jats:p>In part I, at single acadesine dose, the Cmax levels for ZMP in whole blood obtained at 315 mg/kg were similar to the ones obtained at the previous dose (210 mg/kg), suggesting that the saturation plateau was reached, which was confirmed by the PK modeling. In 5 patients treated with acadesine at 210 mg/kg and 315 mg/kg a decrease in absolute B cell count was observed, ranging from 6% to 54% with respect to the B cell count prior to acadesine administration. Reversible asymptomatic hyperuricaemia was observed in four patients in cohorts 1 to 3, probably due the metabolism of acadesine to ZMP and uric acid. Prophylactic allopurinol was used in cohorts 4 and 5 and it has significantly reduced the incidence of hyperuricaemia.</jats:p> <jats:p>Acadesine 315 mg/kg was the dose limiting toxicity (DLT) dose with 2 of 3 patients having DLTs-Tumour Lysis Syndrome (TLS) and clinically significant acute renal failure (CTCAE V3.0 Grade 3-chronic dialysis not indicated).</jats:p> <jats:p>We started Part II of the study, with two consecutive doses at 210 mg/kg (Optimal Biological Dose). Three patients have been included to date. No DLT nor grade 3 or 4 Adverse Events related to acadesine were observed, and in all of treated patients a decrease in absolute B cell count was observed ranging from 6% to 35% with respect to the B cell count prior to acadesine administration.</jats:p> <jats:p>In the following cohort, we will administer 5 consecutive doses of acadesine at 210 mg/kg, at days 1, 4, 8, 11 and 15.</jats:p> <jats:p>In conclusion, a MTD was found at one single acadesine dose. Two consecutive doses have already been tested without safety concerns and 5 consecutive doses are currently planned in part II of this ongoing study. Results for this cohort and additional safety, pharmacokinetics and efficacy data will be presented at the meeting.</jats:p> <jats:sec> <jats:title>Disclosures:</jats:title> <jats:p>Saunders: Advancell: Consultancy. de Frias:Advancell: Employment. Campàs:Advancell: Employment.</jats:p> </jats:sec>
|
[
{
"section_content": "B-cell chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in the Western world, affecting mainly the elderly [1].CLL follows an extremely variable clinical course, with overall survival times ranging from months to decades.The symptoms and signs of the disease arise from a clonal excess of B cells caused mainly by defects that prevent programmed cell death (apoptosis) [2]. Available treatments for CLL generally induce remission, although nearly all patients relapse and CLL remains an incurable disease [1,3].Fludarabine-based regimens have significantly improved the efficacy of CLL treatment, and recently, a large randomized trial showed that the addition of the anti-CD20 monoclonal antibody rituximab to fludarabine and cyclophosphamide (FC) improved progression-free survival (PFS) and overall survival (OS) in treatment-naive patients (as first-line therapy) and PFS in relapsed patients [4][5][6].However, current chemotherapy is not selective for CLL cells, and attendant T-cell apoptosis leads to adverse reactions, including severe and prolonged immunosuppression.Conventional treatment of CLL is also associated with temporary myelosuppression with a concomitant increased risk of infection, anemia, and bleeding/bruising [7,8].Almost all patients relapse or become resistant to existing therapies, especially those with genomic alterations such as deletion 17p and/or mutations in TP53.Both have been shown to predict for poor response to chemotherapy and to be associated with poor OS [9,10]. Acadesine (Acadra Ò ), 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside or AICA-riboside, is a water soluble nucleoside with a different mechanism of action compared to currently approved nucleoside analogs (e.g., fludarabine) [11].When added to cell cultures or administered to animals or humans, acadesine is phosphorylated to its ribotide, AICAribotide (ZMP) [12,13].ZMP is a natural endogenous intermediate in the de novo purine nucleotide biosynthesis.Acadesine induces apoptosis of CLL cells cultured ex vivo in a dose-dependent manner over the concentration range 50 lM to 1 mM, and with an IC50 of 380 ± 60 lM [14,15].Incorporation of acadesine into the cell and its subsequent phosphorylation to ZMP are necessary to induce apoptosis.Moreover, acadesine is not reliant on the p53 tumor suppressor or ataxia telangiectasia mutated (ATM) proteins to drive apoptosis, and hence, the absence/loss of function of these proteins in patients with CLL should not affect the activity of acadesine.This is an important issue because alterations of either p53 or ATM are related to resistance to chemotherapy in CLL [9,16].Also, no significant differences in acadesine-induced apoptosis have been observed in ex vivo analysis between unmutated and mutated CLL samples and either between ZAP70 positive and ZAP70 negative cases [17].Ex vivo, B cells were found to be much more sensitive to acadesine-induced apoptosis than T cells.Given that fludarabine causes both B-and T-cell apoptosis [7], the latter leading to the common and severe complication of immunosuppression, the B cell targeted mode of action of acadesine could provide a safety benefit over other commonly used cytotoxic chemotherapeutic regimens and a valuable treatment alternative to certain groups of patients with CLL who are refractory to other treatments. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "This phase I/II study was approved by the competent institutional review board and conducted in accordance with the International Conference on Harmonization Good Clinical Practice guidelines (ClinicalTrials.govidentifier: NCT00559624). ",
"section_name": "Patients and methods",
"section_num": null
},
{
"section_content": "A total of 24 patients were enrolled in the study.All of them provided written informed consent, prior to any study related procedure not part of the patient's normal medical care.Eligible patients had a diagnosis of CLL according to National Cancer Institute (NCI) Working Group Criteria [18], with refractory or relapsed disease, Eastern Cooperative Oncology Group (ECOG) Performance Status B2, and a life expectancy of at least 3 months.They had received one or more prior lines of treatment which must have included either a fludarabine-or cladribine-based regimen or an alkylator-based regimen.Refractoriness was defined as any patient who had failed to achieve a complete response (CR), nodular partial response (nPR), or partial response (PR) according to the NCI Working Group Guidelines for CLL [18].Fludarabine refractoriness also included patients who achieved a CR, nPR, or PR of B6 months duration.Other inclusion criteria were adequate renal function, defined by serum creatinine B1.5 9 upper limit of normal (ULN) and a calculated creatinine clearance of C60 mL/min. ",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "This was a Phase I/II, open-label, study to evaluate an escalating dose and number of doses of acadesine in patients with CLL.The primary endpoint of the study was to demonstrate the safety and tolerability of acadesine in CLL patients, and the secondary endpoints were to determine the pharmacokinetics (PK) of acadesine and its metabolite, ZMP, and to determine the optimal biological dose (OBD) of acadesine in patients with CLL.OBD for single-dose administration of acadesine was considered as the dose enabling a plasma concentration in the range of that causing apoptosis in in vitro models determined from the PK and safety data, and was defined as the dose below that at which dose escalation is stopped in Part I of the study and this dose will be the starting dose used in Part II of the study which will assess repeat dosing with acadesine. Patients were enrolled in the study in cohorts of three patients or more, depending upon the occurrence of doselimiting toxicities (DLTs).Day 1 dosing was staggered between all patients in each cohort by a minimum of 48 h.In Part I, patients received a single dose of acadesine on Day 1.In Part II, patients received up to 5 doses of acadesine over a period of up to 15 days starting on Day 1.The starting dose for Part I was 50 mg/kg given as a 4 h (±30 min) intravenous (iv) infusion on Day 1 only.Patients were assessed for safety, PK, and pharmacodynamics (PD) for up to 3 weeks after dosing (to Day 22).Investigators were advised to treat patients with prophylactic allopurinol to prevent hyperuricemia in all patients in Part I from Cohort 2 onward; additionally, a specific dose and duration of treatment of allopurinol was specified for Part II.The decision to escalate to the next dose in a separate cohort of patients was based on the assessment of safety, including any DLTs, PK modeling of exposure to ZMP, and PD response data, where available, by the independent Data Monitoring Board (DMB). Dose escalation in Part I of the study followed a modified Fibonacci dose escalation design, with 100 % dose escalations allowed until a confirmed grade 2 toxicity (as defined by NCI-Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 for all toxicities except anemia and thrombocytopenia where CTCAE version 2.0 adapted for leukemia studies was used) considered related to treatment occurred.Once this occurred, future dose escalations were incremental (67, 50, 40, 33 %, etc.).In Part II, patients were treated with 2 or 5 consecutive doses at the MTD/OBD identified in Part I of the study. ",
"section_name": "Treatment plan",
"section_num": null
},
{
"section_content": "",
"section_name": "Assessments",
"section_num": null
},
{
"section_content": "Incidence, causality, and severity of adverse events (AE) and serious adverse events (SAE), local tolerability, changes in laboratory values (including liver enzymes, blood glucose and uric acid) and vital signs were assessed.They were assessed for their relationship to acadesine and classified for severity according to the CTCAE v3.0 for all events except anemia and thrombocytopenia which were assessed according to CTCAE v2.0 (which uses % changes relative to study baseline/entry). ",
"section_name": "Safety",
"section_num": null
},
{
"section_content": "In Part I of the study, blood samples for PK analysis (for both acadesine and its metabolite ZMP) were taken predose and 0, 30, 60 min, 2, 6, 20, 72, 96, and 168 h, 14 and 21 days post-dose in all cohorts.In Part II, PK samples were also taken at pre-dose and 0 min and 20 h post-dose for any interim doses, and 72, 96, and 168 h and 14 and 21 days after completion of the last dose for each patient.Acadesine and ZMP concentrations were determined by a validated bioanalytical HPLC-MS/MS method, with lower limits of quantification (LLOQ) set at 20 ng/mL and 150 ng/mL for acadesine in human plasma and ZMP in whole blood, respectively.A noncompartmental PK analysis of the acadesine and ZMP concentrations was undertaken using the WinNonlin Ò software, Professional Version 5.3 (Pharsight Corporation, Mountain View, CA). ",
"section_name": "Pharmacokinetics",
"section_num": null
},
{
"section_content": "Although no International Working Group (IWG) disease response assessments were included in the protocol, bidimensional nodal area (sum of right neck, left neck, right axillary, left axillary, right inguinal and left inguinal nodes) and uni-dimensional lymph nodes (liver and spleen length) were measured. ",
"section_name": "Anti-leukemic activity",
"section_num": null
},
{
"section_content": "The PD effect of acadesine in patients with CLL was assessed by B-cell and T-cell counts in peripheral blood.Samples were taken at pre-dose and 20, 72, 168 h, and 21 days post-dose in all cohorts.In Part II, PD samples were also taken at pre-dose and 20 h post-dose for any interim doses, and 72, 168 h and 21 days after completion of the last dose for each patient. ",
"section_name": "Pharmacodynamics",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "From January 2008 to January 2011, 24 CLL patients with refractory or relapsed disease were enrolled onto this phase I/II clinical trial.The pretreatment characteristics are listed in Table 1.Patients were between 56 and 79 years old, all diagnosed with CLL between 4 and 21 years (inclusive) prior to study entry.The majority were males (15 patients, 63 %) and had received between 1 and 13 previous lines of therapy (mean 2.7).All patients had an ECOG score of B2 and had received either a fludarabine-or cladribine-based regimen or an alkylator-based regimen, in accordance with the eligibility criteria.Individual Rai and Binet staging and relapsed/refractory status at screening are displayed in Table 2. ",
"section_name": "Patient characteristics",
"section_num": null
},
{
"section_content": "In Part I of the study, patients received a single dose of acadesine on Day 1 at escalating doses: 50 mg/kg (6 patients), 83.5 mg/kg (3 patients), 139.5 mg/kg (3 patients), 210 mg/kg (3 patients), and 315 mg/kg (3 patients).At the 315 mg/kg dose level, 2 of the 3 patients experienced a DLT.Also, PK data indicate that at 315 mg/kg, a plateau in acadesine conversion to ZMP was reached.Therefore, 210 mg/kg was designated per protocol MTD and OBD for single acadesine administration based on the decision of the DMB.The dose administered in Part II (210 mg/kg) was based on the results of Part I.In view of the possible risk of acadesine-related renal adverse events or laboratory evidence of creatinine elevation, either as part of a clinical tumor lysis syndrome or occurring as isolated renal impairment, acadesine in Part II Cohort 1 was to be administered with a minimum of a 72-h interval between each dose.Thus, 3 patients received 2 doses (on Days 1 and 4) and 3 patients received 5 consecutive doses (on Days 1, 4, 8, 11, and 15). ",
"section_name": "Treatment",
"section_num": null
},
{
"section_content": "Acadesine infusions were well tolerated.No acadesinerelated deaths occurred in the study, and no patients withdrew from the study due to AEs (all causality, either related or not related to the study drug), in either the single or repeated dose part of the study.In Part I, a total of 78 AEs were reported in 15 (83 %) patients, and approximately half of all AEs were reported as drug-related (42 AEs, 54 %.See Table 3).Six SAEs were reported in 2 patients in the 50 mg/kg (3 SAEs) and in 2 patients in the 315 mg/kg (3 SAEs) cohorts.In Part II, a total of 48 AEs were reported in 6 patients, over half of all AEs were reported as drug-related (31 AEs, 65 %.See Table 3), and there were no SAEs.Among all study patients, a single CTCAE grade 4 AE was reported (hyperuricemia) and 9 CTCAE grade 3 events (in 6 patients) were reported. In part I of the study, one patient at 50 mg/kg experienced hyperuricemia (CTCAE grade 4, DLT but not reported as an SAE).As a result, prophylactic allopurinol was administered to patients in the higher dose cohorts, and and diarrhea, reported in 2 patients, of CTCAE grade 1 (see Table 3).At 315 mg/kg, 2 out of 3 patients experienced DLTs: the first patient experienced a tumor lysis syndrome (classed as a SAE, CTCAE grade 3), which resolved within 13 days with rasburicase treatment.The second patient, diagnosed with small lymphocytic lymphoma and a large abdominal lymphomatous mass, reported renal impairment/increased creatinine (both reported as SAEs and CTCAE grade 3), which resolved quickly (returning to grade 1 within 14 days) with conservative treatment (no dialysis required). In the repeat dose phase, no SAEs were observed.There was evidence of transient increases in creatinine after dosing in the 5 9 210 mg/kg repeat dose cohort: one DLT was reported in one patient (increased creatinine; CTCAE grade 2) and an AE was reported in another patient (increased creatinine; CTCAE grade 1), both patients having normal creatinine levels at baseline.One patient treated with 2 doses at 210 mg/kg reported acadesinerelated hypotension (CTCAE grade 2), which resolved spontaneously within 24 h, without treatment and without clinical sequelae.In Part II, the most commonly reported drug-related AEs during repeat dosing were anemia (CTCAE grade 1 or 2; reported in 5 patients, 4 of them with hemoglobin values below the normal range at predose), thrombocytopenia (CTCAE grade 1 or 2, reported by 2 patients, one of them with platelets pre-dose levels below the normal range), and nausea (reported by 3 patients; CTCAE grade 1 or 2). ",
"section_name": "Safety and tolerability results",
"section_num": null
},
{
"section_content": "There was reduction in the size of palpable lymph nodes after single and repeat dosing with acadesine, although it should be noted that formal IWG response criteria [18] assessments were not planned in this phase I/II study.In Part I, 11/18 patients showed evidence of anti-leukemic activity, in terms of decreases in B cells and/or decreases in the size of lymph nodes (decreases[20 % at any time after dosing with respect to pre-dosing values in either B-cell counts or lymph nodes size.See Table 4).In Part II, 5/6 patients showed evidence of anti-leukemic activity according to the same criteria.For example, a patient with Del13q14 and Del17p, treated at 50 mg/kg, experienced a sustained reduction in B-cell counts, which at Day 22 posttreatment was 58 % lower than at baseline.Also, one patient treated with five doses at 210 mg/kg experienced a reduction in B-cell counts up to 65.7 % on Day 8 postbaseline.This patient had lymph nodes involvement and paraneoplastic skin manifestations.The lymphocyte count has continued to drop, and 18 months after acadesine time points in some cohorts (see Table 4).In cohort 1, decrease in B cells at Day 2 was statistically significant using parametric (p = 0.012, Student t test) or nonparametric (p = 0.043, Wilcoxon signed rank test) method.Similarly, when pre-dose B cells were compared to Day 2 results in all 15 patients from the single-dose cohort where both values are available, a significant difference was found by both statistical methods (p = 0.038 and p = 0.027, respectively).This suggests a trend in decrease in B cells and tends to confirm the results from in vitro experiments, where acadesine induced apoptosis selectively in B cells [14]. ",
"section_name": "Pharmacodynamic and anti-leukemic activity results",
"section_num": null
},
{
"section_content": "Results showed that acadesine and ZMP were rapidly distributed in the bloodstream, and acadesine was rapidly converted into ZMP.Maximum acadesine concentrations were observed at the end of the infusions and from then on concentrations started to decrease.The LLOQ for acadesine was reached between 20 h and 15 days.Maximum concentrations of ZMP were found between the end of the infusion and 30 min-1 h later due to the time elapsed between acadesine administration and its transformation into ZMP.Blood ZMP concentrations started decreasing progressively from 1 h on (after the end of the infusion), reaching the LLOQ between 5 and 22 days after administration (Fig. 1).At the OBD (210 mg/kg single dose), maximum concentration (C max ) for acadesine was 38,736 ng/mL and 270,988 ng/mL for ZMP (median of the 3 patients treated in the cohort).The Area under the curve (AUC 0?24 ) was 123,124 ng h/mL for acadesine and 2,066,170 ng h/mL for ZMP (median of 3 patients treated in the cohort).Considering that the sum of the C max molar plasma concentrations of acadesine and ZMP, obtained at the end of the 4-h infusion, would be equivalent to the dose used in an in vitro cell culture, at the OBD dose (210 mg/ kg single dose) target acadesine concentrations were achieved (median acadesine ?ZMP concentration was 0.9 mM, higher than the 0.5 mM concentrations used to induce apoptosis in in vitro cell cultures) [14,15]).Time of maximum drug concentration (T max ), maximum drug concentration (C max ), area under the time-concentration curve from 0 to 24 h (AUC 0?24 ), and half life (t) for all cohorts are summarized in Table 5. The PK analysis of both acadesine and ZMP demonstrates a high degree of inter-subject variability.For both cohorts in Part II, there was no important accumulation of acadesine or ZMP upon multiple dosing. Following single-dose infusion of acadesine, there was a trend toward a plateau in ZMP levels after increasing the acadesine dose from 139.5 to 210 mg/kg.PK data at 315 mg/kg and PK modeling indicate a plateau in acadesine conversion to ZMP.Thus, the OBD determined from the PK and safety data was 210 mg/kg. ",
"section_name": "Pharmacokinetic results",
"section_num": null
},
{
"section_content": "This study was the first administration of acadesine in cancer patients-specifically CLL.A manageable and predictable safety profile was demonstrated for acadesine in patients with CLL at single doses between 50 and 210 mg/kg.The key safety findings with regard to AEs and clinical laboratory results were asymptomatic hyperuricemia, transient renal impairment/increased creatinine, and infusion-related hypotension.Hyperuricemia has been documented in previous studies with acadesine administered to prevent ischemic reperfusion injury, and given that acadesine is metabolized to uric acid [12,13], hyperuricemia was not unexpected in patients with CLL.Prophylactic allopurinol adopted from cohort 2 (Part I) resulted in no further hyperuricemia occurring in the study.Four patients had renal impairment during the study (two received 315 mg/kg and two 5 9 210 mg/kg).All of them recovered with appropriate management and none of them required dialysis.Two independent nephrologists reviewed data from all patients and conclude that the renal dysfunction associated with acadesine treatment exhibited functional characteristics rather than nephrotoxic features, based on the rapid reversibility and full recovery of the renal dysfunction even after several acadesine consecutive doses.They proposed renal function monitoring and prophylaxis measures that will be implemented in the following clinical trials with acadesine.Modest (grade 1 or 2), transient anemia and/or thrombocytopenia, with recovery to baseline levels occurring in most patients within last days of acadesine administration, were reported in 3/18 and 5/6 patients treated in Part I and Part II of the study, respectively.All but one these AEs were drug-related, and none of them were clinically significant.Acadesine-related transient hypotension occurred in 3 patients in this trial.The hypotension typically occurred after acadesine infusion and lasted several hours with no clinically significant sequellae.Recovery occurred either spontaneously or with conservative (iv fluid administration) management.Of note, there was no evidence of renal toxicity or hypotension in pre-clinical studies or in cardiac studies in more than 2,000 patients who were administered acadesine [19,20].This is the first time that the PK of acadesine has been investigated in patients with CLL.The results indicate a high degree of inter-subject variability.Several possible factors may be contributing to this observation, for example: the underlying disease, associated co-morbidities, and co-medication.The OBD, that is, the dose enabling a plasma concentration in the range of that causing apoptosis in in vitro models determined from the PK and safety data, was 210 mg/kg. In terms of disease activity, there were no conventional IWG responses in this study; such assessments were not prospectively planned as is typical in a phase I trial.However, there was evidence of anti-leukemic activity in 16/24 patients based on a more than 20 % reduction in either B-cell count or size of palpable lymph nodes.In vitro anti-leukemic activity independent of genetic alterations has been previously described with acadesine by Pairet and collaborators [17].Our study size is too small to draw definitive conclusions, but indications of anti-leukemic activity were seen in patients with poor prognosis genetic alterations, suggesting that acadesine might be a potential treatment option in this sub-group. The absence of significant reduction in T-cell counts with acadesine in this study is in agreement with ex vivo data which showed B cells were more sensitive to acadesine-induced apoptosis than T cells [14].This is in contrast with current chemotherapy agents, such as fludarabine, which cause long-lasting T-and NK-cell depression.[7].Although early indications from this study are that acadesine does not induce T-cell apoptosis to the same extent as existing treatments, larger scale studies will be required to confirm this in the wider population. In conclusion, this study has defined the DLT, MTD, and OBD of acadesine and also shows provisional evidence of acadesine anti-leukemic activity, with an acceptable and manageable safety profile.Assessment and monitoring of renal function is essential in patients receiving acadesine.The modest myelosuppressive profile observed with acadesine (if confirmed in larger trials) may make it either an attractive combination partner for standard of care agents used in CLL patients or a candidate for post-induction intervention.The p53-independent mechanism of action of acadesine makes it an attractive compound in patients bearing alterations of this pathway, which represent a Fig. 1 Acadesine mean plasma levels (ng/mL) (a) and ZMP mean whole blood levels (ng/mL) (b) are represented at 4 h pre-dosing (-4) and 0, 0.5, 1, 2, 6, and 20 h post-acadesine administration.For 50 mg/kg, n = 6, for 83.5 mg/kg, 139.5 ng/kg, and 315 ng/kg n = 3, and for the optimal biological dose 210 mg/kg, data from 9 patients is shown (3 patients at 210 mg/kg single dose, 3 patients at 2 9 210 mg/kg and 3 patients at 5 9 210 mg/kg).For the patients that received several acadesine administrations, only data from the first dose have been used to calculate the mean acadesine and ZMP levels ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "treatment, lymphocyte count is below 5,000/lL, and there are no signs of disease in lymph nodes or skin. Although we only had data from 3 patients per cohort (6 in cohort 1), we compared the B-and T-cell mean values at every post-treatment time with values at pre-treatment.Changes in T-cell counts were inconsistent across the cohorts and no overall significant trend was apparent.For B cells, statistically significant decreases occurred at some Tumor lysis syndrome 1 (1) significant group in advanced patients, and for whom few therapeutic options are available.Preliminary data also show that acadesine has in vitro toxicity toward other B-cell malignancies, such as mantle cell lymphoma (especially when combined with monoclonal antibodies [21]) and multiple myeloma [22].Thus, acadesine could be a worth candidate to be developed in other aggressive leukemic indications in the near future.Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. ",
"section_name": "",
"section_num": ""
}
] |
10.1172/jci170169
|
Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies
|
The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques - BH3 profiling and high-throughput kinase activity mapping - we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.
|
[
{
"section_content": "The incorporation of the B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax into the treatment paradigm for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) has revolutionized the treatment of these diseases (1,2).However, this therapy is not thought to be curative, and with longer-term follow-up, resistance has been increasingly recognized as a major challenge.Furthermore, common lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax (3).Thus, there is an urgent need to identify potential resistance mechanisms and alternative therapeutic approaches to overcome them.While acquired venetoclax resistance is explained in part by genetic mechanisms, such as the BCL2 p.G101V that reduces binding of venetoclax to BCL-2, these mutations are only observed in about half of the patients with CLL on long-term venetoclax therapy, and when these mutations do occur, they are often found at a low variant allele frequency (VAF) (4)(5)(6).Thus, the venetoclax resistance mechanism(s) in 2 of the most common lymphoid malignancies (CLL and DLBCL) remain incompletely understood, particularly with regard to functional (i.e., nongenetic) resistance mechanisms. The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time.Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax.Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases.Using 2 complementary functional precision medicine techniques -BH3 profiling and high-throughput kinase activity mapping -we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL.Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins.Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression. Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies phosphorylation levels and resistance to venetoclax.We observed a selective increase in T163pMCL-1 (antiapoptotic activation) and S112pBAD (proapoptotic inhibition) in intrinsically resistant Su-DHL4, TOLEDO, and TMD8 cells, and a further increase in T163pMCL-1, S70pBCL-2 (antiapoptotic activation), and S112pBAD in the acquired-resistance Su-DHL4-R and OCI-Ly1-R lines.There was, however, no consistent increase in S184pBAX (proapoptotic inhibition) (Figure 1B).These findings suggest that the intrinsically higher T163pMCL-1 and S112pBAD levels in Su-DHL4, TOLEDO, and TMD8 cells may have contributed to intrinsic venetoclax resistance, whereas a further increase in T163pMCL-1, S70pBCL-2, and S112pBAD levels was required for acquired venetoclax resistance following continuous exposure to venetoclax.We also observed an increase in MCL-1 protein levels across all intrinsic and acquiredresistance lines (Figure 1B), supporting previous observations of MCL-1 stabilization by T163pMCL-1 (13)(14)(15).No obvious changes in protein expression of PP2A subunits (B56/PP2A catalytic subunits) were observed in these cell lines (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI170169DS1). We next utilized high-throughput kinase activity mapping (HT-KAM), a functional assay that detects the phosphorylation of 11 mer peptides corresponding to their protein phosphorylation sites by kinases from cell lysates and confirmed the increase in MCL-1, BCL-2, and BAD phosphorylation in both the intrinsically and acquired-resistance Su-DHL4 and OCI-Ly1-R, in comparison with OCI-Ly1-S (Supplemental Figure 1B).Importantly, we also observed significant increases in S70pBCL-2, T163p MCL-1, MCL-1, and S112pBAD levels, but not S184pBAX levels, in primary CLL patient samples at the time of progression while on venetoclax compared with their pre-venetoclax samples (Figure 1C and Supplemental Figure 1, C andD).These findings from primary CLL samples from patients treated with venetoclax support our hypothesis that BCL-2 family phosphorylation contributes to venetoclax resistance in lymphoid malignancies. Sensitivity to BH3 mimetics, such as venetoclax, is directly related to the specific survival dependence of cancer cells on antiapoptotic BCL-2 family proteins.To evaluate antiapoptotic dependencies, we used the functional BH3 profiling technique, which relies on the selective binding of BH3-only peptides or BH3 mimetics to antiapoptotic proteins to induce cytochrome c (Cytc) release, thereby identifying the antiapoptotic protein(s) the cell functionally depends on for survival (Figure 2A) (31).Indeed, we observed higher MCL-1 dependence (MS-1 peptides) and lower BCL-2 dependence (BAD peptides and ABT199) across the intrinsically resistant lymphoid lines in comparison with OCI-Ly1-S (Figure 2B).In particular, we detected a clear switch of dependence from BCL-2 to MCL-1 in OCI-Ly1-R and Su-DHL4-R cells compared with their parental lines (Figure 2B and Supplemental Figure 1E).The delta (Δ) changes in Cytc loss between these resistant and sensitive cell lines further indicated a substantial increase in MCL-1 dependence and a concomitant decrease in BCL-2 dependence (Figure 2, C and D).These findings suggest that the cells were intrinsically resistant to venetoclax at least in part because of dependence on antiapoptotic protein(s) other than BCL-2, or may have acquired venetoclax resistance by switching dependence from BCL-2 to a different antiapoptotic protein. Prior studies have suggested that posttranslational modification(s) of the BCL-2 family proteins could alter their functions in governing the intrinsic apoptotic pathway.In particular, phosphorylation of antiapoptotic BCL-2 at serine 70 (S70pBCL-2) has been shown to promote cancer cell survival in response to proapoptotic stimuli and to enhance its sequestration of the proapoptotic effector BAX (7)(8)(9)(10)(11)(12).Phosphorylation of the antiapoptotic protein myeloid leukemia 1 (MCL-1) at threonine 163 (T163pMCL-1) has been shown to increase MCL-1 protein stability, thereby increasing the sequestration of proapoptotic proteins and inducing chemoresistance in lymphoid malignancies (13)(14)(15)(16).In contrast, phosphorylation of BCL-2 agonist of cell death (BAD) at serine 112 (S112pBAD) or BCL-2 associated X, apoptosis regulator (BAX) at serine 184 (S184pBAX) inhibits its proapoptotic function (17)(18)(19)(20).Given their relevance in promoting survival and chemoresistance, we hypothesized that increased S70pBCL-2, T163pMCL-1, S112pBAD, and/or S184pBAX may be present in resistant tumor cells from patients with lymphoid malignancies and, if so, may be an important factor in driving venetoclax resistance. The protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that dephosphorylates its target proteins.Numerous studies have demonstrated a tumor suppressor role of PP2A, and PP2A inactivation contributes to disease progression in several hematologic malignancies (12,(21)(22)(23)(24)(25)(26).Recent work has linked PP2A inactivation to increased S70pBCL-2 and suggested a strong association between S70pBCL-2 and poor prognosis in certain lymphomas (12).Similarly, the stability and function of MCL-1, BAD, and BAX are governed in part by PP2A (18,19,(27)(28)(29)(30).Thus, we hypothesized that drugs that activate PP2A could reverse venetoclax resistance via dephosphorylation of BCL-2, MCL-1, BAD, and BAX, thereby resensitizing tumor cells to BCL-2 inhibition. Here, we report data comparing venetoclax-sensitive with venetoclax-resistant malignant lymphoid cells, including a panel of cell lines, a lymphoid mouse model, and primary samples from patients with CLL before venetoclax treatment and at the time of progression while on venetoclax.We found that hyperphosphorylated BCL-2 family proteins were a crucial mechanism of intrinsic and acquired venetoclax resistance in lymphoid malignancies and we provide evidence supporting future clinical investigation of a therapeutic strategy utilizing PP2A-activating drugs (PADs) to resensitize resistant cells to venetoclax. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To evaluate the potential contribution of BCL-2 family protein phosphorylation to venetoclax resistance, we examined lymphoid malignant cell lines that were intrinsically sensitive and resistant to venetoclax.We first identified 3 DLBCL cell lines -double-hit Su-DHL4, germinal center B cell TOLEDO, and activated B cell TMD8that were intrinsically resistant to venetoclax in relation to the intrinsically sensitive DLBCL cell line OCI-Ly1-S (Figure 1A).We also used 2 cell lines with acquired venetoclax resistance, generated from their parental OCI-Ly1-S and Su-DHL4 (OCI-Ly1-R, Su-DHL4-R) lines (Figure 1A). Next, we measured the phosphorylation status of the BCL-2 family members to evaluate for an association between their found that cell lines that possessed a high MCL-1 to BCL-2 dependence ratio were likely to be less sensitive to BCL-2 inhibition with venetoclax (Figure 3B), and more sensitive to MCL-1 inhibition with S63845 (Figure 3C).Our findings therefore suggest that sensitivity to venetoclax or S63845 in lymphoid malignancies relied critically on the interplay between BCL-2 and MCL-1 dependence. We also observed that venetoclax-resistant cell lines such as OCI-Ly1-R and Su-DHL4-R became more sensitive to the selective MCL-1 inhibitor S63845, consistent with their greater functional dependence on MCL-1 (Figure 3A).This provides a proof of concept that a change in antiapoptotic dependence may lead to a change in sensitivity to the various BH3 mimetics.Moreover, we Figure 1.Venetoclax-resistant malignant lymphoid cells display hyperphosphorylated BCL-2 family proteins.(A) Cell viability of intrinsically resistant Su-DHL4 (n = 3), TOLEDO (n = 4), and TMD8 (n = 4) cells or acquired-resistance OCI-Ly1-R (n = 3) and Su-DHL4-R (n = 3) cells in comparison with OCI-Ly1-S or Su-DHL4 cells following treatment with increasing concentrations of ABT199/venetoclax (Ven) at 48 hours, measured by CTG assay.(B) Western blots showing increased T163pMCL-1, S70pBCL-2, S112pBAD, and MCL-1 levels in intrinsically resistant and acquired-resistance malignant lymphoid cells.Bands were quantified by ImageJ software (NIH).Normalized expression values derived from T163pMCL-1/β-actin, S70pBCL-2/BCL-2, S112pBAD/BAD, and MCL-1/β-actin are displayed below the targets in this and subsequent specific figures.(C) Western blots showing increased T163pMCL-1 (n = 8), S70pBCL-2 (n = 9), S112pBAD (n = 6), and MCL-1 (n = 9) levels in primary CLL samples from patients on venetoclax with progressive disease (PD) compared with paired pre-venetoclax primary CLL patient samples (human, in vivo).Quantification is displayed in Supplemental Figure 1C.The sample number is different due to sample availability.PT, patient. Su-DHL4-R cells by switching their functional dependence from BCL-2 to MCL-1 (Figure 3H). Targeting BCL-2 family protein phosphorylation restores BCL-2 dependence.Although MCL-1 inhibition effectively kills venetoclax-resistant cell lines that are functionally dependent on MCL-1, direct targeting of MCL-1 in patients has to date proven to be challenging because of cardiotoxicity (32,33).Therefore, alternative approaches are needed to safely and effectively treat lymphoid malignancies that are resistant to venetoclax.On the basis of our initial experiments, we hypothesized that pharmacologically decreasing BCL-2 family protein phosphorylation would resensitize resistant cells to venetoclax.We previously demonstrated that a PAD known as FTY720 (fingolimod), an FDA-approved drug for multiple sclerosis (34), reduces S70pBCL-2 (11).Therefore, we next evaluated whether FTY720 could simultaneously reduce S70p-BCL-2, T163pMCL-1, S112pBAD, and S184pBAX in venetoclaxresistant cells and thereby rewire their dependence on BCL-2. Increasing doses of FTY720 at various time points showed a clear reduction of S70pBCL-2, T163pMCL-1, total MCL-1, and To further evaluate whether these changes in BCL-2 and MCL-1 dependencies were mechanistically related to their protein phosphorylation, such as S70pBCL-2 and T163pMCL-1, we induced transient overexpression of the phosphomimetic (p.S70E) or nonphosphorylatable (p.S70A) mutants of S70p-BCL-2 in OCI-Ly1-S cells (Supplemental Figure 1F).We observed a drop in BCL-2 dependence in cells with the p.S70E mutation but not in those with the p.S70A mutation (Figure 3D).These observations were consistent with the enhanced resistance to venetoclax observed in OCI-Ly1-S cells transfected with p.S70E (Figure 3E).In a reciprocal manner, overexpression of WT MCL-1 further increased MCL-1 dependence in Su-DHL4 cells but not in the nonphosphorylatable mutant of T163pMCL-1 (p.T163A), which destabilized the protein expression of MCL-1 (Figure 3F and Supplemental Figure 1G).These observations were again associated with increased venetoclax resistance for WT MCL-1 but not p.T163A (Figure 3G).Collectively, these findings demonstrate that enforced phosphorylation of BCL-2 family proteins triggered cells to mimic the resistant phenotypes of OCI-Ly1-R or ing the proteasome with MG132 and observed that FTY720 still reduced T163pMCL-1 (Figure 4D).FTY720 thus likely reduced T163pMCL-1 prior to destabilizing MCL-1. Since several kinases are known to phosphorylate BCL-2 family proteins (7), we used the HT-KAM assay to evaluate whether changes in the BCL-2 family protein phosphorylation seen with acquired venetoclax resistance are further coordinated by changes in the kinome enzymatic activity landscape.We specifically chose to assess the activity of kinases and kinase families (MAPKs, PKA/ C/D, CDKs, and AMPK) known to include at least 1 documented member that could affect the phosphorylation and stability of the BCL-2 family (7,(13)(14)(15)(35)(36)(37)(38)(39)(40).We observed a significant change in the kinome enzymatic activity landscape following acquisition of venetoclax resistance in OCI-Ly1-R cells compared with their parental OCI-Ly1-S cells (Figure 4E).This altered kinome enzymatic activity landscape in the OCI-Ly1-R cells mimicked that of the intrinsically venetoclax-resistant Su-DHL4 cells (Figure 4E, R = 0.8633, P < 0.0001), thus suggesting that venetoclax resistance is associated with a distinct pattern of altered kinome activity.Strikingly, we observed a clear reprogramming of the kinome enzymatic activity following FTY720 treatment, reverting the resistant kinome back to a sensitive state (Figure 4E, R = -0.4503,P < 0.0035 and R = -0.5939,P < 0.0001).We also independently verified the activity of some kinases by confirming the inactivation of AKT (reduced Ser473 phosphorylation), downstream activation of GSK3β (reduced Ser9 phosphorylation) and inactivation of ERK (reduced Thr202/Tyr204 phosphorylation) in both the acquired and intrinsic venetoclax-resistant cell lines (Supplemental Figure 2C).To measure the antiapoptotic dependence of the venetoclaxresistant cells following treatment with FTY720, we used dynamic BH3 profiling (DBP), a variation of BH3 profiling that measures the net effect of ex vivo drug treatments on the antiapoptotic dependence of cells (Figure 5A).Treatment with FTY720 was capable S184pBAX in all resistant cell lines (Figure 4, A and B, and Supplemental Figure 2, A andB).Similarly, S112pBAD was decreased in these resistant cell lines (Figure 4C).To rule out the possibility that the reduction in T163pMCL-1 was due to a reduction in total MCL-1 protein levels, we prevented MCL-1 degradation by inhibit- of rewiring dependence from MCL-1 to BCL-2 in all resistant lymphoid cell lines (Figure 5, B and C, and Supplemental Figure 3, A-C), consistently showing a net decrease in MCL-1 dependence and a reciprocal increase in BCL-2 dependence (Figure 5D). To confirm that these changes in dependencies were governed by the reduction of BCL-2 family protein phosphorylation, we induced transient overexpression of the mutants of S70p BCL-2 and T163pMCL-1 in OCI-Ly1-R and Su-DHL4 cells, respectively.Indeed, the p.S70E, but not p.S70A, mutant prevented the increase in BCL-2 dependence following FTY720 treatment, while WT MCL-1, but not the p.T163A mutant, prevented the decrease in MCL-1 dependence following FTY720 treatment (Figure 5, E and F, and Supplemental Figure 3D).These observations were corroborated by the diminished effect of FTY720 in reducing phosphorylation of S70pBCL-2 and T163pMCL-1 in p.S70E-mutant and WT MCL-1-transfected cells, respectively (Supplemental Figure 3, E andF).These findings thus suggest that the rewiring of dependence from MCL-1 to BCL-2 following treatment with FTY720 was directly related to reducing phosphorylation of BCL-2 family proteins.We also noted that FTY720 could still destabilize MCL-1 protein in MCL-1 stabilizes MCL-1, which in turn enhances the sequestration of BIM (9,10,13,16,41).Given that the mechanism of action of venetoclax is to bind to BCL-2 and dissociate BAX, thereby leading to mitochondrial pore formation, Cytc release, and apoptosis, we next examined whether a mechanism underlying functional venetoclax resistance involves an altered interactome of apoptotic proteins.Indeed, we observed less effective dissociation of BAX from BCL-2 in OCI-Ly1-R or Su-DHL4 cells compared with OCI-Ly1-S when these cells were treated with venetoclax (Figure 6, A andB). p.T163A-mutant-transfected cells, even when p.T163A did not have a phosphate group (Supplemental Figure 3F).This was likely due to the inactivation of AKT, which led to the downstream activation of GSK3β (Supplemental Figure 2C) and subsequent phosphorylation of S159pMCL-1 (Supplemental Figure 3G), which tags MCL-1 for proteasomal degradation (Supplemental Figure 3H) (38). Changes in survival dependence are governed by an altered apoptotic protein interactome.It was previously shown that S70pBCL-2 enhances the interaction between BCL-2 and BAX, while T163p- reduction by FTY720, allowed MCL-1 to still sequester BIM (Figure 7B).These findings improve our understanding of the mechanisms underlying the ability of FTY720 to restore the apoptotic interactome through a reduction of BCL-2 family protein phosphorylation, thereby rewiring venetoclax-resistant cells from MCL-1 dependence to BCL-2 dependence. Restoring BCL-2 dependence resensitizes resistant cells to venetoclax.Given that venetoclax-resistant cells were now BCL-2 dependent following FTY720 treatment, we hypothesized that the FTY720 and venetoclax combination would kill the previously resistant cells.We first pretreated resistant cells with FTY720 for 4 hours (to rewire cells toward BCL-2 dependence) and subsequently cotreated them with venetoclax for 24 or 48 hours.Indeed, we observed that both intrinsic and acquired-resistance lymphoid lines were sensitized to venetoclax following FTY720 pretreatment (Figure 8, A andB).In an unbiased manner, synergy score heatmaps were computed using 4 different models, including ZIP, HSA, Loewe, and Bliss, all of which demonstrated that the combination of FTY720 plus venetoclax was synergistic in both intrinsic and acquired resistance lines (Figure 8, C and D, and Supplemental Figure 4, A andB).We further demonstrated that this synergism requires the presence of phosphorylated BCL-2 family proteins, as relatively lower-to-undetectable S70p-BCL-2, T163pMCL-1, and BAX expression in Jurkat cells displayed a more additive cytotoxic effect, as shown by 2 (ZIP and Bliss) synergy models and cell death curves when treated with the drug combination (Figure 9, A-C, and Supplemental Figure 4C).Further supporting this observation, FTY720 did not further enhance venetoclax-induced cell death in OCI-Ly1-S cells (Supplemental Figure 5A), which had lower basal levels of BCL-2 family protein phosphorylation (Figure 1B). To demonstrate that this combination is cytotoxic (rather than cytostatic), we chased this treatment combination for 6 days with a trypan blue exclusion assay using the drug combination doses with the best synergy scores and found that the combination effectively reduced live cell counts of Su-DHL4 and OCI-Ly1-R (Figure 9, D and E), while reciprocally increasing their dead cell counts (Supplemental Figure 5, B andC), thus indicating that this treatment combination was indeed cytotoxic.These observations were further corroborated by an annexin-V/Hoechst assay (Supplemental Figure 5, D andE). To further confirm that the cytotoxic effects of this combination require the reduction of BCL-2 family protein phosphorylation, we demonstrated that p.S70E, but not p.S70A, transfection of OCI-Ly1-R cells and WT MCL-1, but not p.T163A, transfection of Su-DHL4 cells resulted in resistance to the treatment combination, as measured by trypan blue exclusion and CellTiter-Glo (CTG) assays (Figure 9, F and G, and Supplemental Figure 5, F andG).Collectively, our findings indicate that FTY720 reprogrammed the kinome enzymatic landscape, reduced the levels of phosphorylation in BCL-2 family proteins, and rewired the apoptotic interactome and venetoclax-resistant lymphoid cell dependence from MCL-1 to BCL-2, thus resensitizes them to venetoclax-induced killing. Targeting hyperphosphorylated BCL-2 family proteins involves PP2A.Since FTY720 is a PAD (42) and hyperphosphorylated BCL-2 family proteins were all decreased by FTY720, we next sought to assess whether the effects of FTY720 were indeed related to PP2A The stronger interaction between BCL-2 and BAX also corresponded to higher S70pBCL-2 in the acquired-resistance OCI-Ly1-R cells (Figure 6A, input).As MCL-1 protein is more stabilized in OCI-Ly1-R cells, we observed a greater interaction between MCL-1 and all BIM isoforms (Figure 6C).Reciprocally, baseline interaction between BIM and BCL-2 was markedly reduced in OCI-Ly1-R cells as compared with OCI-Ly1-S cells (Figure 6D), therefore corroborating the heightened sequestration of BIM by increased MCL-1 expression (Figure 6C).Additionally, venetoclax could substantially reduce BIM and BCL-2 interaction in OCI-Ly1-S cells but not in OCI-Ly1-R cells, albeit the latter had lower interaction at baseline (DMSO) with potentially negligible functional consequences (Figure 6D).These observations suggest that hyperphosphorylated antiapoptotic proteins in resistant cells possess a higher capability of sequestering and thereby inhibiting proapoptotic proteins.These findings corroborate our baseline BH3-profiling results (Figure 2, B-D), as venetoclax-resistant cells were more sensitive to MS1 peptide and S63845 but not to BAD peptide and venetoclax because of their greater dependence on MCL-1. Importantly, FTY720 was able to modulate this resistant apoptotic interactome by reducing the interaction between BCL-2 and BAX as well as between MCL-1 and BIM, in conjunction with reduced S70pBCL-2, T163pMCL-1, and MCL-1 (Figure 6, A-C).Again, transient overexpression of p.S70E could resist the effects of FTY720 in dissociating BAX from BCL-2 (Figure 7A).Similarly, WT MCL-1 overexpression, which could partially prevent MCL-1 and D, and Supplemental Figure 6F).To further validate these findings, we demonstrated that a different PAD, perphenazine (PPZ) (43), showed a similar effect in resensitizing venetoclaxresistant cells (Supplemental Figure 6G). As a negative control to demonstrate that these effects were PP2A specific and involved FTY720-induced kinome enzymatic reprogramming, we showed that exposure of OCI-Ly1-R or Su-DHL4 cells to a single kinase inhibitor of MEK/ERK1/2 activation.We pretreated Su-DHL4 or OCI-Ly1-R cells with okadaic acid (OA), a PP2A inhibitor, or an siRNA of the PP2A catalytic (siPP2Ac) subunit prior to treatment with FTY720 and venetoclax.Pretreatment with OA or siPP2Ac modestly reduced the cytotoxic effect of FTY720 and venetoclax combination (Figure 10, A andB, and Supplemental Figure 6, A-E).We also observed that OA or siPP2Ac was able to prevent the reduction of S70pBCL-2, T163p-MCL-1, MCL-1, and S184pBAX levels by FTY720 (Figure 10, C altered enzymatic kinome, PP2A, and the use of PADs in resensitizing resistant cells to venetoclax. Targeting phosphorylation of BCL-2 family proteins sensitizes primary cells from treatment-naive CLL patients to venetoclax.To assess whether our cell line findings could be recapitulated in primary CLL cells from treatment-naive patients, we used PBMCs from 28 patients with CLL with various disease characteristics (Supple-(PD98059) or JNK (SP600125) failed to reduce S70pBCL-2, T163pMCL1, and MCL-1, rewire cellular dependence to BCL-2, or resensitize cells to venetoclax to the degree observed with FTY720 (Figure 11, A-E, and Supplemental Figure 7, A andB).These effects were intensified when PD98059 and SP600125 were used simultaneously to inhibit more than 1 kinase (Supplemental Figure 7, C-E).These findings therefore support the involvement of an the clinic.We again confirmed that these effects of FTY720 were related to PP2A, as OA could reverse the cytotoxic effects of the treatment combination and prevent the reduction of S70pBCL-2, T163pMCL-1, and MCL-1 levels (Figure 12, E andF, and Supplemental Figure 10A).Additionally, we observed that the combination of FTY720 and venetoclax enhanced the dissociation of BAX from BCL-2 (Figure 12G and Supplemental Figure 10B).Collectively, these ex vivo results from treatment-naive primary CLL samples were highly consistent with our cell line findings, suggesting that the mechanisms we have elucidated were likely relevant to the biology of CLL in patients. Comparison of paired samples from CLL patients at pretreatment to progression while on/off venetoclax supports the potential utility of PADs in enhancing sensitivity to venetoclax.To further explore this functional mechanism of venetoclax resistance, we studied 13 paired samples from patients with CLL to compare pretreatment baseline with the time of progression while on venetoclax (Fig- 1), treated the cells ex vivo with OA, FTY720, and/ or venetoclax, and performed DBP, Western blotting, cell viability testing, and co-IP (Figure 12A).Similar to what we observed in the in vitro setting, S70pBCL-2, T163pMCL-1, and MCL-1 levels were reduced in CLL patient samples treated ex vivo with FTY720 (Figure 12B and Supplemental Figure 8A).S112pBAD and S184pBAX were omitted because of an insufficient amount of primary samples from patients.FTY720 also increased BCL-2 dependence in these treatment-naive primary CLL cells (Figure 12C), which subsequently increased sensitivity to venetoclax (Figure 12D).This cytotoxic effect was observed at concentrations as low as 1 μM FTY720 (Supplemental Figure 9A), a clinically achievable concentration.Furthermore, combination treatment with FTY720 (1 μM) and venetoclax minimally affected the viability of normal cells (PBMCs, T cells, and B cells) from healthy donors compared with that of primary CLL cells (Supplemental Figure 9, A-D).This further supports the feasibility of exploring this combination in S70pBCL-2, S112pBAD, S184pBAX, and MCL-1 levels (Figure 13C), rewired dependence from MCL-1 to BCL-2 (Figure 13D), and thereby resensitized venetoclax-resistant primary CLL cells to venetoclax (Figure 13E).To explore whether these resistant primary samples would also undergo kinome enzymatic reprogramming following FTY720 treatment, like we observed in the cell lines (Figure 4E), we examined the kinome enzymatic activity of 2 venetoclax progression samples from patients with CLL following ex vivo FTY720 treatment using the HT-KAM assay.Indeed, we observed an altered landscape in these samples from patients with CLL whose disease was progressing on venetoclax, in which many of the suppressed kinases were from the MAPK family that affects the phosphorylation of BCL-2, MCL-1, BAD, and BAX (Figure 13F).We further confirmed the suppression of the activity of several kinases by showing reduced phospho-activation sites of AMPK, AKT, and ERK via Western blotting (Figure 13F). A venetoclax-resistant aggressive B cell lymphoma mouse model further supports the ability of PADs to resensitize venetoclax resis-ure 13A and Supplemental Table 2).Compared with their paired pre-venetoclax samples, the progression samples had increased MCL-1 and reciprocally reduced BCL-2 dependencies (Figure 13B).This finding is consistent with the significant increases in T163pMCL-1, S70pBCL-2, S112pBAD, and MCL-1 observed in progression samples relative to their paired pre-venetoclax samples, as shown earlier (Supplemental Figure 1C and Figure 1C, samples from the same patients).We next treated these paired pre-venetoclax and progression CLL samples with venetoclax ex vivo and observed that the progression samples were indeed more resistant to venetoclax-induced cell death (Supplemental Figure 11A).These observations support the hypothesis that venetoclax resistance in lymphoid malignancies is due at least in part to hyperphosphorylated BCL-2 family proteins, which leads to a switch from BCL-2 dependence to MCL-1 dependence. We next tested whether ex vivo treatment with FTY720 could resensitize these progression samples to venetoclax.Indeed, we found that FTY720 simultaneously reduced T163pMCL-1, cells to venetoclax.The consistency of our results across paired sensitive and resistant cell lines, an in vivo mouse model, and in primary samples from patients with CLL speaks to the robustness of these findings. Our findings build on the results of our prior work that first detected elevated levels of AMPK and MCL-1 in venetoclax-resistant lymphoid malignant cells (44).We believe our new data are particularly noteworthy because AMPK has been demonstrated to positively regulate the phosphorylation and expression of BCL-2 and MCL-1, respectively (36,37,(45)(46)(47).Further supporting the involvement of AMPK are our HT-KAM results showing enhanced AMPK activity (PRKAA1/2) in resistant cells, which was abrogated in the presence of FTY720 (Figure 4E).Additionally, as AKT and ERK also phosphorylate and inactivate BAX (S184pBAX) and BAD (S112pBAD), whereas GSK3β phosphorylates and destabilizes MCL-1 (S159pMCL-1) (17,38,40), our findings from Western blotting and HT-KAM assays demonstrating the inactivation of AKT and ERK as well as activation of GSK3β further corroborate the downstream changes in S184pBAX, S112pBAD, and S159p MCL-1. A detailed analysis of the mechanisms underlying the drastic change in the kinome enzymatic activity landscape for venetoclax resistance is beyond the scope of this work.But given that venetoclax resistance has previously been shown to increase ROS, and because ROS could regulate the enzymatic activity of kinases and S70pBCL-2 levels (11,21,44,48), it is possible that ROS are a key upstream contributor to the drastic change in the kinome enzymatic landscape and BCL-2 family protein phosphorylation, leading to venetoclax resistance. While genomic mechanisms including BCL2 mutations have a critical role in some cases of venetoclax resistance, these mutations were often detected at low VAF in patients with resistance to venetoclax (4,5).Moreover, BCL-2 inhibitors that could overcome such genomic resistance mechanisms have not yet emerged.Hence, it is imperative to minimize the occurrence of such mutations by efficiently and thoroughly eliminating tumor cells in a time-limited venetoclax therapy approach.One such approach being utilized in CLL is time-limited venetoclax plus anti-CD20 monoclonal antibody therapy, which could potentially reduce the risk of acquisition of BCL2 mutations by minimizing the duration of venetoclax exposure (49).This approach therefore provides a rationale for the use of venetoclax plus PADs as another time-limited therapeutic option.Nonetheless, the functional resistance mechanism we have identified here may provide more straightforward therapeutic opportunities for patients with relapsed/refractory disease while on venetoclax due to the \"druggability\" of S70pBCL-2, T163pMCL-1, S112pBAD, and S184pBAX with several promising drug candidates such as PADs.Additionally, it is likely that functional and genomic resistance mechanisms are not mutually exclusive, and our data do not exclude the possibility that BCL2 mutations could exist concomitantly with hyperphosphorylation of BCL-2 family proteins.An important remaining question is whether BCL2 mutations are related in some way to BCL-2 phosphorylation (S70pBCL-2) at the BCL-2 flexible loop, or whether the co-occurrence of S70pBCL-2 and BCL2 mutation(s) may further distort the conformation of BCL-2, leading to a further decrease in venetoclax binding. Our data also suggest that venetoclax-resistant lymphoid cells may be sensitive to MCL-1 inhibition, as the resistant cells in our tance in vivo.We used an acquired venetoclax-resistant OCI-Ly1-R murine model to evaluate whether FTY720 could overcome venetoclax resistance in vivo.A stable, Luc-tagged OCI-Ly1-R cell line was implanted into NOD/SCID/Il2Rγ -/-(NSG) mice via tail-vein injection, and the mice were randomized and subjected to bioluminescence imaging (BLI) for luciferase signals on day 14.Mice without tumors were removed, while the rest were then subjected to specific treatment schedules on day 16 (Figure 14A).BLI on day 23 showed that, whereas single-agent venetoclax or FTY720 failed to induce any appreciable therapeutic effect, the combination of the 2 agents resulted in a significantly lower tumor burden (Figure 14, B andC).The resistance of OCI-Ly1-R murine model to venetoclax was also verified in vivo when compared with the sensitive OCI-Ly1-S murine model (Supplemental Figure 11B).Importantly, mice harboring venetoclax-resistant cells had a significantly longer survival in the combination arm compared with controls, particularly for mice in the venetoclax monotherapy arm (Figure 14D and Supplemental Figure 11, C andD).These in vivo data corroborate our in vitro and ex vivo data and collectively suggest that FTY720 has the potential to overcome venetoclax resistance in patients with lymphoid malignancies. ",
"section_name": "BCL-2 family protein phosphorylation contributes to intrinsic and acquired venetoclax resistance in lymphoid malignancies.",
"section_num": null
},
{
"section_content": "We have uncovered a functional mechanism of venetoclax resistance in lymphoid malignancies driven by hyperphosphorylation of the BCL-2 family proteins BCL-2, MCL-1, BAD, and BAX.We demonstrate that heightened BCL-2 family protein phosphorylation modulated the apoptotic protein interactome and altered tumor cell survival dependence from BCL-2 to MCL-1, thus diminishing the sensitivity of malignant lymphoid cells to venetoclax.Importantly, we demonstrate an approach that addresses this resistance mechanism through the use of PADs to reprogram the resistance-state kinome enzymatic activity and dephosphorylate BCL-2 family proteins, thereby rewiring the apoptotic interactome and survival dependence back to BCL-2 and resensitizing resistant Our data suggest that targeting only 1 kinase, such as ERK1/2 or JNK, to overcome resistance may not be sufficient (Figure 11, A-E and Supplemental Figure 7, A andB).The lower cytotoxic activity of these combinations compared with that achieved with PADs could relate to the various redundant kinases that could sustain the phosphorylation of S70pBCL-2, T163pMCL-1, S112pBAD, and S184pBAX in a compensatory manner (7,(13)(14)(15)(36)(37)(38)(39)(40).Given that multiple individual kinases have recently been reported to affect the efficacy of venetoclax by different research groups, it is likely that venetoclax resistance relies on more than 1 hyperactivated kinase, as we found with the HT-KAM assay and simultaneous inhibition of JNK and ERK (Figure 4E and Supplemental Figure 7, C-E).This suggests that targeting 1 kinase at a time may only serve to inhibit cancer growth transiently, with subsequent tumor progression being related to a variety of other kinases.Inhibiting each of these kinases with individual drugs would be prohibitively challenging in the clinical setting, thus highlighting the therapeutic potential of PADs as a way to target multiple kinases using a single drug as a combination partner for venetoclax.Nonetheless, we do not dismiss the possibility of identifying combinations of kinase inhibitors with tolerable safety profiles, particularly with individual kinase inhibitors that are already in clinical use.HT-KAM will be an efficient way to functionally screen, identify, and evaluate the many possible combinations to determine which kinase inhibitors would be most promising to preclinically and explore further in the clinic in combination with venetoclax.Collectively, our current findings support the further exploration of PADs and venetoclax in prospective clinical trials involving patients with lymphoid malignancies. Finally, our work has the potential to open up new treatment approaches and to identify new therapeutic vulnerabilities for patients with lymphoid malignancies that are resistant to venetoclax.For example, the biological signatures we identified here allow the stratification of patients with lymphoid malignancies into different categories of resistance mechanisms, thus paving the way to more personalized therapeutic strategies (e.g., combi-experiments generally depended on MCL-1 for survival.Dual targeting of both MCL-1 and BCL-2 could, in theory, overcome such a resistance mechanism; however, the development of direct MCL-1 inhibitors in the clinic has been limited by cardiotoxicity.Our findings here suggest that an alternative approach to MCL-1 inhibition that may have a better safety profile is to use PADs.In particular, we found that FTY720, a fingolimod compound that is FDA approved to treat multiple sclerosis, is a promising candidate to be studied in the clinic as a partner for venetoclax.Moreover, venetoclax is also being studied in solid tumors.For example, in breast cancer, in which BCL-2 family protein phosphorylation is implicated in tumor progression and drug resistance (50-52), the resistance mechanism of hyperphosphorylation of BCL-2 family proteins we identified here may provide a shared insight into venetoclax resistance in this disease.The addition of PADs could therefore also be explored in this context. The significant changes we observed in the kinome enzymatic activity of venetoclax-resistant cells represent another important finding from our HT-KAM assay that supports the use of PADs.Statistics.Statistical analyses were performed using GraphPad Prism, version 9 (GraphPad Software).Data are presented as the mean ± SD (or as otherwise specified in the figure legends) of individual values, scatter plots, heatmaps, and bar graphs with triplicates or more for cell lines and single runs for primary samples.Statistical tests included a 2-tailed t test, 1-way ANOVA, 2-way ANOVA, and multiple-comparison tests, with statistical significance set at a P value of less than 0.05.Pearson's correlation coefficient R was computed for correlations between 2 variables. Study approval.Animal studies were performed under a protocol approved by the IACUC of the Dana-Farber Cancer Institute (DFCI).Patient samples were used with written informed consent and a protocol reviewed and approved by the Dana-Farber/Harvard Cancer Center IRB.nation of PAD plus venetoclax or kinase inhibitors plus venetoclax in lymphoid malignancies expressing hyperphosphorylated BCL-2 family protein[s]).Coupled with the functional assays such as BH3-profiling and HT-KAM assays, our biological signatures could further assist in identifying lead compounds or repurposing FDA-approved drugs (e.g., FTY720) via compound/drug library screenings based on their potential to activate PP2A/inhibit kinases, reduce BCL-2 family protein phosphorylation, and rewire survival dependencies toward BCL-2 to facilitate venetoclax-induced cell death. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Additional details on methods and materials can be found in the Supplemental Materials. Primary cells and cell lines.PBMCs were isolated from whole blood obtained from consented patients with CLL using the Ficoll-Paque (GE Healthcare) density gradient centrifugation method.Isolated primary cells were either immediately used for experiments or viably frozen in freezing media (90% FBS, 10% DMSO) for subsequent experiments.The cell lines used in this study were OCI-Ly1, Su-DHL4, TOLEDO, TMD8, Jurkat, and Stroma-NKTert.The acquired-resistance cell lines OCI-Ly1-R and Su-DHL4-R were generated from parental cells following continuous culturing in venetoclax starting from a sublethal dose of 10 nM and then doubling this dose when cells could grow at a rate similar to that of its parental cells, up to 1 μM (44).Acquired venetoclax-resistant cells were previously tested through wholeexome sequencing and confirmed to not have BCL2 mutations that have been reported to affect binding to venetoclax (4,44).All primary cells and cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.Routine mycoplasma testing was performed to ensure the absence of mycoplasma contamination. BH3-profiling technique.BH3 profiling is a functional technique that measures the proximity of cells to apoptosis and the dependence of cells on specific antiapoptotic proteins for survival.Additional details are included in the Supplemental Methods. HT-KAM assay.HT-KAM is a high-throughput functional platform that measures cellular kinase activity (57).Additional details are included in the Supplemental Methods. Cell viability.Three different cell viability assays were used in this study: CTG, trypan blue exclusion, and annexin/Hoechst.The cell density used was approximately 0.75 million cells/mL.Additional details are included in the Supplemental Methods. Animal studies.NOD/SCID/Il2Rγ -/-(NSG) mice were purchased from The Jackson Laboratory.Luc-tagged OCI-Ly1-S or OCI-Ly1-R cells (1 million cells) were injected into the tail vein of 6-to 8-weekold female NSG mice, which were randomized to the treatment arms (n = 8 mice per arm).Treatments were administered until euthanization.BLI was performed for cell engraftment and tumor burden on mice using the Xenogen imager, and analysis was done using Living Image 4.2 software (PerkinElmer).Mice with no cell engraftment or tumor development were removed.Venetoclax was administered at 50 mg/kg/day p.o. (dissolved in 10% ethanol, 60% PHOSAL 50PG, and 30% PEG400), and FTY720 (dissolved in saline) was administered at 4 mg/kg/day i.p. Synergy analysis.Synergy analyses for drug combinations were computed by SynergyFinder (https://synergyfinder.org/#!/) (58). ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "SJFC is a recipient of a Simeon J. Fortin Charitable Foundation Postdoctoral Fellowship from the Charles A. King Trust Postdoctoral Fellowship Program and acknowledges funding support from the Simeon J. Fortin Charitable Foundation with Bank of America Private Bank as co-trustee for this study.MSD is a Scholar in Clinical Research for the Leukemia and Lymphoma Society and acknowledges funding support from the NIH (1R01CA266298-01A1), a DFCI Lymphoma Pilot Grant, the DFCI Medical Oncology Grant Program, and the Pan-Mass Challenge Team FLAMES.MSD and CSM acknowledge funding support from Novartis Drug Discovery and Translational Research Program for this study.JBL acknowledges funding support from the NIH (K12CA090354) and the Conquer Cancer Foundation/Sontag Foundation.We acknowledge Jennifer Smith and Richard Siu and the assistance and support of the ICCB-Longwood screening facility at Harvard Medical School for the generation of the BH3-profiling plates.Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA.Phone: 617.632.6331;Email: [email protected]'s present address is Molecular Diagnostics Laboratory, Department of Hematopathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "Data availability.All supporting data values are provided in the Supplemental Supporting Data Values file.Materials are available from the authors on reasonable request. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "SJFC and MSD conceptualized the study.SJFC, RG, CSM, JPC, and MSD designed the study methodology.SJFC, FZ, OD, RM, JXHL, LH, CER, MCC, JP, RC, YCH, MB, and DPM performed experiments.SJFC, FZ, OD, RM, JXHL, RC, YCH, MB, DPM, YCL, CSM, JPC, and MSD performed data analysis and interpretation.SJFC, LH, CER, MCC, JBI, CJW, SP, BCG, RRF, JNA, CSM, JPC, and MSD provided administrative, technical, material, or funding support.MSD supervised the study.SJFC, JPC, and MSD wrote the original draft of the manuscript.All authors reviewed and edited the manuscript. ",
"section_name": "Author contributions",
"section_num": null
}
] |
10.3390/ijms23126712
|
A Novel Triple-Action Inhibitor Targeting B-Cell Receptor Signaling and BRD4 Demonstrates Preclinical Activity in Chronic Lymphocytic Leukemia
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<jats:p>B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.</jats:p>
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{
"section_content": "Chronic lymphocytic leukemia (CLL) is characterized by the expansion of mature B-cells that accumulate in the blood, bone marrow (BM), and secondary lymphoid organs such as the lymph node (LN) and spleen.Hallmark features of CLL include amplified B-cell receptor (BCR) signaling, defective apoptosis, profound tumor microenvironment (TME) dependency, and immune dysfunction [1,2].Novel therapies targeting BCR signaling (e.g., ibrutinib and idelalisib) and B-cell leukemia/lymphoma-2 (BCL-2) family proteins (e.g., venetoclax) provide significantly improved outcomes for both previously untreated 2 of 17 CLL patients and those with adverse disease (e.g., 17p deletion and/or TP53 mutation or complex karyotype).Unfortunately, patients continue to progress on these agents by developing acquired resistance through multiple mechanisms [3]. Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib, acalabrutinib, zanubrutinib) disrupt effective BCR signaling, which is vital to CLL cell proliferation and supportive TME interactions [4,5].BTK inhibition elicits high response rates and durable remission in CLL patients, including those with 17p deletion [del(17p)], but does not fully eliminate the disease [3,6].Studies have shown that the persistent activation of PI3K-AKT-mTOR, NF-κB, and/or MYC signaling contributes to acquired resistance to ibrutinib and residual disease [7][8][9][10].Relapse on ibrutinib is frequently due to BTK mutations that prevent inhibitor binding and often lead to the development of more aggressive diseases such as relapse/refractory CLL or Richter Transformation (RT) [11,12] from CLL into diffuse large Bcell lymphoma (DLBCL-RT) [13].Other inhibitors targeting phosphatidylinositol-3 kinases (PI3K) or anti-apoptotic pathways through BCL-2 inhibition can be used to further disrupt proliferation/survival signaling and defective apoptosis in CLL [12].However, these therapies often require combination with other agents, including monoclonal antibodies, to elicit durable responses with varied toxicity [3,12].Despite these advances, CLL remains an incurable malignancy, and relapse post-BTK and/or BCL-2 inhibitor therapy is a major clinical challenge in need of novel therapies. Combination strategies targeting different facets of CLL biology used earlier in disease management may result in lower rates of emergence of drug-resistant clones [14,15].Epigenetic dysregulation plays a central role in cancer pathogenesis [16][17][18], making strategies that therapeutically target abnormal epigenetic factors an area of active investigation.For instance, aberrations in epigenetic modifiers (e.g., BRD4, CREBBP) have been identified as early driving events in lymphomagenesis and relapse-associated events including chemoresistance and immune escape [19].Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family of epigenetic reader proteins, binds to acetylated histones to facilitate the recruitment of transcriptional machinery [20].Cancer cells display exceptionally higher BRD4 binding at super-enhancers of genes that are crucial to maintaining cancer cell identity and promoting oncogenic gene transcription [21,22].By doing so, BRD4 regulates the expression of genes that govern cell growth and evasion of apoptosis in cancer (e.g., MYC, BCL6, BCL2, and CDK4/6) [22][23][24][25].Furthermore, histoneindependent roles for BET proteins are relevant in leukemia/lymphoma cell survival, as BRD4 interacts with acetylated RELA [26], which augments NF-κB signaling, a central mediator of both external TME triggers and cell-intrinsic aberrations in CLL [27].Currently available small-molecule BET inhibitors competitively bind to the acetyl-lysine recognition pocket of BET bromodomains, displacing BET proteins from active chromatin, which in turn reduces associated gene expression [28,29]. Recently, BRD4 was reported to be overexpressed in primary CLL cells compared to normal B-cells [30].In that study, BRD4 was enriched at hallmark genes implicated in CLL disease biology and progression such as genes associated with BCR pathways (e.g., BTK, BLK, SYK, PLCG2, and PIK3CG), ZAP70, CXCR4, MIR155, IL4R, IL21R, IKZF1, and TCL1A [30].BET inhibition has demonstrated preclinical activity in various leukemia and lymphoma models [31], including CLL.Novel BET inhibitors (e.g., PLX51107, OTX015, and GS-5829) were reported to reduce CLL cell proliferation, induce cell cycle arrest, and promote cell apoptosis in vitro even in the presence of TME protection [30,32].Moreover, BET inhibition significantly decreased tumor burden and prolonged survival in murine models of aggressive CLL [30].Despite the remarkable preclinical activity of BET inhibitors in hematological malignancies, studies to identify beneficial combination therapy approaches to enhance efficacy have been reported [31].As expected, synergism was observed when combining BET inhibitors (e.g., BAY 1238097, GS-5829, OTX015) with inhibitors of BTK (ibrutinib), SYK (entospletinib), PI3K (copanlisib, idelalisib), or BCL2 (venetoclax) in preclinical models of CLL [32][33][34] and DLBCL-RT [35].Interestingly, the anti-leukemic activity of the BET inhibitor, GS-5829, proved to be synergistic in combination with ibrutinib or idelalisib in primary CLL/nurse-like cell co-cultures reflective of the LN TME in CLL [32].Simultaneous targeting of different components of BCR signaling and BET proteins may permit less opportunity for resistance mechanisms to develop, which could translate into more durable patient responses and disease control. We recently introduced thienopyranone (TP) scaffold-based chemotypes [36] for the combinatorial inhibition of BTK, PI3K-AKT, and BRD4-MYC in a single compound (i.e., SRX3262 and SRX3305) and demonstrate impressive preclinical activity in mantle cell lymphoma (MCL) [37,38].BTK/PI3K/BRD4 triple-inhibitors present a potential solution to the toxicity that often plagues combination therapeutic strategies that are necessary to overcome CLL progression and drug resistance.Importantly, our studies demonstrated that these novel multi-action inhibitors yield potent anti-tumor effects but spare healthy bystander cells and are less toxic to healthy donor B-cells than the combination of single-target drugs required to effectively inhibit the same three targets.In this study, we extend our evaluation of SRX3305 to include preclinical models of CLL and DLBCL, including ibrutinibresistant CLL, further validating the efficacy of a multi-target single-molecule approach to overcome TME-and drug-induced resistance mechanisms in B-cell malignancies. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "The anti-proliferative properties of SRX3305 or SRX3262 (first-generation BTK/PI3K/BRD4 triple-inhibitor) were first evaluated in a panel of B-cell non-Hodgkin lymphoma (B-NHL) cell lines representative of CLL (OSU-CLL, HG-3, MEC-1, and MEC-2) and DLBCL (OCI-LY3 and SU-DHL-6).Key cell line characteristics/genetic aberrations are summarized in Supplementary Table S1 [39][40][41][42][43][44][45][46][47].SRX3305 and SRX3262 significantly inhibited CLL cell proliferation in a dose-dependent manner (Figure 1A-D).We report an average IC 50 of 1.38 µM for SRX3305 and 2.98 µM for SRX3262 across the CLL lines evaluated.SRX3305 appeared to be superior to SRX3262 (on average 2.5-fold more potent, p < 0.05) and hence was used for all further studies.SRX3305 is at least equipotent to common single-target inhibitors (ibrutinib, idelalisib, JQ1, or OTX015) evaluated on this panel of B-NHL cells.Similarly, SRX3305 reduced cell proliferation in DLBCL cells (Supplementary Figure S1) with IC 50 values of ~290 nM in OCI-LY3 cells (activated B-cell DLBCL subtype) and ~920 nM in SU-DHL-6 cells (germinal center DLBCL subtype).We next demonstrate that SRX3305 induces apoptosis in HG-3 and OSU-CLL cells in a dose-dependent manner at 24 h (Figure 1E,F), and a similar trend was witnessed at 48 h (data not shown).This indicates that SRX3305 not only induces a cytostatic effect, but also a cytotoxic effect in CLL cells. ",
"section_name": "SRX3305 Inhibits Proliferation and Induces Apoptosis of Malignant B-Cell Lines",
"section_num": "2.1."
},
{
"section_content": "Two major targets of SRX3305, BTK and PI3K, are vital to BCR survival and prolifer ation signaling [49].The third, BRD4, is critical for the transcriptional regulation of nu merous factors driving CLL pathogenesis, including MYC [23,30].We show that SRX330 effectively inhibits the phosphorylation of BTK and PRAS40 (indicative of PI3K/AKT sig naling) and reduces MYC expression in OSU-CLL, MEC-1 and MEC-2 cells (Figure 2A) Single-target inhibitors (ibrutinib, idelalisib, JQ1) were used as controls. ",
"section_name": "SRX3305 Inhibits Critical BCR Survival Signaling in CLL",
"section_num": "2.2."
},
{
"section_content": "Two major targets of SRX3305, BTK and PI3K, are vital to BCR survival and proliferation signaling [48].The third, BRD4, is critical for the transcriptional regulation of numerous factors driving CLL pathogenesis, including MYC [23,30].We show that SRX3305 effectively inhibits the phosphorylation of BTK and PRAS40 (indicative of PI3K/AKT signaling) and reduces MYC expression in OSU-CLL, MEC-1 and MEC-2 cells (Figure 2A).Single-target inhibitors (ibrutinib, idelalisib, JQ1) were used as controls. Next, we sought to examine the effects of SRX3305 under activation-induced BCR signaling.Following inhibitor treatment for 4 h, OSU-CLL and MEC-1 cells were stimulated with anti-IgM in the last 15 min.In parallel, inhibitor washout experiments were conducted to further evaluate whether inhibitor-induced target modulation could be maintained.CLL cells were subject to inhibitor treatment for 1 h prior to inhibitor washout.Three hours later, the cells were stimulated with anti-IgM (last 15 min) to elicit BCR crosslinking.In these studies, ibrutinib, a covalent irreversible BTK inhibitor, and the noncovalent reversible BTK inhibitor BMS-935177 were used as controls.As expected, continuous inhibitor treatment resulted in decreased phosphorylation of BTK (p-BTK) and PRAS40 (p-PRAS40) and reduced MYC expression in BCR-activated CLL cells.CLL cells treated with SRX3305 or ibrutinib (covalent BTK inhibitor) also retained the inhibition of p-BTK following treatment washout, while cells treated with the reversible inhibitor BMS935177 regained BTK phosphorylation.Notably, SRX3305-treated cells maintained marked inhibition of p-PRAS40 and MYC expression after treatment washout (Figure 2B), whereas these effects were lost in ibrutinib-treated cells.In comparison to OSU-CLL cells, MEC-1 cells that harbor del(17p) (Supplementary Table S1) appeared less sensitive to maintained SRX3305-mediated downregulation of p-PRAS40 in the washout condition.This may be due to reduced sensitivity of del(17p) CLL cells to PI3K/AKT inhibition as previously reported [49].In spite of the modest modulation of p-PRAS40 in the MEC-1 washout conditions, SRX3305 still maintained prominent modulation of other oncogenic factors (p-BTK, MYC), indicating the therapeutic benefit of this triple-inhibition in high risk del(17p) CLL.Next, we sought to examine the effects of SRX3305 under activation-induced BCR signaling.Following inhibitor treatment for 4 h, OSU-CLL and MEC-1 cells were stimulated with anti-IgM in the last 15 min.In parallel, inhibitor washout experiments were conducted to further evaluate whether inhibitor-induced target modulation could be maintained.CLL cells were subject to inhibitor treatment for 1 h prior to inhibitor washout. ",
"section_name": "SRX3305 Inhibits Critical BCR Survival Signaling in CLL",
"section_num": "2.2."
},
{
"section_content": "To mimic conditions within pseudo-proliferation centers where CLL expands [50], we evaluated SRX3305 in primary CLL cells stimulated with CpG oligodeoxynucleotides (CpG ODN), a well-known Toll-like receptor 9 agonist that promotes ex vivo CLL cell proliferation [51].Characteristics of the patients' samples used are summarized in Supplementary Table S2.SRX3305 markedly decreased CpG ODN-induced proliferation of primary CLL cells in a dose-dependent manner (Figure 3A).Immunoblot analysis showed that SRX3305 reversed CpG ODN-mediated proliferation reflected by reduced MYC levels in primary CLL cells (Figure 3B).In parallel, SRX3305 induced the accumulation of P21 (cyclin-dependent kinase inhibitor), indicative of cell cycle arrest [52] (Figure 3B).SRX3305 did not influence the proliferation of patient-derived CLL cells under unstimulated/basal conditions (data not shown).We next investigated SRX3305-induced cytotoxicity on tumor cells derived from murine models of aggressive B-cell leukemia and lymphoma.We utilized malignant B-cells isolated from Eµ-TCL1 [53,54] (CLL model) and Eµ-Myc/TCL1 mice [55,56] (concurrent CLL and B-cell lymphoma model).Primary murine spleen-derived tumor samples (comprising >90% malignant B-cells) were cultured ex vivo under mitogenic stimulation (PMA/ionomycin).SRX3305 treatment induced significant cytotoxicity in Eµ-TCL1 and Eµ-Myc/TCL1-derived malignant cells in a dose-dependent manner (Figure 3C), suggesting promising therapeutic benefits in aggressive CLL and associated lymphomas (e.g., DLBCL-RT).As expected, SRX3305 was more effective that the single-target inhibitors (ibrutinib, idelalisib, JQ1) in primary malignant B-cells. ",
"section_name": "SRX3305 Inhibits Primary Malignant B-Cell Survival and Proliferation",
"section_num": "2.3."
},
{
"section_content": "Given the importance of the TME in CLL pathogenesis and therapy resistance [57, 59,59], we sought to evaluate the efficacy of SRX3305 in the presence of stroma protection.The clinical efficacy of inhibitors targeting BCR signaling is partially attributed to disrupting CLL-cell interactions with its TME [60].As expected, the co-culture of primary CLL cells on BM-derived stromal cells protects them from spontaneous apoptosis [59], resulting in a significant increase in CLL cell viability.Notably, treatment with SRX3305 reduced CLL cell viability in a dose-dependent manner despite stroma protection (Figure 4A).This was comparable to CLL therapeutics, ibrutinib, or idelalisib.Of note, SRX3305 was not toxic to the stromal cells (Supplementary Figure S2), indicating that SRX3305-induced CLL cell cytotoxicity is not a function of reduced stroma viability. CLL cell migration, homing to supportive TME niches, and retention therein have been reported to support leukemic cell survival and disease progression [61,62].We therefore investigated if SRX3305 could inhibit CLL cell migration towards key chemokines secreted by protective stromal cells in the BM (CXCL-12) and secondary lymphoid tissues (CXCL-13) [63][64][65].Using a trans-well assay system, we demonstrate that SRX3305 reduced the migration of MEC-1 cells towards CXCL-12 or CXCL-13 (Figure 4B,C), further indicating that SRX3305 can disrupt CLL cell trafficking to supportive niches.Similar findings were seen in preclinical studies of the highly active BCR-targeting CLL therapeutic ibrutinib [4,66].These findings suggest that SRX3305 can overcome TME-induced survival signaling, an established mediator in resistance to therapy in CLL. duced CLL cell viability in a dose-dependent manner despite stroma protection 4A).This was comparable to CLL therapeutics, ibrutinib, or idelalisib.Of note, S was not toxic to the stromal cells (Supplementary Figure S2), indicating that S induced CLL cell cytotoxicity is not a function of reduced stroma viability.CLL cell migration, homing to supportive TME niches, and retention there been reported to support leukemic cell survival and disease progression [62,63].W fore investigated if SRX3305 could inhibit CLL cell migration towards key che secreted by protective stromal cells in the BM (CXCL-12) and secondary lymphoi (CXCL-13) [64][65][66].Using a trans-well assay system, we demonstrate that SRX duced the migration of MEC-1 cells towards CXCL-12 or CXCL-13 (Figure 4B,C) indicating that SRX3305 can disrupt CLL cell trafficking to supportive niches. ",
"section_name": "SRX3305 Disrupts Stroma Survival Support and Chemokine-Induced Migration in CLL",
"section_num": "2.4."
},
{
"section_content": "We hypothesized that because SRX3305 selectively targets PI3K and BRD4 in addition to BTK, this inhibitor would remain effective against ibrutinib-resistant CLL.To evaluate this in the context of non-genetic acquired drug resistance, we generated an ibrutinibresistant CLL cell line model by prolonged culture of HG-3 cells with increasing amounts of ibrutinib (Supplementary Figure S3).Remarkably, SRX3305 decreased cell proliferation in ibrutinib-resistant HG3 (IR-HG3) cells (Figure 5A), while the anti-proliferative effects of ibrutinib were attenuated in IR-HG3 cells.These data suggest that SRX3305 has the potential to overcome acquired ibrutinib resistance.Immunoblot analysis revealed that SRX3305 consistently decreased MYC expression and p-PRAS40 (PI3K target) and increased P21 levels in IR-HG3 cells, while ibrutinib demonstrated a partial response and failed to reduce p-PRAS40 (Figure 5B).Overall, BTK activation (phosphorylation) is reduced in the IR-HG3 cell line, suggesting that adaptive kinome reprogramming bypasses the effect of ibrutinib and an increased reliance on alternative survival mechanisms such as PI3K/AKT/ERK [7,67].Hence, as expected, we observed minimal decrease in BTK phosphorylation upon treatment with SRX3305 or ibrutinib.creased P21 levels in IR-HG3 cells, while ibrutinib demonstrated a partial response and failed to reduce p-PRAS40 (Figure 5B).Overall, BTK activation (phosphorylation) is reduced in the IR-HG3 cell line, suggesting that adaptive kinome reprogramming bypasses the effect of ibrutinib and an increased reliance on alternative survival mechanisms such as PI3K/AKT/ERK [7,68].Hence, as expected, we observed minimal decrease in BTK phosphorylation upon treatment with SRX3305 or ibrutinib. ",
"section_name": "SRX3305 Is Active in Ibrutinib-Resistant CLL",
"section_num": "2.5."
},
{
"section_content": "",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "Aberrant activities of diverse signaling pathways, including BTK, PI3K-AKT, and MYC-BRD4, contribute to CLL pathogenesis and the persistence of residual disease with treatment.Each of these signaling pathways can be disrupted by the novel single small molecule BTK/PI3K/BRD4 inhibitor, SRX3305.In this study, we evaluated the preclinical efficacy of SRX3305 in CLL.We found that SRX3305 inhibits CLL cell proliferation at significantly lower doses than individual inhibitors of BTK, PI3K, and BRD4.SRX3305 proved to be not only cytostatic but also cytotoxic to CLL cells with marked induction of apoptosis.This response is in part due to irreversible binding of BTK, inhibiting its kinase function and thus significantly impairing BCR signaling.However, malignant Bcells can prioritize other signaling pathways to survive under consistent BTK inhibition.Importantly, SRX3305 also potently and irreversibly impairs phosphorylation of PI3K targets (e.g., PRAS40) and expression of BRD4 targets (e.g., MYC).Remarkably, SRX3305 sustains impressive anti-leukemic properties in ibrutinib-resistant CLL cells.SRX3305 also exhibited potent anti-tumor activity toward primary CLL cells ex vivo.The triple-inhibitor dose-dependently impaired the proliferation of stimulated primary CLL patient-derived B-cells and significantly reduced BTK phosphorylation and MYC expression in CLL cells while inducing P21 expression, indicative of cell cycle arrest.Lastly, SRX3305 significantly inhibited the proliferation of malignant B-cells from Eµ-TCL1 and Eµ-Myc/TCL1 mice ex vivo, suggesting promising therapeutic benefits in aggressive CLL and lymphomas. CLL cells are highly dependent on diverse supportive stimuli produced by surrounding TME cells, including stromal cells in the BM and secondary lymphoid tissue niches [59].Crosstalk between malignant and stromal cells in the TME can occur through direct cell-to-cell contact (via adhesion molecules) or indirectly by soluble factors (e.g., CXCL-12 and CXCL-13) [68].These interactions lead to the reciprocal activation of BCR and NF-κB pathways, gene expression changes (e.g., increase in MYC and anti-apoptotic proteins), and chemotaxis, resulting in sustained growth/proliferation of leukemic cells and resistance therapeutic agents [69].In our study, primary CLL cells co-cultured with stromal cells maintained significantly greater viability ex vivo, and this was markedly reduced in the presence of SRX3305.Bidirectional signaling networks within the TME are integral to CLL disease progression and drug response.For instance, CXCL-12/CXCR4 and CXCL-13/CXCR5 are key chemokine networks that promote CLL cell homing to protective lymphoid tissues [63][64][65].Inhibiting the BCR signaling pathway with ibrutinib [70] or idelalisib [71] is known to impair BCR-and chemokine-mediated cell adhesion and migration in CLL.Using a trans-well system, we showed that treatment with SRX3305 significantly impairs CLL cell migration toward both CXCL-12 and CXCL-13.Preclinical studies of BET inhibitors in CLL [30,32] suggest that their efficacy may be partially attributed to interference with key CLL/TME interactions including those mediated by chemokine/cytokine networks.Interestingly, BRD4 was enriched at key CLL cell-trafficking genes (CCR7, CXCR4) in primary CLL cells [30].Our preclinical findings demonstrate that SRX3305 can overcome TME-mediated survival signals, suggesting its efficacy within TME sanctuaries in CLL. Selective pressure of ibrutinib monotherapy often leads to the development of drug resistance in CLL.Ibrutinib can promote the mutation of cysteine 481 of BTK required for irreversible binding of ibrutinib to BTK.We previously demonstrated that SRX3262 and SRX3305 exhibited cytotoxic effects in malignant B-cells harboring the BTK-C481S mutation [37,38].In addition to mutations in drug-targeting proteins, several non-genetic mechanisms rendering CLL cells resistant to ibrutinib have been described and usually manifest as upregulation of alternative survival signaling pathways, such as the PI3K/AKT/ERK [67].Here, we evaluated SRX3305 in the context of acquired resistance to prolonged ibrutinib treatment.Notably, SRX3305 was almost equipotent in ibrutinib-resistant CLL cells and their parental ibrutinib-sensitive counterpart, whereas ibrutinib was significantly less cytotoxic in ibrutinib-resistant cells.Our ibrutinib-resistant CLL cells downregulate BTK activation, suggesting an increased reliance on alternative survival pathways.The phosphorylation of PI3K target PRAS40 was inhibited in ibrutinib-resistant and parental CLL cells following SRX3305 treatment.Similarly, MYC expression was equally downregulated in ibrutinib-resistant cells with SRX3305 treatment, illustrating the potential for a triple-inhibitor to bypass the reciprocal activation of alternative survival signaling and overcome single-target drug resistance.These results suggest further evaluation of this novel chemotype in patients with CLL where responses to ibrutinib are partial or have relapse/refractory disease. Numerous combination therapy approaches have been pursued for the treatment of CLL to combat emergence of drug resistance [14,15].Preclinical studies have demonstrated that repressing MYC by targeting BET proteins enhances lymphoma cell vulnerability to PI3K inhibitors through the upregulation of several PI3K pathways genes and increased GSK3β phosphorylation, resulting in increased β-catenin protein abundance [72,73], suggesting that combinatory targeting of PI3K with SRX3305 would further promote MYC degradation by allowing GSK3β-dependent MYC phosphorylation [74] and stabilization of the MYC antagonist, MAD1 [75].Our recent preclinical studies with the first-generation BTK/PI3K/BRD4 inhibitor SRX3262 in MCL demonstrated reduced phosphorylation of MYC at Ser62 and Thr58 [37], which are critical mediators of MYC protein stability and degradation in cancer [74].Furthermore, earlier studies have reported BET inhibition to be synergistically lethal with ibrutinib in MCL and CLL models [32,76].PI3K and BTK inhibitors have also been combined to intercept constitutive BCR pathway signaling at multiple points [77].While combination approaches have shown promising preclinical efficacy, the increased risk of toxicity when combining multiple small-molecule-targeted agents remains an evident concern [38,78].Individual drugs used to inhibit BTK, PI3K, and BET proteins each have unique side-effect profiles, and as such, additive side-effects from simultaneous inhibition of these pathways may occur.BTK inhibitors are associated with platelet effects and bleeding, as well as cardiovascular toxicities [79], and PI3K inhibitors carry the risk of inflammatory pneumonitis, liver toxicity, and inflammatory diarrhea [80].BET inhibition is associated with anemia, thrombocytopenia, and neutropenia in advanced malignancies [81,82].Combining multiple agents/drugs presents challenges in tailoring therapeutic regimens based on each drug's unique pharmacokinetic profile (adsorption, distribution, metabolism, and elimination) and additive adverse toxicities [83,84].A multitarget inhibitor such as SRX3305 provides the advantage of multi-axis inhibition with a single compound that may result in a better toxicity profile and reduced pill burden, which is a major issue impacting the adherence of patients to multi-drug therapeutic regimens.Future studies evaluating the pharmacokinetic and pharmacodynamic effects of this novel class of inhibitors in vivo are needed to delineate the associated toxicities and translational potential of such promising TP-scaffold multi-action inhibitors for the treatment of B-cell malignancies. ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "Malignant B-cell lines (MEC-1, MEC-2, HG-3, OCI-LY3) were purchased from DSMZ (Braunschweig, Germany), while SU-DHL-6 cells were purchased from ATCC (Gaithersburg, MD, USA).The OSU-CLL cell line [85] was provided by the Human Genetics Sample Bank of The Ohio State University (OSU; Columbus, OH, USA).The murine 9-15c stromal cell line [86] was obtained from RIKEN (Ibaraki, Japan).To generate ibrutinib-resistant cells, HG-3 cells were treated with progressively increasing ibrutinib concentrations (up to 25 µM) over the course of 3 months, as further detailed in the Supplementary Materials. CLL patient samples (peripheral blood mononuclear cells; PBMCs) were obtained from the Leukemia Tissue Bank (OSU) in accordance with the Declaration of Helsinki following informed consent and under a protocol approved by the Institutional Review Board at OSU.In brief, PBMCs from CLL patients were isolated from whole blood through ficoll density gradient centrifugation.Prior to use in experiments, CLL patient-derived samples were confirmed to contain more than 90% CD5 + /CD19 + cells by flow cytometric analysis on a NovoCyte 2060R flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA). Lymphocytes isolated from Eµ-TCL1 and Eµ-Myc/TCL1 mouse spleens were obtained from the Animal Research Facility of the University of Nebraska Medical Center (UNMC), adhering to institutional animal care guidelines.Spleens were harvested from terminally ill mice (i.e., moribund at humane endpoints), and the percentage of malignant B-cells was confirmed via flow cytometry to exceed 90% CD5 + /CD19 + prior to experimental use.Fluorochrome-conjugated CD19 and CD5 antibodies were ordered from BD Biosciences (Franklin Lakes, NJ, USA). Ibrutinib, idelalisib, JQ1, and OTX015 were purchased from Cayman Chemicals.SRX3262 and SRX3305 were synthesized and provided by SignalRx Pharmaceuticals, Inc. (Cumming, GA, USA). ",
"section_name": "Cell Lines, Primary Samples, and Inhibitors",
"section_num": "4.1."
},
{
"section_content": "Cell lines and primary CLL samples were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (hi-FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (P/S), and 2 µM L-glutamine except for OCI-LY3, which was maintained in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% hi-FBS, P/S, and 55 µM 2-mercaptoethanol.Primary murine splenocytes were cultured in RPMI-1640 supplemented with 10% hi-FBS, P/S, 2 mM L-glutamine, 55 µM 2-mercaptoethanol, 100 µM MEM non-essential amino acid solution, 1 mM sodium pyruvate, and 10 mM HEPES buffer.Hi-FBS was purchased from Avantor ® (Radnor, PA, USA).Basal media and supplements were obtained from Life Technologies (Gaithersburg, MD, USA). ",
"section_name": "Cell Culture",
"section_num": "4.2."
},
{
"section_content": "MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium] assays were used to determine inhibitor-induced cytotoxicity.Briefly, primary cells (~0.7 × 10 6 /well) or cell lines (~25,000/well) were treated with vehicle (DMSO) or increasing inhibitor concentrations for up to 72 h in 96-well plates and then the CellTiter 96 ® AQ ueous assay (Promega, Madison, WI, USA) was performed according to manufacturer's instruction to determine cell proliferation.Absorbance signal from each well was acquired at 490 nm on a Tecan Infinite ® M1000 Pro microplate reader (Männedorf, Switzerland).In addition, cell viability and/or apoptosis was measured by flow cytometry using annexin V/propidium iodide (PI) assay kit from Leinco Technologies (Fenton, MO, USA) as per manufacturer's protocol.Stromal cell co-culture experiments were performed as previously described by plating a 75 cm 2 flask (90% confluent) in 48-well plate 24 h before adding patient-derived CLL cells (1 × 10 7 /mL). ",
"section_name": "Cytotoxicity and Flow Cytometric Studies",
"section_num": "4.3."
},
{
"section_content": "To induce B-cell receptor crosslinking, OSU-CLL and MEC-1 were treated with 10 µg/mL of goat F(ab')2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA) for the final 15 min of treatment.Cells were then harvested and lysed for immunoblot analyses. ",
"section_name": "BCR Pathway Activation",
"section_num": "4.4."
},
{
"section_content": "OSU-CLL and MEC-1 cells were treated with indicated concentrations of inhibitors or vehicle for 1 h, washed three times with PBS, and incubated in complete culture medium for 3 h.For continuous treatment, the inhibitors or vehicle control remained for the entire treatment duration (4 h).In the final 15 min of treatment, cells were treated with anti-IgM to induce BCR crosslinking as described above and lysed for immunoblot analyses. ",
"section_name": "Inhibitor Washout Assay",
"section_num": "4.5."
},
{
"section_content": "Primary CLL cells were cultured with 3.2 µM CpG 2006 oligodeoxynucleotides (Integrated DNA Technologies) to induce proliferation for the duration of treatment.For murine samples, 1× phorbol 12-myristate 13-acetate (PMA)/ionomycin cell stimulation cocktail (eBioscience, San Diego, CA, USA) was added during treatments. ",
"section_name": "Ex Vivo Stimulation of Primary Malignant B-Cells",
"section_num": "4.6."
},
{
"section_content": "Cells were treated with indicated concentrations of inhibitors or vehicle control for 4 h under different stimuli.Thereafter, lysates were prepared, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and probed for select proteins.See Supplementary Methods for details. ",
"section_name": "Immunoblot Analyses",
"section_num": "4.7."
},
{
"section_content": "Following 1 h pre-treatment with DMSO, SRX3305, or ibrutinib, MEC-1 cells were placed onto 5-micron trans-well inserts (Corning, Tewksbury, MA, USA) resting in wells containing 200 ng/mL CXCL-12 or 1000 ng/mL CXCL-13 (PeproTech, Cranbury, NJ, USA).No chemokine control wells were included for each treatment condition.After incubating for 6 h, trans-well inserts were carefully removed, and the number of cells that migrated through the insert towards the chemokines were counted by flow cytometry.Data were analyzed using NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA). ",
"section_name": "Migration Assay",
"section_num": "4.8."
},
{
"section_content": "Differences in cell viability and apoptosis between conditions of interest were assessed using ANOVA models.Dunnett's test was used to compare conditions and dose levels with vehicle control.A test of linear trend for SRX3305 dosing was performed by employing orthogonal polynomial contrast coefficients.Log-transformations of the data were applied, when necessary, before modeling to stabilize variances specifically for data analyzed as raw absorption.Percent proliferation data or percent viable data were analyzed without transformation.Data are represented as mean ± SEM.Analyses were conducted using SAS/STAT software, version 9.4 of the SAS System for Windows (SAS Institute Inc., Cary, NC, USA).p-values < 0.05 were considered statistically significant. ",
"section_name": "Statistical Analyses",
"section_num": "4.9."
},
{
"section_content": "The novel BTK/PI3K/BRD4 inhibitor, SRX3305, demonstrates marked anti-tumor properties in preclinical models of CLL.Importantly, the pro-survival, proliferative, therapyresistant, and activation effects promoted by TME sanctuaries were abrogated by SRX3305, which furthermore blocked CLL cell chemotaxis.Additionally, SRX3305 sustained impressive anti-tumor effects in ibrutinib-resistant CLL cells and effectively attenuated alternative/downstream survival signaling pathways.Together, these findings provide strong rationale for the clinical development of this distinct triple-action inhibitor for treating B-cell lymphoproliferative diseases, especially in the relapse/refractory setting. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23126712/s1. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "We thank SignalRx Pharmaceuticals, Inc. for providing SRX3305 and SRX3262 and Donald L. Durden for discussion.The authors also thank The Ohio State University Leukemia Tissue Bank staff for primary patient sample procurement services that was supported by the National Cancer Institute Grant P30 CA016058.The authors acknowledge Biorender.com,which was used to create the graphical abstract. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This work was supported by institutional funds from the University of Nebraska Medical Center (D.E.-G.). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The structures of SRX3305 or SRX3262 used in this study are proprietary information of SignalRx Pharmaceuticals, Inc. (Patent number WO2020023340A1, 2020) [36] and are not publicly available.All other data will be made available from the authors upon reasonable request. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: Primary PBMCs from CLL patients were obtained from the Leukemia Tissue Bank at The Ohio State University Comprehensive Cancer Center in accordance with the Declaration of Helsinki following informed consent under approved Institutional Review Board protocol (#1997C0194).The samples were de-identified and used for the in vitro experiments outlined in this study.Primary murine tumor samples were obtained through an approved institutional animal care and use committee protocol at UNMC (#1816901FC). Patients provided informed consent for the use of their biologic specimens for research purposes according to institutional guidelines. ",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: Primary PBMCs from CLL patients were obtained from the Leukemia Tissue Bank at The Ohio State University Comprehensive Cancer Center in accordance with the Declaration of Helsinki following informed consent under approved Institutional Review Board protocol (#1997C0194).The samples were de-identified and used for the in vitro experiments outlined in this study.Primary murine tumor samples were obtained through an approved institutional animal care and use committee protocol at UNMC (#1816901FC). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Patients provided informed consent for the use of their biologic specimens for research purposes according to institutional guidelines. ",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "",
"section_name": "Conflicts of",
"section_num": null
}
] |
10.3390/cells10061488
|
Novel Genetic and Molecular Pathways in Pulmonary Arterial Hypertension Associated with Connective Tissue Disease
|
<jats:p>Pulmonary Arterial Hypertension (PAH) is a severe complication of Connective Tissue Disease (CTD), with remarkable morbidity and mortality. However, the molecular and genetic basis of CTD-PAH remains incompletely understood. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH, using a PAH-specific panel of 35 genes. During recruitment, 79 patients were studied, including 59 Systemic Sclerosis patients (SSc) and 69 females. Disease-associated variants were observed in nine patients: 4 pathogenic/likely pathogenic variants in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 Variants of Unknown Significance (VUS) in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). One patient with mixed CTD had a frameshift pathogenic variant in TBX4. Two patients with SSc-PAH carried variants in ABCC8. A patient diagnosed with Systemic Lupus Erythematous (SLE) presented a pathogenic nonsense variant in GDF2/BMP9. Another patient with SSc-PAH presented a pathogenic variant in KCNA5. Four patients with SSc-PAH carried a VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. These findings suggest that genetic factors may contribute to Pulmonary Vascular Disease (PVD) in CTD patients.</jats:p>
|
[
{
"section_content": "Pulmonary Arterial Hypertension (PAH) is a feared complication of Connective Tissue Diseases (CTD), with remarkable morbidity and mortality [1].Systemic sclerosis (SSc) is most commonly associated with PAH, but it can be present in other CTD such as Systemic Lupus Erythematous (SLE) or Mixed CTD (MCTD) [2].CTD-associated PAH (CTD-PAH) is present in up to 12% of patients with SSc and it is one of the leading disease-related Cells 2021, 10, 1488 2 of 10 causes of death [3].Moreover, CTD-PAH represents 15-30% of cases in PAH registries [4,5].Despite major advances in PAH therapy, survival in CTD-PAH remains poor, with a three-year survival of 40-50% [4]. To date, 12 genes have been associated with PAH with a high level of evidence, and 5 have been associated with a low level of evidence [6].Furthermore, high-throughput sequencing (HTS) technologies have led to the identification of novel associated genes [7].The main gene involved in PAH encodes the bone morphogenic protein receptor type 2 (BMPR2), a receptor belonging to the transforming growth factor beta (TGF-β) superfamily [8].Other genes have also been identified: potassium channel genes (KCNK3, KCNA5, ABCC8), T-box transcription factor 4 (TBX4), and other genes in the TGF-β/BMP signaling pathway (BMP9/GDF2, SMAD1, SMAD4, SMAD9, BMPR1B) [6].Previous studies have demonstrated that rare coding mutations are present in ~80% of familial forms and ~20% of sporadic cases [8]. Currently, the molecular and genetic basis of PAH in CTD has not been fully addressed.Previous studies have suggested that genetic factors may play a significant role in the development of Pulmonary Vascular Disease (PVD) in other conditions, such as congenital heart disease [9].However, the role of genetic abnormalities in CTD-associated PVD remains unclear. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Since November 2011, genetic testing has been offered to all patients with idiopathic, hereditable and associated forms of PAH, and Pulmonary Venooclusive Disease (PVOD), included in the Spanish Registry of Pulmonary Arterial Hypertension (REHAP).A full list of REHAP centers and investigators is provided in the Supporting Information (See Table S1). Pulmonary Arterial Hypertension was defined according to the 2015 ERS/ESC Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension [10].Routine diagnostic workup included medical history, physical examination, 6-min walking test (6MWT), echocardiogram, multidetector computed tomography (MDCT), ventilation/perfusion lung scan, pulmonary function tests (PFT), and screening of connective tissue disease, HIV infection and portal hypertension.Right Heart Catheterism (RHC) at diagnosis includes Right Atrium Pressure, Mean Pulmonary Artery Pressure, Pulmonary Wedge Pressure, Cardiac Output, Cardiac Index and Pulmonary Vascular Resistance.Pulmonary vasoreactivity testing was performed in Idiopathic PAH (IPAH), Hereditable PAH (HPAH) and drug-induced PAH.Routine diagnostic workup included medical history, physical examination, 6-min Walking Test (6MWT), echocardiogram, Multidetector Computed Tomography (MDCT), ventilation/perfusion lung scan, pulmonary function tests (PFT), and screening of Connective Tissue Disease, HIV infection and Portal Hypertension.PFT included the diffusing capacity for carbon monoxide (DLCO), which was considered moderately reduced when DLCO 43-62% of predicted values and severely reduced when DLCO < 43% of predicted values [11].Therapeutic management is left to the discretion of individual physicians. All patients or legal tutors included in the analysis gave their written informed consent and the project was approved by the ethical committee for scientific research of the participant centers.We obtained written parental consent from the parents or guardians of minors included in this study. ",
"section_name": "Study Patients",
"section_num": "2.1."
},
{
"section_content": "A PAH-specific HTS panel of 35 genes was designed, including all PAH-associated genes at that date with a variable level of evidence [6].Review, classification and interpretation of variants were carried out according to the American College of Medical Genetics and Genomics guidelines [12].The ethical principles of the European Board of Medical Genetics and the 2015 ERS/ESC guidelines for the diagnosis and treatment of pulmonary Cells 2021, 10, 1488 3 of 10 hypertension offer accurate information on the range of options available to make informed decisions, and allow equal access to genetic counseling and testing [10].Pre-and post-test genetic counseling was provided.In the pre-test visit, family history information was collected, but only probands were studied.When a positive result was observed, a genetic study was offered to first degree relatives where available.Cascade or co-segregation genetic tests were also performed.When an unaffected carrier was identified, a complete diagnostic was performed, including electrocardiogram, echocardiogram, N-terminal probrain natriuretic peptide (NT-proBNP) and 6 Minute Walking Test.This evaluation is periodically repeated.When a sustained suspicion of early-stage PAH was observed, RHC was performed to rule out the condition. ",
"section_name": "Molecular Analysis",
"section_num": "2.2."
},
{
"section_content": "During patients' enrolling, 79 CTD-PAH patients were recruited: 59 SSc, 11 Systemic Lupus Erythematous (SLE) and 9 other CTD (Figure 1).Baseline characteristics are shown in Table 1.Sixty-nine patients were female, mean age was 55.6 ± 1.9 years, mean pulmonary vascular resistance (PVR) was 8.6 ± 0.5 wood units (WU) and mean diffusing capacity of the lung for carbon monoxide (DLCO) was 47.5 ± 2% of predicted value. A PAH-specific HTS panel of 35 genes was designed, including all PAH-associated genes at that date with a variable level of evidence [6].Review, classification and interpretation of variants were carried out according to the American College of Medical Genetics and Genomics guidelines [12].The ethical principles of the European Board of Medical Genetics and the 2015 ERS/ESC guidelines for the diagnosis and treatment of pulmonary hypertension offer accurate information on the range of options available to make informed decisions, and allow equal access to genetic counseling and testing [10].Pre-and post-test genetic counseling was provided.In the pre-test visit, family history information was collected, but only probands were studied.When a positive result was observed, a genetic study was offered to first degree relatives where available.Cascade or co-segregation genetic tests were also performed.When an unaffected carrier was identified, a complete diagnostic was performed, including electrocardiogram, echocardiogram, Nterminal pro-brain natriuretic peptide (NT-proBNP) and 6 Minute Walking Test.This evaluation is periodically repeated.When a sustained suspicion of early-stage PAH was observed, RHC was performed to rule out the condition. ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "During patients' enrolling, 79 CTD-PAH patients were recruited: 59 SSc, 11 Systemic Lupus Erythematous (SLE) and 9 other CTD (Figure 1).Baseline characteristics are shown in Table 1.Sixty-nine patients were female, mean age was 55.6 ± 1.9 years, mean pulmonary vascular resistance (PVR) was 8.6 ± 0.5 wood units (WU) and mean diffusing capacity of the lung for carbon monoxide (DLCO) was 47.5 ± 2% of predicted value.Disease-associated variants were observed in nine patients.Four of them were classified as pathogenic or likely pathogenic in four different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9), and five as variants of unknown significance (VUS) in four genes (ABCC8, NOTCH3, TOPBP1 and CTCFL).Clinical characteristics of patients with pathogenic or likely pathogenic variants and variant analyses are shown in Tables 2 and3, respectively., but a reduction in DLCO was observed (61% of predicted value).Small Patella Syndrome was also ruled out.Up-front oral combination therapy was prescribed.After six years of follow-up, she has a low-risk profile under double oral combination therapy. Two patients carry variants in ABCC8.Patient 2 is a Caucasian female with SSc, diagnosed with PAH at 27 years of age.She carries a splicing variant in ABCC8: (NM_000352.6):c.2694+1G>A, classified as likely pathogenic.Her mother was diagnosed with PAH, associated with a repaired atrial septal defect, at 61 years of age.In the genetic testing, no variants were observed in ABCC8 or other PAH genes.ILD was also ruled out in patient 2. However, a mild reduction in diffusion capacity was observed at diagnosis (DLCO 71% of predicted value).Monotherapy was initiated.During follow-up, goal-oriented PAH therapy was applied, and risk profile was assessed periodically.Eighteen years after diagnosis, she presents a low-risk profile under triple combination therapy, including systemic prostanoids.In this time, diffusion capacity progressively worsened (current DLCO 45% of predicted value), without signs of ILD.Patient 3 is a Caucasian male, with clinical suspicion of PVOD associated with SSc and HIV infection.PAH was diagnosed at 57 years of age.He presented a missense variant in ABCC8 (NM_000352.6):c.298G>Ap.(Glu100Lys), located in a gating regulatory region, and classified as VUS.His sister was also diagnosed with PVOD associated with SSc at 48 years of age.Referred to the lung transplant unit, she died on the waiting list.No blood or tissue samples are available for histological or genetic analysis.In patient 3, DLCO at diagnosis was 22% of predicted value.MDCT showed the typical PVOD triad, consisting of ground grass opacification, interlobular septal thickening or mediastinal lymphadenopathy.He also had respiratory insufficiency when resting and a significant drop in oxygen desaturation during exercise.Although referred to the lung transplant unit, he was not eligible due to advanced age and comorbidities.The clinical course was progressive, and he died 4.5 years after the diagnosis. Patient 4 is a Latin American female with SLE, diagnosed with PAH at 25 years of age.She presented a nonsense variant in GDF2/BMP9: (NM_016204.4):c.642G>A: (p.Trp214*) which causes the appearance of a premature stop codon, classified as pathogenic.Three years after diagnosis, she presented a low-risk profile under dual oral therapy. Patient 5 is a Caucasian female with SSc, diagnosed with PAH at 70 years of age.She presented a pathogenic variant in KCNA5: (NM_002234.3):c.1685delC(p.Phe563fs*21).During follow-up, goal-oriented PAH therapy was applied.She died 8.5 years after diagnosis due to progressive heart failure. Another patient with SSc carries a VUS in NOTCH1.Three patients show variants in novel PAH-related genes.Patients 9 and 10 had a previous diagnosis of SSc and carry a VUS in CTCFL.Patient 11 was diagnosed with SSc-PAH and carries a VUS in TOPBP1. ",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The pathobiology of PAH-CTD remains incompletely understood.On the one hand, it is speculated that this complication may be triggered by immune dysregulation present in CTD [2,13,14].On the other hand, an imbalance in the TGF-β/BMP axis might also contribute to CTD-PAH development [15,16].However, despite progress in our knowledge of CTD-PVD, neither genetic theory nor inflammatory theory have been proven.In hereditable and idiopathic forms of PAH, BMPR2 haploinsufficiency is the most common inherited molecular mechanism [8,17].However, the penetrance of the disease phenotype is incomplete and additional stimuli are necessary [18].Female sex is the single most important factor influencing the development of PAH in mutation carriers [19].Other factors might be genetic (a second variant in another gene), epigenetic or environmental [17,20].Furthermore, inflammatory cells and their mediator also contribute to pulmonary vascular remodeling in idiopathic forms [21].Whether the proinflammatory state in CTD is a trigger in genetically susceptible individuals remains unclear [22][23][24][25].One might speculate that the simultaneous occurrence of genetic and inflammatory factors might explain PAH in CTD [6].Nevertheless, the genetic basis of CTD-PAH has not been well elucidated to date. In our CTD-PAH cohort, four patients (5.1%) carried a pathogenic or likely pathogenic variant in a PAH-related gene.Furthermore, VUS were observed in another five patients (6.3%).Functional assays must be performed in order to confirm or discard the possible role of these variants in protein function, and whether this can be related to the phenotype.Some previous studies failed to observe variants in PAH-related genes throughout this population [26].The most likely explanation for this is that only one or a small number of genes were included.Furthermore, current guidelines for the management of PAH do not recommend genetic testing in associated forms, and most previous studies have excluded them [10].However, a recent study by Zhu et al. studied 722 CTD-PAH patients [7].Rare coding variants were observed in 5.26% of the cohort.However, phenotype and clinical information are not provided.For our study, a PAH-specific HTS panel was designed (35 genes), and associated forms of PAH and PVOD were also included.As a result, we also obtained a significant number of variant carriers. Our study highlights different molecular pathways involved in CTD-PAH.Half of the pathogenic or likely pathogenic variants were located in potassium channel genes (KCNA5, ABCC8).Only one gene was included in the TGF β pathway (GDF2/BMP9) and no variants were observed in BMPR2.Another pathogenic variant was present in TBX, whose mutations express an ever-expanding phenotype.With this in mind, one might speculate that screening genetic variants may be a practical non-invasive tool to identify high risk CTD patients.Furthermore, it might be especially useful in rare forms of CTD-PAH: early-onset SSc or CTD other than SSc.However, the presence of a genetic defect does not seem to influence clinical course or prognosis. TGF-β signaling pathway expression and activity is reduced in both idiopathic and hereditable PAH, regardless of the presence of germline mutations [27].Restoration of the BMPR2 axis is a promising therapy to prevent or treat PAH, by restoring the balance between proliferative and anti-proligerative pathways [13,28,29] Recently, PULSAR trial has demonstrated the benefit of sotatercept in PAH, including a high proportion of CTD-PAH patients.BMP9/GDF2 is a ligand of the BMP signaling pathway, recently identified as a PAH gene [30].Furthermore, the application of recombinant BMP9 reversed pulmonary hypertension in animal models [31].Restoring BMPR2 expression is also a promising treatment target [13,32,33].The presence of germline mutations in BMP pathway genes suggests that this mechanism might contribute to PVD in CTD patients. TBX4 is involved in the regulation of embryonic developmental processes and its haploinsufficiency was classically associated with Small Patella Syndrome.Pathogenic variants in this gene are a common cause of hereditable PAH in infants and children [34,35].Recently, our group has demonstrated that PAH associated with TBX4 variants shows a wide spectrum of clinical presentations, with overlapping forms of PAH [36].A severely reduced DLCO was a common finding.However, TBX4 mutations are not an established cause of CTD-PAH.Thus, the relationship between TBX4 variants and connective tissue diseases remains unclear.Further investigation is necessary to address this issue. In our cohort, three patients carry a variant in a potassium channel gene.There is growing evidence supporting the role of potassium channel dysfunction in PAH [37].KCNK3 and KCNA5 are considered to play a predominant role in pulmonary vascular tone [38].ABCC8 codifies for SUR1, a subunit of the ATP-sensitive potassium channel that is mostly present in the β pancreatic cells.This explains why pathogenic variants in ABCC8 have been widely related to diabetes mellitus and congenital hyperinsulinism.However, how ABCC8 can cause PAH remains unclear.Previous studies done by our group and others have observed rare coding mutations in ABCC8 in idiopathic, familial and CHD-associated PAH [39,40].However, the role of potassium channels in PAH associated with CTD is not well researched.Further investigation might explain the importance of resting membrane potential in the development of pulmonary vasculopathy in this population. ",
"section_name": "Discussion",
"section_num": "4."
}
] |
[
{
"section_content": "Acknowledgments: This project was supported by the Spanish Pulmonary Arterial Hypertension Registry (REHAP), Registry of Pediatric Pulmonary Hypertension Patients (REHIPED), Xunta de Galicia and Centro de Investigación Biomédica en Red de Enfermedades Cardiovascular (CIBERCV).In Addition: we would like to thank our patients for making this study possible. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Cells 2021, 10, 1488 8 of 10 These findings suggest that genetic testing might be a useful tool in screening or initial diagnosis work-up of CTD-PAH.The discovery of rare variants in these patients forces us to take a comprehensive approach and provide accurate genetic counseling.Further research is still necessary to confirm these findings and help to provide a personalized medicine approach to these patients. The following are available online at https://www.mdpi.com/article/10.3390/cells10061488/s1,Table S1: Spanish Registry of Pulmonary Arterial Hypertension Centers and Investigators.Funding: This research was funded by project \"Bases Genético Moleculares de la Medicina de Precisión en la Hipertensión Arterial Pulmonar\".Funder: Instituto de Salud Carlos III.Ministerio de Economía y Competitividad.Grant number: PI 18/01233. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethical committee for scientific research of the participant centers.A full list of REHAP centers and investigators is provided in the Supporting Information. Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study. 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": "Conclusions",
"section_num": "5."
},
{
"section_content": "Cells 2021, 10, 1488 8 of 10 ",
"section_name": "",
"section_num": ""
},
{
"section_content": "These findings suggest that genetic testing might be a useful tool in screening or initial diagnosis work-up of CTD-PAH.The discovery of rare variants in these patients forces us to take a comprehensive approach and provide accurate genetic counseling.Further research is still necessary to confirm these findings and help to provide a personalized medicine approach to these patients. ",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following are available online at https://www.mdpi.com/article/10.3390/cells10061488/s1,Table S1: Spanish Registry of Pulmonary Arterial Hypertension Centers and Investigators.Funding: This research was funded by project \"Bases Genético Moleculares de la Medicina de Precisión en la Hipertensión Arterial Pulmonar\".Funder: Instituto de Salud Carlos III.Ministerio de Economía y Competitividad.Grant number: PI 18/01233. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "",
"section_name": "Author",
"section_num": null
},
{
"section_content": "The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethical committee for scientific research of the participant centers.A full list of REHAP centers and investigators is provided in the Supporting Information. Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study. ",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"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
}
] |
10.1186/s13039-019-0417-5
|
Genomic instability in a chronic lymphocytic leukemia patient with mono-allelic deletion of the DLEU and RB1 genes
|
BACKGROUND: The most frequent cytogenetic abnormality detected in chronic lymphocytic leukemia (CLL) patients is the presence of a deletion within the chromosome band 13q14. Deletions can be heterogeneous in size, generally encompassing the DLEU1 and DLEU2 genes (minimal deleted region), but at times also including the RB1 gene. The latter, larger type of deletions are associated with worse prognosis. Genomic instability is a characteristic of most cancers and it has been observed in CLL patients mainly associated with telomere shortening. CASE PRESENTATION: Cytogenetic and fluorescence in situ hybridization studies of a CLL patient showed a chromosomal translocation t(12;13)(q15;q14), a mono-allelic 13q14 deletion encompassing both the DLEU and RB1 genes, and genomic instability manifested as chromosomal breaks, telomeric associations, binucleated cells, nucleoplasmic bridges, and micronucleated cells. In conclusion, our CLL patient showed genomic instability in conjunction with a 13q14 deletion of approximately 2.6 megabase pair involving the DLEU and RB1 genes, as well as other genes with potential for producing genomic instability due to haploinsufficiency.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a B-cell lymphoproliferative disorder commonly affecting elderly people [1].The most frequent cytogenetic abnormality detected by interphase fluorescence in situ hybridization (FISH) is the presence of a deletion within the chromosome band 13q14.Deletions can be heterogeneous in size, generally encompassing the DLEU1 and DLEU2 genes (minimal deleted region), but at times also including the RB1 gene.The latter, larger type of deletions are associated with worse prognosis [1][2][3]. Genomic instability is present in most cancers.It is characterized by a high frequency of mutations occurring within the cell genome.Alterations in several pathways involved in detecting and repairing DNA damage, telomere maintenance, and chromosomal mitotic segregation will cause increased frequencies of base pair mutation, microsatellite instability, telomere shortening, and chromosome instability mainly manifested as numerical and structural chromosomal abnormalities, micronuclei, and nucleoplasmic bridges (NPB) [4][5][6][7][8].Several forms of genomic instability has been observed in CLL patients [9][10][11][12][13][14].We here report a CLL patient with genomic instability and a large mono-allelic 13q14 deletion encompassing the DLEU1, DLEU2 and RB1 genes. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "A 61-year-old male patient with bilateral adenomegaly in the neck showed in his peripheral blood a leukocyte count of 49.1 X10 9 /L, with 90% of lymphocytes.Immunophenotyped cells were positive for CD20, CD5, and CD23 surface antigens; therefore, after being diagnosed with CLL (Rai IV), the hematologist administered chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisone, but the patient's disease was refractory to such treatment.Next, the patient was started on fludarabicin and rituximab but an adverse reaction was later reported.Another cycle of treatment with cyclophosphamide and prednisone was administered with no response since leukocytosis remained during the 3 years that preceded his demise. ",
"section_name": "Case presentation",
"section_num": null
},
{
"section_content": "Peripheral blood lymphocytes obtained before therapy were cultured in RPMI-1640 medium and stimulated with a mixture of phorbol-12-myristate-13-acetate plus pokeweed mitogen at concentrations previously described [15,16].After 72 h of incubation, metaphase cells were obtained from cell cultures harvested by standard methods.Chromosomes were stained following the Giemsa-trypsin banding protocol and analyzed under the microscope.Results were interpreted following the ISCN (2016) recommendations [17]. ",
"section_name": "Cytogenetic studies",
"section_num": null
},
{
"section_content": "Three fluorescent in situ hybridization (FISH) analyses were performed separately.In a first analysis, we used a mixture of the dual color 13q14.3-deletionprobe (Cytocell, LPH 006), which covers the DLEU1 and DLEU2 genes (labeled in red) and the 13q subtelomere sequence (labeled in green), plus the RB1 (13q14) probe (labeled in green; Kreatech, KI-40001).According to information published by the providers, the red labeled probe targeted to the DLEU genes is conformed of two separated fragments of 215 and 93 kb, which together span a sequence from chr13:49962705 to 50,671,242 (hg38; 700 kb).As for the RB1 (13q14) probe, it covers a continuous sequence approximately from chr13:48062708 to 48,801,516 (hg38; ~740 kb).A second FISH examination was performed using the MDM2 Amplification probe (Cytocell, LPS 016).We also performed a third FISH study with the dual color P53/ATM probe (Cytocell, LPH 052).In all these FISH studies, cells were counterstained with 4′,6-diamino-2-phenylindole. ",
"section_name": "FISH studies",
"section_num": null
},
{
"section_content": "The Giemsa-trypsin banded metaphase analysis displayed the karyotype 46,XY,t(12;13)(q15;q14) [25]/46,XY [2] (Fig. 1 a andb).Seven out of these 25 cells carrying the translocation t(12;13) showed other single-cell abnormalities as chromosomal breaks, translocations, marker chromosomes, and telomeric associations (Fig. 1 a-b).In addition, while performing the chromosomal banding analysis we observed micronucleated and binucleated cells (Fig. 1 c-k).Micronuclei were observed in 68 out of the 1434 scored cells (4.7%), which is within the range of 2.23 to 4.8% of basal micronucleus frequency reported by [13].Moreover, thirty out of the 1434 scored cells (2.1%) were binucleated cells; and, eleven of them (0.77%) displayed NPB (Fig. 1 c-g), which is statistically different (p < 0.001; Fisher's exact test) from the overall baseline NPB frequency reported by Cai et al. in the peripheral blood lymphocytes of 121 healthy individuals from the general population (0.46 ± 0.20 per 1000 binucleated cells) [18]. The FISH study with the mixture of DLEU, 13q subtelomere, and RB1 probes aimed to reveal the status of the DLEU and RB1 genes in the derivative chromosomes of the translocation.We found a heterozygous 13q14 deletion of approximately 2.6 megabase pair (nearby from chr13:48062708 to 50,671,242, (hg38)), which included both the DLEU and RB1 genes (Fig. 2 a).Such a deletion was observed in 92% of the 200 scored nuclei.Strikingly, all binucleated cells, as well as cells having micronuclei, analyzed in this FISH experiment, were positive for that deletion (Fig. 2 b-d).Subsequent FISH analysis with the MDM2 probe was done in order to explore the location of the 12q breakpoint in the t(12;13) translocation; such a breakpoint was located centromeric to the MDM2 gene (Fig. 2 e-f ).In addition, no evidence of trisomy for chromosome 12 was found after analyzing 200 interphase cells, which consistently showed two MDM2 and two D12Z1 probe signals.Furthermore, FISH study with the dual color P53/ATM probe for searching deletions of these genes disclosed normal results in the 200 scored nuclei (not shown). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The translocation t(12;13)(q15;q14) observed in our patient caused a heterozygous deletion of the DLEU and RB1 genes.There are other translocations registered in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer [19] that affect the 13q14 chromosomal band.However, there are only four cases of translocation t(12;13) sharing the breakpoints observed in our case [20][21][22][23].FISH analysis in two of these four cases showed 13q14 deletions of D13S319 or D13S25 markers, which are within sequence of the DLEU1 and DLEU2 genes [22,23].The 13q14.3 chromosomal band is the minimal deleted region for B-cell CLL and it is known for its potential tumor-suppressing function.It contains various tumor suppressor gene candidates, mainly the DLEU1 and DLEU2 genes whose loss has been considered as an early step in the development of the disease [24,25]. It is hard to explain the genomic instability present in our patient since there are several mechanisms that Fig. 2 FISH observations.a-d FISH study performed with a mixture of DLEU, 13q subtelomere, and RB1 probes.In a, the normal chromosome 13 shows the three expected signals (RB1 (green), DLEU (red) and 13q subtelomere (green)), whereas, only one 13q subtelomere signal is observed on the der(12) chromosome.All analyzed binucleated cells with NPB (b and c), as well as micronucleated cells (d), displayed a signal pattern concordant with RB1-DLEU deletion.e GTG-banded metaphase with translocation t(12;13).f The same metaphase was studied by FISH with the MDM2 amplification probe.The MDM2 red signal is observed on the der(13) chromosome evidencing that the breakpoint on the der(12) occurred centromeric to the MDM2 gene.MN = micronucleus could be related with its presence, as telomere shortening suggested by the presence of telomeric associations, as well as the gene content of the 13q14 region.There are several genes mapping in the deleted segment observed in our patient (chr13:48062708 to 50671242, hg38) whose haploinsufficiency has the potential for causing genome instability.The Mir-16 gene is involved in the DNA damage signaling pathway [26]; the RCBTB2 and SETDB2 genes play a role in chromosome condensation and segregation during mitosis [27,28]; and, the KPNA3 gene is involved in the nuclear import of protein MeCP2 that has important roles in regulating chromatin structure [29].Regarding the RB1 gene, its protein plays a well-known role in G1 to S phase progression.Alteration of this mechanism of regulation causes a replicative stress, resulting in the production of DNA double-stranded breaks [30].Interestingly, new roles of the RB1 protein have been recently identified and all of them are directly involved in the maintenance of genome stability [31,32].Coschi et al. [31] linked haploinsufficiency of the RB1 gene with a wide variety of aberrant processes affecting the normal cell cycle as alteration of the number of centrosomes, defects in the mitotic spindle assembly, occurrence of merotelic kinetochore attachments, and failure of cytokinesis generating either binucleated cells or NPB.Therefore, from this perspective, genomic instability could be a variable phenotypic consequence comparable to that occurring in contiguous gene syndromes, where deletions have variable phenotypic manifestations depending mainly on the amount of genetic material lost. On the other hand, telomeric associations observed in our patient could be true chromosomal translocations rendering dicentric chromosomes and could be interpreted as an indicator of telomere shortening, phenomenon that has been directly associated with genome instability in CLL [9][10][11].It is well known that during cell division, dicentric chromosomes can be pulled apart towards opposite spindle poles causing an abortion of cytokinesis, which in turn, produces both binucleated cells and NPB [5,31,33] as was observed in our patient (Fig. 1 c-g). In conclusion, our CLL patient showed genomic instability in conjunction with a 13q14 deletion involving the DLEU and RB1 genes.Although studies of CLL patients include the determination of the 13q14 deletion, most of them focus almost exclusively on the DLEU genes.It would be advisable to determine the status of the RB1 gene in those patients with DLEU deletion in order to determine the size of the deletion and to make a better assessment of the prognosis.In addition, more attention should also be paid to the search for biomarkers of genomic instability in patients with CLL, as it could be a more frequent phenomenon than is currently reported. ",
"section_name": "Discussion and conclusion",
"section_num": null
}
] |
[
{
"section_content": "We thank María de Lourdes Carbajal her review of the writing. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was funded by the \"FIS -Instituto Mexicano del Seguro Social\": # FIS/IMSS/PROT/PRIO/16/061. The funding body did not participate either in the design of the study, collection, analysis, or interpretation of data, nor in the writing or reviewing of the manuscript.MPNR received a scholarship from Coordinación de Investigación en Salud, IMSS.México. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The authors declare that all relevant data are included in the article. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Abbreviations CLL: Chronic lymphocytic leukemia; FISH: Fluorescent in situ hybridization; NPB: Nucleoplasmic bridges Authors' contributions MPNR, MDDC, did laboratory work as karyotyping and FISH, acquired and interpreted data; LBAL and CBG contributed with the diagnosis, management and clinical-hematological follow-up of the patient; MTMT and JRGG interpreted data, designed the figures, and wrote de manuscript.All authors critically read the manuscript.All of them approved and agreed to publish the material contained in this manuscript. The authors declare to have complied with ethical standards.The data presented here are part of a research project approved by our institutional research and ethics committees (Instituto Mexicano del Seguro Social, Project # R-2013-785-071).Written informed consent was obtained from the patient for participating in this study. The patient gave his approval by signing an informed consent. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Abbreviations CLL: Chronic lymphocytic leukemia; FISH: Fluorescent in situ hybridization; NPB: Nucleoplasmic bridges Authors' contributions MPNR, MDDC, did laboratory work as karyotyping and FISH, acquired and interpreted data; LBAL and CBG contributed with the diagnosis, management and clinical-hematological follow-up of the patient; MTMT and JRGG interpreted data, designed the figures, and wrote de manuscript.All authors critically read the manuscript.All of them approved and agreed to publish the material contained in this manuscript. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare to have complied with ethical standards.The data presented here are part of a research project approved by our institutional research and ethics committees (Instituto Mexicano del Seguro Social, Project # R-2013-785-071).Written informed consent was obtained from the patient for participating in this study. ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "The patient gave his approval by signing an informed consent. ",
"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/s12967-019-2005-1
|
SNORD89 promotes stemness phenotype of ovarian cancer cells by regulating Notch1-c-Myc pathway
|
Ovarian cancer is the leading cause of death in gynecological cancer. Cancer stem cells (CSCs) contribute to the occurrence, progression and resistance. Small nucleolar RNAs (SnoRNAs), a class of small molecule non-coding RNA, involve in the cancer cell stemness and tumorigenesis.In this study, we screened out SNORNAs related to ovarian patient's prognosis by analyzing the data of 379 cases of ovarian cancer patients in the TCGA database, and analyzed the difference of SNORNAs expression between OVCAR-3 (OV) sphere-forming (OS) cells and OV cells. After overexpression or knockdown SNORD89, the expression of Nanog, CD44, and CD133 was measured by qRT-PCR or flow cytometry analysis in OV, CAOV-3 (CA) and OS cells, respectively. CCK-8 assays, plate clone formation assay and soft agar colony formation assay were carried out to evaluate the changes of cell proliferation and self-renewal ability. Scratch migration assay and trans-well invasion analysis were used for assessing the changes of migration and invasion ability.High expression of SNORD89 indicates the poor prognosis of ovarian cancer patients and was associated with patients' age, therapy outcome. SNORD89 highly expressed in ovarian cancer stem cells. The overexpression of SNORD89 resulted in the increased stemness markers, S phase cell cycle, cell proliferation, invasion and migration ability in OV and CA cells. Conversely, these phenomena were reversed after SNORD89 silencing in OS cells. Further, we found that SNORD89 could upregulate c-Myc and Notch1 expression in mRNA and protein levels. SNORD89 deteriorates the prognosis of ovarian cancer patients by regulating Notch1-c-Myc pathway to promote cell stemness and acts as an oncogene in ovarian tumorigenesis. Consequently, SNORD89 can be a novel prognostic biomarker and therapeutic target for ovarian cancer.
|
[
{
"section_content": "Ovarian cancer is the leading cause of death in gynecological cancer, since patients with early stage ovarian cancer do not have symptoms of discomfort and 75% of patients have reached advanced stage (stage III or IV) [1].It was estimated about 295,414 new cases and 184,799 deaths for ovarian cancer worldwide in 2018 according to the American Cancer Society [2].Moreover, the treatments for ovarian cancer were limited and the death rates are higher than incidence rates because of the resistance to radiotherapy and chemotherapy in ovarian cancer, especially in advanced stage [3].Numerous studies indicate that the resistance is related to ovarian cancer stem cells, and many researchers proposed to treat ovarian cancer by targeting tumor stem cells [4][5][6]. SNORNAs are a class of non-coding RNAs widely distributed in the nucleolus of eukaryotic cells and are mainly ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Journal of Translational Medicine classified as box C/D SNORNAs and box H/ACA SNOR-NAs [7,8].They are combined with a set of core proteins to form SNORNP. C/D SNORNAs and H/ACA SNOR-NAs serve as guides to the 2′-O-ribose methylation of rRNAs or small nuclear RNA (snRNAs) and isomerization of uridine residues into pseudouridine, respectively [9][10][11].Accumulating evidence has indicated the role of SNOR-NAs in the occurrence and development of various cancers [12][13][14].SNORNAs were once thought to be noise in the process of RNA transcription.However, with further research, they were found to be involved in the process of cancer occurrence and development.Many snoRNAs are highly expressed in tumor cells [15], can be used as candidate diagnostic and prognostic markers of cancers [11,[16][17][18].Also, some reports promote that snoRNA is critical for the growth, metastasis and self-renewal of cancer cells [19,20].SNORA42 expression was associated with expression of stem cell-core transcription factors in lung tumor-initiating cells (TICs) [21].It was reported that the expression levels of C/D box SNORNAs in acutemyelogenous leukaemia (AML) patients were highly related to in vivo frequency of leukaemic stem cells [20]. So far, there are few reports about SnoRNAs related to ovarian cancer.Here, we screened out SNORNAs related to ovarian patient's prognosis by analyzing the data of 379 cases of ovarian cancer patients in the TCGA database, and revealed that high expression of SNORD89 was associated with poor outcomes of ovarian cancer patients and SNORD89 had an important role in the stemness regulation of ovarian cancer cells. ",
"section_name": "Open Access",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Both the clinical data and the RNA-Seq data in ovarian cancer patients shown here were wholly acquired from TCGA ovarian cancer cohort within the Genomic Data Common (GDC) data portal: https ://porta l.gdc.cance r.gov/.In total, 379 ovarian cancer samples logged in TCGA had both the clinical data and RNA-Seq data available for analysis.TCGA barcode ID for samples and patients in different data files was used to associate those data tables, and clinical data were matched to the RNA-Seq data.The Edge R package was applied to acquire the RNA expression matrix. ",
"section_name": "Database analysis",
"section_num": null
},
{
"section_content": "Ovarian epithelial cells (HOSEpiC), Ovarian cancer OVCAR-3 (OV) and CAOV-3 (CA) cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 Medium (HyClone, USA) supplemented with 10% fetal bovine serum (Tian Jin Hao Yang Biological Manufacture CL., LTD, China), 1% Penicillin-Streptomycin Solution (Biosharp Company, China) in a humid atmosphere containing 5% CO2 at 37 °C. OVCAR-3 spheroids (OS) cells were cultured as our previous report [22].In Brief, OVCAR-3 cells were cultured in suspension in serum-free DMEM-F12 medium (HyClone, USA) supplemented with growth factors of 20 ng/mL EGF (Peprotech Corporation, USA), 10 ng/ mL bFGF (Peprotech Corporation, USA), and 2% B27 (Invitrogen Corporation, USA). The cells were washed with phosphate buffer saline (PBS) and then transiently transfected with 4 μg SNORD89 overexpression plasmid (Shanghai Genechem Co., LTD, China) or SNORD89 silence plasmid (Shanghai Genechem Co., LTD, China) using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer's instructions. ",
"section_name": "Cell lines and spheroids culture and transfection",
"section_num": null
},
{
"section_content": "Total RNA samples were extracted from cultured cells using the TRIzol reagent (Tiangen Biotech Company, China) according to the manufacturer's instructions.cDNAs of SNORDs and mRNA were synthesized from total RNAs by using ReverTra Ace qPCR RT Kit (Toyobo Co., LTD, Japan). qRT-PCR of SNORD89 and CD133, CD44, Nanog, Notch1, c-Myc was performed with the SYBR qPCR Mix (Toyobo Co., LTD, Japan).10 μL reaction system was set up according to the manufacturer's instructions and amplified for 40 cycles.The expression levels of SNORD89 and stemness genes were normalized by U6 and GAPDH.The qRT-PCR was performed on qRT-PCR instrument (Applied Biosystems, USA).Melting curve analysis was performed at the end to validate the specificity of the expected PCR product.Relative expression was calculated using the way of 2 -∆∆Ct .Three independent samples were prepared for each assay, and each experiment was performed three times.Primer names and primer sequences are listed in the table.Quantification of SNORD89 and U6 were performed with a stem-loop real time PCR miRNA kit (Ribobio Co., LTD, China). ",
"section_name": "RNA isolation and quantitative real-time PCR (qRT-PCR)",
"section_num": null
},
{
"section_content": "cDNAs of SNORDs and mRNA were synthesized from total RNAs by using ReverTra Ace qPCR RT Kit (Toyobo Co., LTD, Japan), and were amplified with the SYBR qPCR Mix (Toyobo Co., LTD, Japan).The amplification reaction was going on for 30 cycles.0.45 g agarose gel (Gene Company, LTD, China) in 45 mL TAE (0.04 M Tris, 0.02 M acetic acid, 0.002 M EDTA; pH adjusted to 8.5 with acetic acid) plus 4.5 μL nucleic acid dyes (BBI Life Science Corporation, China) was melted by microwave and add to the agarose gel box.After hardening, the samples were added into the gel and run for 30 min at 100 V. Bands were visualized by fluorescence over long wavelength ultraviolet light and photographed by agarose gel-electrophoretic apparatus (Beijing Liuyi Biotechnology Co. LTD, China). ",
"section_name": "Reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis",
"section_num": null
},
{
"section_content": "For CD133 expression analysis, cells were digested and suspended with PBS.The cell suspension was incubated with FITC-conjugated antibody against CD133 (1:20, BD Pharmingen, USA) at 4 °C for 30 min in darkness, and washed with cold PBS twice, finally for the determination. For cell cycle analysis, cells were digested and washed with PBS.The cell suspension was fixed with 3 mL cold ethanol overnight at 4 °C.After that, the cells were incubated with 20μL Rnase A at 37 °C for 30 min, then incubated with 400 μL propidium iodide (PI) at 4 °C for 30 min, and analyzed by Flow Cytometry (ACEA Biosciences Inc., China) with the software \"NovoExpress 1.2.5\" (ACEA Biosciences Inc., China). ",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "The cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumomoto, Japan).Cells were seeded in 96-well plates at a density of 3000 cells/well.After 24 h, 48 h and 72 h of transfection with SNORD89 overexpression plasmid or silence plasmid, CCK-8 solution (10 μL) was added into each well and incubated for 3 h at 37 °C.The OD value of the reaction solution was measured at 450 nm by an Anthos 2010 microplate reader (Anthos Labtec Instruments GmbH, Austria). ",
"section_name": "Cell proliferation assay",
"section_num": null
},
{
"section_content": "Cells were cultured into 6-well plates until 70% confluency, and then transfected with SNORD89 OE plasmid.Linear 'scratches' were created on the monolayer cells in straight lines with sterile tips.The cells were washed three times with PBS, and added serum free medium.The cells were photographed after 0 h, 24 h and 48 h of incubation under a microscope (Nikon Eclipse TE2000-U, Japan).Wound closure was quantified by Image J software. ",
"section_name": "Wound healing assay/in vitro scratch assay",
"section_num": null
},
{
"section_content": "The invasion of cells was measured using transwell plates (8.0 μm pore size, Corning, USA).The OV and CA cells transfected with SNORD89 OE plasmids and OS cells transfected with sh-SNORD89 plasmids were starved overnight, and then suspended in serum free medium.100 μL single cell suspension with 20000 cells was seeded into the upper chamber precoated with Matrigel, and 600 μL medium with 15% fetal bovine serum was added into the lower chamber.48 h later, the cells at the upper side were removed, and the cells that invaded to the lower side were fixed and further stained with crystal violet.The number of cells in three random fields was counted for each filter by Image J software. ",
"section_name": "Cell invasion assay",
"section_num": null
},
{
"section_content": "The OV and CA cells (1000 cells/well) transfected with SNORD89 OE plasmids were placed in 6-well plates and maintained in medium containing 15% FBS.After 14 days, the cells were fixed and stained by crystal violet.Visible colonies were then counted manually.Each well was assessed in triplicate. ",
"section_name": "Clonogenic assay",
"section_num": null
},
{
"section_content": "Soft agar colony formation assay was carried out as described previously [22].Briefly, 1.2% agarose gel (Lonza Rockland, ME USA) was mixed with 1640 medium containing 20% FBS and 5% Penicillin-Streptomycin solution as a bottom layer in 6-well plates.The OV and CA cells transfected with SNORD89 OE plasmids (5000 cells/well) were mixed into a top layer in 0.6% agarose gel and the same medium.After the incubation for 3 weeks at 37 °C, the colonies were stained with MTT and counted. ",
"section_name": "Soft agar colony formation assay",
"section_num": null
},
{
"section_content": "The OS cells transfected with sh-SNORD89 or sh-NC plasmids were seeded onto Ultra-Low Attachment Surface 6-well plates (Guangzhou Jet Bio-Filtration Co., LTD, China) at a density of 2000 cells/well in serum-free DMEM-F12 medium supplemented with 20 ng/mL EGF, 10 ng/mL bFGF, and 2% B27.Fresh medium of 0.5 mL was added into each well every 3 days.After the culture for 2 weeks in a humid atmosphere containing 5% CO2 at Zhu et al.J Transl Med (2019) 17:259 37 °C, the number of the spheres > 50 μm in diameter was counted under an inverted microscope (Nikon TE2000-U, Japan). ",
"section_name": "Spheroid formation assay",
"section_num": null
},
{
"section_content": "Protein was extracted from OV, CA and OS cells after SNORD89 interference, and subjected to SDS-polyacrylamide separating gel and transferred onto a polyvinylidene fluoride (PVDF) membrane.The primary antibodies used in these experiments were c-Myc (1:1000, Cell Signaling Technology, USA), Notch1 (1:1000, Cell Signaling Technology, USA), β-actin (1:1000, Absin Bioscience Inc, China).The bands were visualized by enhanced chemiluminescence (ECL). ",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "The Log-rank (Mantel-Cox) test was used for survival analysis by GraphPad Prism 7.0.The link between SNOR-NAs' expression and clinicopathologic features of ovarian cancer patients was assessed using the Chi square test and unpaired t test by SPSS Statistics 24.0 software.HRs were calculated using the Cox proportional hazard model, and 95% confidence intervals (CI) were also determined by SPSS Statistics 24.0 software.All other analyses were performed with GraphPad prism7.0 using unpaired t test.Differences were considered statistically significant when the P-value was < 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To find which SNORNAs are related to poor prognosis of ovarian cancer, we used TCGA database to analyze the relationship between of SNORNAs expression and overall survival for 379 ovarian cancer patients.Finally, 4 SNORNAs (SNORA2B, SNORD19, SNORD116-4 and SNORD89) associated with poor prognosis of ovarian cancer were screened out by Kaplan-Meier analysis.Low expression of SNORA2B and SNORD19 is related to poor prognosis of ovarian cancer patients (Fig. 1a,c), and high expression of SNORD116-4 and SNORD89 is associated with poor prognosis of ovarian cancer patients (Fig. 1e,g).Next, we analyzed these four SNOR-NAs resort to SNORic database.Based on the retrieval of SNORic database, we found that SNORD89 was correlated with 178 mRNAs and participated in splicing of 43 mRNAs in ovarian cancer.SNORA2B, on the other hand, was correlated with only two mRNAs and not participated in mRNA splicing in ovarian cancer.SNORD19 is involved in splicing of 2 mRNAs and not correlated with any mRNA.Further, there is no information of SNORD116-4 in ovarian cancer in SNORic database. TO avoid the influence of a small number of patients who survived for more than 5 years on survival curve differences, we set a 5-year cut-off data for survival to observe the effect of screened snoRNAs on patients' prognosis.We found that only the SNORD89 dysregulation was significantly correlated with poor prognosis of patients (Fig. 1b,d,f,h).Thus, these results suggest that SNORA2B and SNORD19 may play roles as suppressor genes, while SNORD116-4 and SNORD89 as oncogenes in ovarian cancer. In order to comprehend the correlation between the oncogenes and poor prognosis of ovarian cancer patients, we further selected SNORD89 and SNORD116-4 for our study.The ovarian cancer patients in TCGA database are mainly in stage III and IV.Therefore, we analyzed the relationship between the expression of SNORD89, SNORD116-4 and prognosis of patients in stage III and IV.The survival curves showed that the expression of SNORD89 was an important prognostic factor in stage IV, and patients with high expression of SNORD89 have more poorly prognosis (Additional file 1: Figure S1a,b).Among the ovarian cancer patients in TCGA, 152 and 24 patients had high expression of SNORD116-4 in stage III and stage IV, 139 and 33 patients had low expression of SNORD116-4 in stage III and stage IV, respectively.The patients in stage III with high expression of SNORD116-4 have poor prognosis, however, patients in stage IV with high expression of SNORD116-4 have better prognosis, SNORD116-4 shows a trend for better survival in stage IV (Additional file 1: Figure S1c).These results suggest that SNORD116-4 has different effects on the prognosis of ovarian cancer in stage III and stage IV. Moreover, we further confirmed that the prognosis of patients in stage IV was worse than that in stage III (Additional file 1: Figure S1d).Interestingly, this was reversed by the SNORD89 high expression.From the survival curve, we can see that the patients with SNORD89 high expression in stage III have worse prognosis than the patients with SNORD89 low expression in stage IV (Additional file 1: Figure S1e).In addition, we analyzed the effects of SNORD89, SNORD116-4 and stage on the 5-year survival of patients with ovarian cancer.Unlike the OS curves, SNORD116-4 had no significant effect on the 5-year survival of patients with stage III and IV, and, stage has a strong correlation with the 5-year survival of patients (Additional file 2: Figure S2).However, the prognosis of patients with high SNORD89 expression was worse than that of patients with low SNORD89 expression in OS and 5-year survival curves.The findings suggest that SNORD89 might have an important role in the progress of ovarian cancer. ",
"section_name": "Screening SNORNAs related to prognosis in ovarian cancer patients",
"section_num": null
},
{
"section_content": "The high expression of SNORD89 and SNORD116-4 predicts poor prognosis in ovarian cancer patients.We next analyzed the correlation between the two SNORNAs expression and the clinicopathologic features of ovarian cancer patients in TCGA database by Chi square test.We found that the expression of SNORD116-4 was not correlated with any clinicopathological parameters.However, the expression of SNORD89 was correlated with age (P = 0.03, Additional file 3: Table S1).We also found that the expression of SNORD89 was higher in elderly patients (P = 0.0202) and progressive disease (P = 0.0486) by unpaired t test (Additional file 4: Figure S3a,b). In order to observe SNORD89 expression and the clinical factors affect the prognosis of ovarian cancer, the univariate and multivariate COX's regression analysis were performed (Additional file 3: Tables S2 andS3).Univariate analysis showed that the expression of SNORD89, age, and tumor size was significantly associated with overall survival (OS), and age was also related to progression-free survival (PFS) of ovarian cancer patients.In addition, multivariate COX's regression analysis revealed that race and venous invasion were independent predictors of OS, and venous invasion was also independent predictors of PFS in patients with ovarian cancer.Thus, SNORD89 expression may be associated with other factors affecting the prognosis of ovarian cancer patients. ",
"section_name": "Prognostic factors of ovarian cancer patients",
"section_num": null
},
{
"section_content": "Tumor stem cells cause tumor recurrence by promoting tumor invasion, metastasis, drug resistance, and so on, leading to poor prognosis of patients [23].It has been reported that SNORNAs have important roles in the cancer progress [12].We compared the non-coding RNAs (NC RNAs) expression in ovarian epithelial cells (HOSEpiC), ovarian cancer cells (Ovcar-3, OV) and ovarian cancer stem cells (Ovcar-3-S, OS) by gene chips.Finally, 15 SNORNAs with high expression in HOSEpiC and OS were screened out (Fig. 2a,b, Additional file 3: Table S4).Among them, only SNORD89 and SNORD116-4 were associated with the prognosis of ovarian cancer patients, while SNORD116-4 played an opposite role in stage III and stage IV.Therefore, SNORD89 can better predict the prognosis of ovarian cancer patients.As we know, the poor prognosis of tumors is closely related to cancer stem cells.To our surprise, SNORD89 is not only associated with poor prognosis of ovarian cancer, but also highly expressed in OS vs OV and OS vs HOSEpiC, while SNORD116-4 was not highly expressed in OS.Therefore, we decided to conduct a further research on SNORD89. Next, we measured the expression difference of SNORD89 in HOSEpiC, OV and OS cells by quantitative real-time PCR (qRT-PCR) and reverse transcription PCR (RT-PCR).qRT-PCR results showed that SNORD89 expression was up-regulated 2.38 ± 0.29-fold in OS vs HOSEpiC cells, and 5.93 ± 0.53-fold in OS vs OV cells (Fig. 2c).And the increased expression of SNORD89 was also observed by RT-PCR (Fig. 2d).The data suggest that SNORD89 upregulated in OS versus OV and HOSEpiC. ",
"section_name": "SNORD89 is highly expressed in ovarian cancer stem cells",
"section_num": null
},
{
"section_content": "We compared the expression of genes associated with stemness in OV and OS by qRT-PCR.The expression levels of CD133, CD44, and Nanog were increased by 3.32 ± 0.48, 7.11 ± 0.90 and 6.78 ± 1.10-fold in OS cells relative to OV cells, respectively (Fig. 3a).Also, RT-PCR and agarose gel electrophoresis showed the higher exprssion of CD44 and Nanog in OS cells (Fig. 3b).Additionally, we observed that the CD133 positive cells were significantly increased in OS than those in OV by flow cytometry analysis (Fig. 3c). To investigate whether the SNORD89 interference affects the stemness of ovarian cancer cells, we first examined the SNORD89 expression in OV cells transfected with over expression plasmid (OE) of SNORD89 and in OS cells transfected with silent plasmid (shRNA) of SNORD89 for 24, 48, and 72 h, respectively.The qRT-PCR analysis showed that the SNORD89 overexpression in OV and CA cells was the most efficient at 24 h' transfection (3.81 ± 0.28-fold than NC in OV cells and 20.65 ± 1.195-fold than NC in CA cells, Fig. 3d and Additional file 5: Figure S4a).And the most silence efficiency of three shRNA (shRNA-1, shRNA-2, shRNA-3) was 0.255 ± 0.08, 0.56 ± 0.05 and 0.70 ± 0.04 at 48 h in OS cells (Fig. 3e). Next, we examined the changes of stemness genes in OE-transfected OV cells at 24 h, and in shRNA-transfected OS cells at 48 h.We found that the expression of CD133, CD44, and Nanog was increased by 4.28 ± 0.48, 4.58 ± 0.28 and 1.90 ± 0.49-fold in OE group than NC group in OV cells by qRT-PCR (Fig. 3f ), and increased by 1.80 ± 0.25, 1.40 ± 0.11 and 1.47 ± 0.09-fold in OE group than NC group in CA cells by qRT-PCR (Additional file 5: Figure S4b).RT-PCR and agarose gel electrophoresis also verified the elevated expression of CD44 and Nanog (Fig. 3g).The flow cytometry analysis showed the higher CD133 positive cells in OE group (Fig. 3h).Consistantly, the silence of SNORD89 with shRNA-1 and shRNA-2 at 48 h notably decreased the expression of CD44 and Nanog in OS cells by qRT-PCR (Fig. 3i) and RT-PCR and agarose gel electrophoresis (Fig. 3j).The flow cytometry analysis showed the decreased CD133 in OS cells transfected with SNORD89 silence plasmids (Fig. 3k).These data suggest that SNORD89 can increase the expression of stemness genes in ovarian cancer cells. ",
"section_name": "Effects of SNORD89 interference on the stemness of ovarian cancer cells",
"section_num": null
},
{
"section_content": "We analyzed the expression correlation of SNORD89 with other genes using the data from ovarian cancer patients in TCGA, and found that the expression level of SNORD89 was related to some genes of cell cycle checkpoint and cell cycle arrest (Fig. 4a).So, we suspected that the interference of SNORD89 expression may affect cell cycle.And we examined the changes of cell cycle phases in OV and OS cells after interfering the expression of SNORD89.The flow cytometry analysis showed the proportion of S phase was obviously increased in OV cells transfected with SNORD89 OE plasmids (Fig. 4b).Conversely, the proportion of S phase was obviously decreased in OS cells transfected e The SNORD89 expression in OS cells transfected with silence plasmids of SNORD89 (shRNA-1, shRNA-2, shRNA-3) or shRNA negative control (shRNA-NC) plasmid at 24, 48, and 72 h by qRT-PCR.The SNORD89 expression in OS cells transfected with shRNA-NC plasmids was set as 1. f The mRNA expression of CD133, CD44 and Nanog were detected in OV cells transfected with SNORD89 OE or NC plasmids at 24 h by qRT-PCR.The mRNA expression of these genes in OV cells transfected with NC plasmids was set as 1. g The representative agarose gel electrophoresis photos showed the increased expression of CD44 and Nanog in OV cells transfected with SNORD89 OE. h The increased CD133 positive cells in OV cells transfected with SNORD89 OE by flow cytometry analysis.i The mRNA expression of CD133, CD44 and Nanog were detected in OS cells transfected with shRNA-1, shRNA-2, and shRNA-NC plasmids at 48 h by qRT-PCR.The mRNA expression of these genes in OS cells transfected with shRNA-NC plasmids was set as 1. j The representative agarose gel electrophoresis photos showed the decreased expression of CD44 and Nanog in OS cells transfected with SNORD89 shRNA-1 and shRNA-2.k The decreased CD133 in OS cells after silencing SNORD89 by flow cytometry analysis with shRNA-1 and shRNA-2, while the proportion of G 2 phase was raised (Fig. 4c).These results indicated that SNORD89 may have an effect on cell proliferation in ovarian cancer. Then, we checked the cell proliferation ability changes in OV and CA cells of SNORD89 overexpression and OS cells of SNORD89 silence by Cell Counting Kit-8 (CCK-8) assays at 24 h, 48 h, 72 h and 96 h transfection.CCK-8 assays revealed a significant raise cell proliferation in OE cells compared with the NC cells (Fig. 4d and Additional file 5: Figure S4c), and a notable decreased cell proliferation ability in SNORD89 knockdown-OS cells (Fig. 4e). In addition, both plate clone formation assay and soft agar colony formation assay showed that SNORD89 overexpression significantly increased the number of clone formation in OV and CA cells, and the size was larger in OE group (Fig. 4f, g, Additional file 5: Figure S4d,e).Furthermore, colony formation assays indicated that silencing SNORD89 obviously reduced the number of colony formation in OS cells, and the size was smaller in shRNA-1 and shRNA-2 groups (Fig. 4h).These results suggest that SNORD89 may increase cell proliferation and self-renewal ability of ovarian cancer cells. ",
"section_name": "Effects of SNORD89 interference on cell proliferation and self-renewal ability of ovarian cancer cells",
"section_num": null
},
{
"section_content": "We further detected the effects of SNORD89 interference on invasion and migration ability of ovarian cancer cells by scratch migration assay and cell invasion analysis.The scratch migration assay showed that the overexpression of SNORD89 significantly increased the area wound healed than NC-transfected OV and CA cells at 24 h, 48 h and 72 h (Fig. 5a and Additional file 5: Figure S4f ), suggesting SNORD89 can increase the migration ability of ovarian cancer cells.In addition, the cell invasion analysis indicated that SNORD89 overexpression obviously elevated the invasion ability of OV and CA cells (Fig. 5b and Additional file 5: Figure S4g), while SNORD89 knockdown notably reduced the invasion ability of OS cells (Fig. 5c). ",
"section_name": "Effects of SNORD89 interference on cell migration and invasion of ovarian cancer cells",
"section_num": null
},
{
"section_content": "Many reports have shown that c-Myc binds with NOTCH1 to promote the development of cancer by acting as a target of NOTCH1 to form a NOTCH1-MYC pathway [24,25].We first compared the expression of c-Myc and Notch1 in OV and OS by qRT-PCR, and found that the expression levels of c-Myc and Notch1 were 1.39 ± 0.09 and 2.97 ± 0.37-fold in OS cells than OV cells (Fig. 6a).RT-PCR and agarose gel electrophoresis also verified the increased expression of c-Myc and Notch1 in OS cells (Fig. 6b). Next, we assessed the effects of SNORD89 interference on the expression of c-Myc and Notch1 in ovarian cancer cells.The Notch1 and c-Myc expression levels were elevated 2.56 ± 0.33 and 1.47 ± 0.05-fold in the OV cells transfected with SNORD89 OE plasmids by qRT-PCR (Fig. 6c), and RT-PCR and agarose gel electrophoresis also showed the consistent results (Fig. 6d).The Notch1 and c-Myc expression levels were elevated 1.75 ± 0.06 and 1.58 ± 0.10-fold in the CA cells transfected with SNORD89 OE plasmids by qRT-PCR (Additional file 5: Figure S4h).Additionally, silencing SNORD89 significantly declined the expression of c-Myc and Notch1 in OS cells (Fig. 6e).The similar results were also verified by RT-PCR and agarose gel electrophoresis (Fig. 6f ).We further observed that SNORD89 overexpression obviously increased the protein expression of c-Myc and Notch1 in OV and CA cells (Fig. 6g and Additional file 5: Figure S4i), while SNORD89 knockdown notably decreased the expression of these proteins in OS cells (Fig. 6h). In our experiments, we found that SNORD89 affected the proliferation, self-renewal ability, invasion and migration of ovarian cancer cells related to stemness phenotype.Notch1 is a well-known pathway associated with stemness phenotype in cancer, and c-Myc can bind with NOTCH1 to promote the development of cancer by acting as a target of NOTCH1 to form a NOTCH1/c-Myc pathway.Thus, the data suggest that SNORD89 might regulate the Notch pathway in ovarian cancer. ",
"section_name": "The carcinogenicity of SNORD89 may be related to the NOTCH1-MYC highway",
"section_num": null
},
{
"section_content": "Ovarian cancer is the most lethal of the women tumors, because it is rarely diagnosed at an early stage and most patients are diagnosed at the advanced stage.Therefore, ovarian cancer is historically called the \"silent killer\" [26].Though ovarian cancer is sensitive to chemotherapeutic drugs, about 60% of the advanced ovarian cancer patients will ultimately recue within 5 years, and some will develop resistance [27][28][29][30].Thus, the key to the successful treatment of ovarian cancer is its early detection. Small nucleolar RNA (SnoRNA) is a kind of singlestranded small molecule non-coding RNA widely distributed in nucleolus of eukaryotic cells, with stable metabolism and a length of 60-300 nucleotide sequences [7,31].They interact with ribonucleoproteins to form stable small nucleolar ribonucleoproteins particles (snoRNPs), which are involved in the posttranscriptional modification of ribosomal RNA and other RNAs [32,33].SnoRA42 was reported to have an important role in lung tumorigenesis as an oncogene [34,35].Xu et al. demonstrated that SNORND113-1 functioned as a tumor suppressor in hepatocellular carcinoma (HCC) [36].However, few studies on SNORNAs in ovarian cancer were carried out at present.In the present study, we identified 4 SNORNAs associated with the poor prognosis of ovarian cancer patients from TCGA database.Among them, the high expression of snord116-4 and SNORD89 was associated with patients' poor prognosis.Further analysis revealed that the expression of SNORD89 was higher in elderly patients and progressive disease patients, and SNORD89 high expression reversed the survival curve of stage III in ovarian cancer patients, suggesting the main role of SNORD89 in the progress of ovarian cancer. Cancer stem cells (CSCs) are a small group of dormant cells that have self-renewal, infinite proliferation, invasion and migration abilities, which can lead to the recurrence and metastasis of tumors after conventional therapy [23,37,38].In recent years, ovarian cancer has been described as a kind of stem cell disease [39].Here, we obtained OVCAR-3 (OV) sphere-forming (OS) cells with higher expression of stem cell markers, CD133, CD44, and Nanog, by culturing OVCAR-3 (OV) cells in suspension in serum-free medium.We found that SNORD89 highly expressed in OS than OV and HOSEpiC by gene chips and qRT-PCR analysis.Furthermore, overexpression of SNORD89 upregulated the expression of Nanog, CD44 and CD133, and increased the cell proliferation and self-renewal ability of OV and CA cells.Conversely, silencing SNORD89 resulted in the downregulation of Nanog, CD44 and CD133 expression, and the decreased cell proliferation and self-renewal ability of OS cells.The results showed the main roles of SNORD89 in the stemness regulation of ovarian cancer cells.This is the first report of SNORNAs in regulating stemness of ovarian cancer.SNORD78 was reported to be required for the self-renewal of cancer-stem cells of non-small cell lung cancer (NSCLC) [12].Mannoor et al. also revealed that SNORA42 had important influences in regulating features of lung tumor-initiating cells (TICs) [21]. Additionally, we found the expression level of SNORD89 was related to some genes of cell cycle checkpoint and cell cycle arrest by analyzing TCGA database.Furthermore, SNORD89 overexpression upregulated the proportion of S phase of OV cells, whereas SNORD89 silence downregulated the proportion of S phase of OS cells.There are some other SNORNAs that were reported to regulate cell cycle and proliferation of tumor cells.SNORD78 knockdown could inhibit the proliferation of NSCLC cells via inducing cell cycle arrest at G0/G1 phase [12].Valleron et al. demonstrated that SNORD114-1 variant could regulate G0/G1 to S phase transition to promote cell growth by the Rb/p16 pathways in acute leukemia [40].Our study revealed that SNORD89 might modulate cell cycle to promote proliferation of ovarian cancer cells by regulating stemness. Besides, scratch migration assay and trans-well invasion analysis showed that SNORD89 could promote the migration and invasion ability of ovarian cancer cells in our study.Cui et al. reported that SNORA23 knockdown decreased the invasive potential of Pancreatic Ductal Adenocarcinoma (PDAC) cells [19].Crea et al. found that SNORA55 silencing inhibited cell migration in prostate cancer cell lines [41].Our study suggests that SNORD89 has a potential influence on the progression of ovarian cancer by promoting cell migration and invasion. NOTCH signaling is involved in the regulation of cancer and stem cells.NOTCH1-4 receptors have complex functions in different tissues and tumors.NOTCH1 signaling could regulate self-renewal and resistance of CSCs [24].C-Myc is an important direct target of Notch1 in various cancers, such as T-cell lymphoblastic leukemias [25,42,43], breast cancer [44,45], lung adenomas and head [46] and neck squamous cell carcinoma [47].We found that c-Myc and Notch1 highly expressed in OS cells compared with the parental OV cells.SNORD89 overexpression significantly increased the expression of c-Myc and Notch1 in mRNA and protein levels in OV and CA cells, whereas SNORD89 knockdown notably decreased their expression in OS cells.After the overexpression of SNORD89, the expression levels of c-Myc and Notch1 was increased.The number of combinations of c-Myc and NOTCH1 will raise as the increased expression and then promoting the stemness phenotype and development of ovarian cancer.Therefore, we speculate that the oncogenic effect of SNORD89 may be related to the regulation of Notch1-c-Myc pathway. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, we demonstrate that the expression of SNORD89 was associated with the prognosis of ovarian cancer patients and involved in the stemness regulation of ovarian cancer cells.SNORD89 could influence the stemness of ovarian cancer cells to promote cell proliferation, self-renewal, and invasion of ovarian cancer cells.The stemness regulation of SNORD89 might be mediated by the activation of Notch1-c-Myc pathway.Our study will supply an important clue that SNORD89 may facilitate the development of SNORNA-directed diagnostics and therapeutics against ovarian cancer. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by National Natural Science Foundation of China (No. 81572898, 81673475, 81502996, 81601370), National Natural Science Foundation of China and Liaoning joint fund key program (No. U1608281), The Drug Innovation Major Project (Grant no 2018ZX09711001-008-006), Key Laboratory Foundation from Shenyang S&T Projects (Z18-4-020), Liaoning Province Scientific Research Foundation (20180550500) and Liaoning Province Scientific Research Foundation (LQNK20173, 20170541024). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The datasets analyzed during the current study are available in the TCGA repository, https ://cance rgeno me.nih.gov/. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": ". Correlation between SNORNA89 and SNORD116-4 expression and the clinicopathologic features of ovarian cancer patients in TCGA (Chi square test).Table S2.Univariate and multivariate analysis for predictors of overall survival (OS) of ovarian cancer patients in TCGA.Table S3.Univariate and multivariate analysis for predictors of progression-free survival (PFS) of ovarian cancer patients in TCGA.Table S4.15 CSCs: cancer stem cells; SnoRNA: nucleolar small molecule RNA; snoRNPs: small nucleolar ribonucleoproteins particles; rRNA: ribosomal RNA; TICs: tumor-initiating cells; AML: acute myelogenous leukaemia; NC RNAs: noncoding RNAs; HOSEpiC: ovarian epithelial cells; OV: ovarian cancer cells; CA: CAOV-3; OS: ovarian cancer stem cells; qRT-PCR: quantitative real-time PCR; RT-PCR: reverse transcription PCR; OE: over expression plasmid; shRNA: silent plasmid; NC: negative control; shRNA-NC: shRNA negative control; CCK-8: Cell Counting Kit-8; snRNAs: small nuclear RNA; HCC: hepatocellular carcinoma; CSCs: cancer stem cells; NSCLC: non-small cell lung cancer; PDAC: Pancreatic Ductal Adenocarcinoma. Authors' contributions MJW, JD, MH and WJZ designed the experiments.WJZ, LWZ, XML, FXL and YYY performed the experiments.WJZ, YF analyzed the TCGA data and experimental data.MH, JZ, LYJ, ZJY, LZ, JB, HH and QW gave technical and material support.MJW, MH and WJZ wrote and reviewed the manuscript.All authors read and approved the final manuscript. Ethical approval has been obtained by The Cancer Genome Atlas (TCGA). Not applicable. The authors declare that they have no competing interests. • 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 ?Choose BMC and benefit from: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": ". Correlation between SNORNA89 and SNORD116-4 expression and the clinicopathologic features of ovarian cancer patients in TCGA (Chi square test).Table S2.Univariate and multivariate analysis for predictors of overall survival (OS) of ovarian cancer patients in TCGA.Table S3.Univariate and multivariate analysis for predictors of progression-free survival (PFS) of ovarian cancer patients in TCGA.Table S4.15 ",
"section_name": "Additional file 3: Table",
"section_num": null
},
{
"section_content": "CSCs: cancer stem cells; SnoRNA: nucleolar small molecule RNA; snoRNPs: small nucleolar ribonucleoproteins particles; rRNA: ribosomal RNA; TICs: tumor-initiating cells; AML: acute myelogenous leukaemia; NC RNAs: noncoding RNAs; HOSEpiC: ovarian epithelial cells; OV: ovarian cancer cells; CA: CAOV-3; OS: ovarian cancer stem cells; qRT-PCR: quantitative real-time PCR; RT-PCR: reverse transcription PCR; OE: over expression plasmid; shRNA: silent plasmid; NC: negative control; shRNA-NC: shRNA negative control; CCK-8: Cell Counting Kit-8; snRNAs: small nuclear RNA; HCC: hepatocellular carcinoma; CSCs: cancer stem cells; NSCLC: non-small cell lung cancer; PDAC: Pancreatic Ductal Adenocarcinoma. Authors' contributions MJW, JD, MH and WJZ designed the experiments.WJZ, LWZ, XML, FXL and YYY performed the experiments.WJZ, YF analyzed the TCGA data and experimental data.MH, JZ, LYJ, ZJY, LZ, JB, HH and QW gave technical and material support.MJW, MH and WJZ wrote and reviewed the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Ethical approval has been obtained by The Cancer Genome Atlas (TCGA). ",
"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. • 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 ?Choose BMC and benefit from: ",
"section_name": "Learn more biomedcentral.com/submissions",
"section_num": null
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"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/cancers15194786
|
Could Immune Checkpoint Disorders and EBV Reactivation Be Connected in the Development of Hematological Malignancies in Immunodeficient Patients?
|
<jats:p>Primary immunodeficiencies (PIDs) and secondary immunodeficiencies (SIDs) are characterized by compromised immune function, rendering individuals susceptible to infections and potentially influencing cancer development. Epstein–Barr virus (EBV), a widespread herpesvirus, has been linked to cancer, particularly in those with weakened immune systems. This study aims to compare selected immune parameters, focusing on immune checkpoint molecules (PD-1/PD-L1, CTLA-4/CD86, CD200R/CD200), and EBV reactivation in patients with chronic lymphocytic leukemia (CLL, a representative of SIDs) and common variable immunodeficiency (CVID, a representative of PIDs). We performed a correlation analysis involving patients diagnosed with CLL, CVID, and a healthy control group. EBV reactivation was assessed using specific antibody serology and viral load quantification. Peripheral blood morphology, biochemistry, and immunophenotyping were performed, with emphasis on T and B lymphocytes expressing immune checkpoints and their serum concentrations. Our findings revealed elevated EBV reactivation markers in both CLL and CVID patients compared with healthy controls, indicating increased viral activity in immunodeficient individuals. Furthermore, immune checkpoint expression analysis demonstrated significantly altered percentages of T and B lymphocytes expressing PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200 in CLL and CVID patients. This suggests a potential interplay between immune checkpoint dysregulation and EBV reactivation in the context of immunodeficiency. In conclusion, our study underscores the intricate relationship between immune dysfunction, EBV reactivation, and immune checkpoint modulation in the context of immunodeficiency-associated cancers. The altered expression of immune checkpoints, along with heightened EBV reactivation, suggests a potential mechanism for immune evasion and tumor progression. These findings provide insights into the complex interactions that contribute to cancer development in immunocompromised individuals, shedding light on potential therapeutic targets for improved management and treatment outcomes. Further investigations are warranted to elucidate the underlying mechanisms and to explore potential interventions to mitigate cancer risk in these patient populations.</jats:p>
|
[
{
"section_content": "Primary immunodeficiencies (PIDs) and secondary immunodeficiencies (SIDs) are conditions that affect the immune system's ability to protect the body from infections and can potentially affect the development of cancer [1][2][3][4].PIDs are inherited genetic disorders that cause defects in various components of the immune system.These defects can lead to increased susceptibility to infections, autoimmune diseases, and, in some cases, an increased risk of cancer [5][6][7][8].Some PIDs are associated with a higher risk of certain cancers, mainly due to impaired immune surveillance that normally helps detect and eliminate cancer cells [9,10].One such example is common variable immunodeficiency (CVID).CVID is an immune disorder characterized by low levels of serum immunoglobulins and antibodies, leading to increased susceptibility to infection and a higher risk of autoimmune diseases, cancers, and lymphoproliferative disorders (like lymphomas, gastric cancer, and other malignancies) [11][12][13][14].The pathogenesis of CVID is not fully understood, but it is associated with various abnormalities in B cell differentiation and function.Recent studies suggest that dysregulation of the immune checkpoint may also be involved [15][16][17][18][19]. SIDs are acquired disorders that result from external factors, such as infections, medications, and other medical conditions, and can also affect the immune system's ability to function properly [3,20,21].These factors can sometimes contribute to an increased risk of cancer.One example of a SID that is also an example of a hematologic malignancy is chronic lymphocytic leukemia (CLL).CLL is a type of cancer where the bone marrow produces too many lymphocytes (a type of white blood cell), often leading to dysfunction of the immune system, which the literature suggests may be related to checkpoint molecules such as PD-1 (programmed cell death 1) and its ligand PD-L1, or CTLA-4 (T cell cytotoxic antigen 4), which may play a significant role in the progression of CLL.Overexpression of these inhibitory molecules often leads to immune exhaustion and the formation of a suppressive tumor microenvironment, allowing malignant cells to escape immune surveillance [22][23][24][25][26][27][28][29]. It is important to remember that while immunodeficiency may contribute to an increased risk of certain cancers, not everyone with a PID or SID will develop cancer.The interplay between the immune system and cancer development is complex and depends on various factors, including genetic predisposition, environmental exposure, and the specific nature of the immune defect [30,31].Scientists are increasingly elevating the role of two rather extreme but mutually interacting mechanisms: the deregulation of immune checkpoints and the role of Epstein-Barr virus (EBV) reactivation [32][33][34].EBV belongs to the herpesvirus family and is one of the most common viruses in humans.It is usually associated with causing infectious mononucleosis (commonly known as mononucleosis or glandular fever) in teenagers and young adults [35][36][37].EBV infection is usually mild and self-limiting, but in some cases, it can lead to more serious complications, especially in people with PIDs or SIDs.In addition, research in recent years has linked EBV to the development of several types of cancer, especially in people with a weakened immune system or certain genetic predispositions [38,39].EBV-associated cancers are more common in regions with higher rates of EBV infection and are often linked to the ability of the virus to cause latent (dormant) infections in certain cells [40,41].The interplay between EBV, the immune system, and other environmental and genetic factors is complex and Cancers 2023, 15, 4786 3 of 31 not fully understood.According to researchers, EBV can manipulate the immune system by interfering with immune checkpoint function.For example, latent EBV infection may increase PD-L1 expression, enhancing immune evasion [32,42,43]. Therefore, the purpose of this publication was a comparative analysis of selected parameters of the immune system, with particular emphasis on the role of immune checkpoints and their ligands (PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200) in the course of patients with CLL (representing a group of with SID) and CVID (representing a group with PID) in the context of EBV reactivation.The immunological checkpoints selected for the analysis, such as PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200, play a significant role in regulating the immune response in many diseases, including cancer, and influencing the tumor microenvironment.They are responsible not only for regulating the immune response but also for maintaining self-tolerance to prevent excessive immune reactions against healthy tissues.However, cancer cells can use these checkpoints to evade immune surveillance, allowing tumors to grow and spread.Selected immune checkpoints are among the most common targets of anticancer therapies.Testing immune checkpoints in patients with PID and SID could be extremely important in monitoring cancer development, which will be critical to tailoring therapeutic approaches, predicting responses, and advancing our understanding of the complex interactions between the immune system and hematological malignancies in this particular group of patients. For this purpose, analyses were carried out to assess EBV reactivation (serological profile of specific antibodies and the number of EBV copies in the tested genetic material) among patients with immunodeficiency in relation to healthy volunteers.We focused on selected parameters including morphology, biochemistry, and immunophenotype of peripheral blood of patients included in the study, with particular emphasis on the percentage of T and B lymphocytes positively expressing PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200, and the assessment of their serum concentrations. The performed analyses showed a significant increase in the percentage of these checkpoints and ligands in both groups of patients compared with those without EBV.In particular, the PD-1, PD-L1, CTLA-4, CD86, CD200R, and CD200 pathways showed marked upregulation in response to EBV reactivation.Notably, the observed changes were more pronounced in CLL patients with EBV reactivation compared with CVID patients with the same disease.Our studies suggest that EBV reactivation plays a key role in modifying the immune landscape in both CLL and CVID patients. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "A total of 142 participants were enrolled in this study: 60 individuals with chronic lymphocytic leukemia (CLL), 52 individuals with common variable immunodeficiency (CVID), and 30 healthy volunteers (HVs) who formed the control group.Inclusion and exclusion criteria were applied to both immunodeficient patients and HVs.Selection of participants was conducted by a clinical immunology specialist and based on specific conditions: • age 18 or older; • life expectancy of at least 12 months; • no recent immunosuppressive treatment in the three months prior to study entry; and • willingness to provide written consent for study participation.Patients were excluded from the study based on the following criteria: ",
"section_name": "Characteristics of Participants and Study Materials",
"section_num": "2.1."
},
{
"section_content": "Ongoing viral, bacterial, and fungal infections; Presence of tumor metastases in the central nervous system or mental illness. In addition, all participants were age-matched for CLL (median age: 57; range: 37-75; 24 women and 36 men); CVID (median age: 55; range: 31-77; 27 women and 25 men); HVs (median age: 54; range: 32-71; 18 women and 12 men).The age of the patients shown is the age at which the diagnosis was made.None of the patients included in this study had undergone any therapy prior to analysis.Patients with CLL were classified according to Rai Stage 0 (36 subjects) and I (24 subjects); the patients did not show splenomegaly or hepatomegaly and had not undergone any therapy at the time of this analysis or before it was performed.This study utilized 10 mL of peripheral blood drawn from the basilic vein into EDTA (Strasted) tubes for immunophenotypic analysis and 5 mL of blood collected in a clot tube to obtain serum for quantitative measurement of the tested molecules.The research protocol received ethical approval from the Bioethics Committee at the Medical University of Lublin (KE-0254/186/06/2023). ",
"section_name": "•",
"section_num": null
},
{
"section_content": "EBV genomic copy quantification was conducted utilizing the ISEX variant of the EBV PCR assay by GeneProof (Brno, Czech Republic).Duplicate assessments were carried out for all samples, with the inclusion of a negative control containing DNA elution buffer.Amplification targeted the conserved DNA sequence specific to the EBNA1 gene of EBV, using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in strict accordance with the ISEX EBV PCR kit protocol.The final viral DNA copy concentration, expressed per µg of extracted DNA, was normalized considering the DNA isolation efficiency.A detection threshold was established at ten EBV DNA copies/µL, and samples falling beneath this level were deemed EBV-negative. ",
"section_name": "Quantification of EBV Genomic Copies in PBMC-Derived DNA",
"section_num": "2.2."
},
{
"section_content": "Lymphocyte immunophenotypic profiling from peripheral blood was undertaken utilizing flow cytometry.Blood samples were subjected to a comprehensive panel of human-specific monoclonal antibodies, which encompassed: anti-CD45 AF700 (cat.no.: 368514, clone: 2D1), anti-CD4 BV421 (cat.no.: 300532, clone: RPA-T4), anti-CD3 PerCp (cat.no.: 344814, clone: SK7), anti-CD8 BV605 (cat.no.: 344742, clone: SK1), and anti-CD19 FITC (cat.no.: 302206, clone: HIB19), alongside antibodies targeting immune checkpoints and other relevant markers such as anti-PD-1 APC (cat.no.: 329908), anti-PD-L1 PE (cat.no.: 329706, clone: 29E.2A3), anti-CTLA-4 PE (cat.no.: 349906, clone: L3D10), anti-CD86 APC (cat.no.: 374208, clone: BU63), anti-CD200 PE (cat.no.: 399804, clone: A18042B), and anti-CD200R APC (cat.no.: 329308, clone: OX-108), all sourced from BioLegend (San Diego, CA, USA).For precise gating during cytometric analysis, FMO controls were incorporated for immune checkpoint antibodies.Subsequent to the antibody incubation phase, erythrocytes were lysed using a specific lysing buffer from BD (Franklin Lakes, NJ, USA).The cell suspension post-lysis was then subjected to a wash step and interrogated using a CytoFLEX LX cytometer (Beckman Coulter, Indianapolis, IN, USA).For downstream data processing, Kaluza Analysis v 2.1 software, also from Beckman Coulter (Indianapolis, USA) was used.Routine quality assurance for the CytoFLEX LX system was maintained using CytoFLEX Ready to Use Daily QC Fluorospheres reagents (Beckman Coulter, Indianapolis, USA).Figures 1 and2 depict the resultant analysis from the samples.Flow cytometric analysis and gating strategy.This depiction showcases the gating approach utilized to discern subpopulations of T and B lymphocytes.Specifically, we quantified the events expressing CD4+ CD3+ (represented in violet), CD8+ CD3+ (highlighted in green), and CD19+ CD3-(shown in orange) phenotypes.These subpopulations were further examined for expression of molecular markers including CD200, CD200R, CTLA-4, CD86, PD-1, and PD-L1.To bolster the precision of our gating and data interpretation, fluorescence minus one (FMO) controls were incorporated as negative benchmarks, helping to define the optimal gating parameters. ",
"section_name": "Lymphocyte Immunophenotyping",
"section_num": "2.3."
},
{
"section_content": "We executed a qualitative analysis to detect the presence of specific IgA, IgM, and IgG antibodies targeted against selected Epstein-Barr virus (EBV) antigens: viral-capsid antigen (VCA), early antigen (EA), and Epstein-Barr nuclear antigen 1 (EBNA1) in the sera of both study and control cohorts.We leveraged commercial ELISA kits designed to discern antibodies of classes IgA, IgG, and IgM specific to these EBV antigens.Protocols followed the stipulated guidelines of the kit manufacturer.The assay used a suite of kits: EBV VCA IgA, EBV EA IgA, EBV EBNA1 IgA, EBV VCA IgG, EBV EA IgG, EBV EBNA1 IgG, EBV VCA IgM, EBV EA IgM, and EBV EBNA1 IgM (Demeditec Diagnostics GmbH, Germany).Absorbance readings were captured using a VictorTM3 microplate reader (PerkinElmer, Waltham, MA, USA).Antibody titers, quantified from these readings, were represented as U/mL, adhering to the calibration guidelines provided by the manufacturer.A titer value surpassing 11 was designated as positive. ",
"section_name": "Serological Profiling of Anti-EBV Specific Antibodies",
"section_num": "2.4."
},
{
"section_content": "The concentration of all tested molecules was evaluated with immunoenzymatic assays using serum samples collected from all participants.Commercial kits were used according to manufacturer instructions: Measurements were conducted using a VictorTM3 reader (PerkinElmer, Waltham, MA, USA). ",
"section_name": "Assessment of Soluble Immune Checkpoint and Ligand Concentrations in Serum",
"section_num": "2.5."
},
{
"section_content": "The acquired data underwent statistical analysis utilizing Tibco Statistica 13.3 software, Palo Alto, CA, USA.The distribution normality of data was assessed using the Shapiro-Wilk test.Differences between groups were analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test.The p-values for Dunn's test were adjusted for multiple comparisons using the Bonferroni method.Spearman's correlation coefficients were used to explore relationships between pairs of variables.ROC curves were used to assess the diagnostic performance of laboratory tests for patient-related parameters.Data visualizations were generated using GraphPad Prism Software v. 9.4.1,San Diego, CA, USA. ",
"section_name": "Statistical Analysis of Obtained Data",
"section_num": "2.6."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "In order to assess selected parameters of immune system functioning, with particular emphasis on the relationship between PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200 in the context of EBV reactivation, all patients included in this study were tested for the assessment of serological profiles and the number of EBV copies in the tested genetic material, which was the basis for the classification of patients into EBV+ (showing EBV reactivation) and EBV-(patients without reactivation).The analysis of serological profiles of antibodies against specific EBV antigens such as EA (early antigen), VCA (viral-capsid antigen), and EBNA-1 (Epstein-Barr virus nuclear antigen 1) in classes IgA, IgM, and IgG showed EBV reactivation in 60% of patients with CLL and 57.69% of patients with CVID.No EBV reactivation was observed in healthy volunteers enrolled in this study.Detailed antibody serum concentration data and their statistical analyses are presented in Table 1 and Figure 3A-I.The observed concentration levels of individual antibodies directed against specific EBV antigens were extremely diverse between the individual groups of the analyzed patients.However, all antibody concentrations tested in all classes were higher in EBV+ patients than in EBV-patients, both in CLL and CVID patients.Moreover, the statistical analysis of serological profiles between EBV+ CLL and EBV+ CVID patients showed a statistically significant increase in anti-EBA EA IgA (1.41-fold); anti-EBV EA IgG (1.28-fold); anti-EBV VCA IgA (1.38-fold); anti-EBV VCA IgG (1.29-fold); and anti-EBV EBNA-1 IgA (1.20-fold) in CLL patients compared with CVID (Figure 3A,C,D,F,G).The analysis of the obtained results for this experience of patients in the study group in relation to healthy volunteers is presented in Supplementary Materials Table S1. The next step was to assess the number of EBV copies in the genetic material, where EBV copies were present only in CLL EBV+ and CVID EBV+ patients.In the remaining cases, the number of EBV copies extracted with the test was below the detection limit of the test used (i.e., below 10 copies).This means that patients below the detection limit of less than 10 copies were labeled as EBV+ patients, while all patients with more than 11 copies were labeled as EBV+.(Table 2).The mean values obtained for patients with CLL EBV+ were 794.21 ± 56.61 virus copies, and for patients with CVID EBV+, the mean values were 633.98 ± 53.69.The analysis of medians obtained for individual groups of patients showed an increase in the number of viral copies in patients with CLL EBV+ relative to CVID EBV+ by more than 25% (Figure 4).The next step was to assess the number of EBV copies in the genetic material, where EBV copies were present only in CLL EBV+ and CVID EBV+ patients.In the remaining cases, the number of EBV copies extracted with the test was below the detection limit of the test used (i.e., below 10 copies).This means that patients below the detection limit of less than 10 copies were labeled as EBV+ patients, while all patients with more than 11 copies were labeled as EBV+.(Table 2).The mean values obtained for patients with CLL EBV+ were 794.21 ± 56.61 virus copies, and for patients with CVID EBV+, the mean values were 633.98 ± 53.69.The analysis of medians obtained for individual groups of patients showed an increase in the number of viral copies in patients with CLL EBV+ relative to CVID EBV+ by more than 25% (Figure 4).In the next stage of our study, we decided to take a closer look at the selected parameters of peripheral blood, including analyses of morphology, biochemistry, and immunophenotype of patients, taking into account EBV reactivation.In Table 3, we present a comparative analysis of the morphology and biochemistry of peripheral blood from all analyzed patients.As we can see, patients with EBV+ have significantly changed parameters compared with EBV-, both in patients with CLL and CVID.In the case of the first group, we observed an increase in WBC, LYM, MON, and IgM and a decrease in HGB and PLT (Table 3).However, for patients with CVID, we noted a significant decrease in WBC, LYM, MON, HGB, PLT, and IgG (Table 3).The analysis between patients with CLL EBV+ and In the next stage of our study, we decided to take a closer look at the selected parameters of peripheral blood, including analyses of morphology, biochemistry, and immunophenotype of patients, taking into account EBV reactivation.In Table 3, we present a comparative analysis of the morphology and biochemistry of peripheral blood from all analyzed patients.As we can see, patients with EBV+ have significantly changed parameters compared with EBV-, both in patients with CLL and CVID.In the case of the first group, we observed an increase in WBC, LYM, MON, and IgM and a decrease in HGB and PLT (Table 3).However, for patients with CVID, we noted a significant decrease in WBC, LYM, MON, HGB, PLT, and IgG (Table 3).The analysis between patients with CLL EBV+ and CVID EBV+ also showed a significant increase in almost all analyzed parameters, except HGB and IgA in patients with CLL in relation to CVID (Table 3).Of course, we also noted a number of significant changes in selected parameters between individual groups of patients and healthy volunteers, which are presented in detail in Supplementary Materials Table S2.The performed analysis of peripheral blood immunophenotype of all patients also provided extremely valuable information about the state of the immune system for each of the analyzed groups.Of course, some results of peripheral blood immunophenotyping are characteristic of the analyzed disease subunits, such as a higher number of CD19+ B cells in CLL patients and a higher number of TCD3+ cells in CVID patients.The obtained test results and their statistical analysis are presented in Table 4 and Supplementary Materials Table S3 (in relation to healthy volunteers). EBV reactivation significantly increases the percentage of CD3+ lymphocytes and CD8+ T lymphocytes or CD19+ B lymphocytes in patients diagnosed with CLL.In patients with CVID, no statistically significant changes were observed in the percentage of tested immune cell populations in the context of EBV reactivation (Table 4).However, a comparative analysis of both groups of EBV+ patients with each other showed statistically significant changes in almost every cell population, with the exception of the CD4+/CD8+ ratio. ",
"section_name": "Classification and Characteristics of Selected Peripheral Blood Parameters of Patients with CLL and CVID in the Context of EBV Reactivation",
"section_num": "3.1."
},
{
"section_content": "After a detailed characterization of the basic parameters of the patients included in this study, we decided to take a closer look at the impact of EBV reactivation on the percentage of selected T and B lymphocyte subpopulations positive for PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200.The test data obtained are presented collectively in Table 5 and graphically for individual molecules in Figures 567. Cancers 2023, 15, x FOR PEER REVIEW 16 of 35 ",
"section_name": "Analysis of the Effect of EBV Reactivation on the Percentage of Lymphocytes Expressing Positive Immune Checkpoints and Their Ligands",
"section_num": "3.2."
},
{
"section_content": "After a detailed characterization of the basic parameters of the patients included in this study, we decided to take a closer look at the impact of EBV reactivation on the percentage of selected T and B lymphocyte subpopulations positive for PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200.The test data obtained are presented collectively in Table 5 and graphically for individual molecules in Figures 567.Analysis of EBV reactivation in CLL patients showed a significant increase in the percentage of all analyzed checkpoints and their ligands in all T and B lymphocyte subpopulations compared with EBV-patients (Table 5).In the case of PD-1, the increase was correspondingly higher by 2.20 times for CD4+; 2.55-fold for CD8+; and 2.47-fold for CD19+ (Figure 5A-C), while PD-L1 was 1.86-fold for CD4+, respectively; and 3.00-fold for CD8+ and 2.28-fold for CD19+ (Figure 5D-F).Consecutive changes in CTLA-4 expression were: 2.97-fold for CD4+; 2.07-fold for CD8+; and 2.96-fold for CD19+ (Figure 6A-C), and for CD86: 1.77-fold for CD4+; 1.72-fold for CD8+; and 1.42-fold for CD19+ (Figure 6D-F).For CD200R, these values were 2.74 times, 3.07-fold, and 2.34-fold higher for CD4+, CD8+, and CD19+, respectively (Figure 7A-C); and for CD200: 3.13-fold for CD4+; 2.62-fold for CD8+; and 1.26-fold for CD19+ (Figure 5D-F).These changes emphasize that EBV reactivation significantly increases the expression of the tested immunological checkpoints and their ligands, which may affect the deregulation of the functioning of the immune system.Similar changes were observed in patients with CVID showing EBV reactivation.The following changes were noted for this group of patients: 1.69-fold CD4+ PD-1+; 3.48-fold CD8+ PD-1+; 2.62-fold CD19+ PD-1+ (Figure 4A-C); 2.22-fold CD4+ PD-L1+; 5.58-fold CD8+ PD-L1+; and 3.01-fold CD19+ PD-L1+ (Figure 5D-F).For the values observed for CTLA-4: 2.77-fold for CD4+; 4.77-fold for CD8+; and 4.28-fold for CD19+ (Figure 6A-C), and for CD86: 1.51-fold for CD4+; 1.97-fold for CD8+; and 1.25-fold for CD19+ (Figure 6D-F).For the CD200R/CD200 pathway, these changes were: 2.49-fold CD4+ CD200R; 2.74 times CD8+ CD200R+; and 2.04 times CD19+ CD200R+ (Figure 7A-C) and 1.95 times CD4+ CD200+; 3.17-fold for CD8+ CD200+; and 1.51-fold for CD19+ CD200+ (Figure 7D-F). Comparative analysis of patients with CLL EBV+ and CVID EBV+ also showed a number of significant statistical changes in almost all analyzed percentages of T and B lymphocytes positively expressing all tested immunological checkpoints and their ligands, with the exception of CD8+ CD200R+ (Table 5).Moreover, almost most of the analyzed parameters were higher in patients with CLL EBV+, with the exception of CD8+ PD-1+ and CD8+ CTLA-4+, which were 1.16 times and 1.32 times higher, respectively, in patients with CVID EBV+. The analysis of the percentage of T and B lymphocytes positively expressing all tested immunological checkpoints and their ligands in the compared groups of patients in relation to healthy volunteers is presented in Supplementary Materials Table S4. ",
"section_name": "Analysis of the Effect of EBV Reactivation on the Percentage of Lymphocytes Expressing Positive Immune Checkpoints and Their Ligands",
"section_num": "3.2."
},
{
"section_content": "Due to such significant changes observed in the immunophenotypic picture, we also decided to assess the serum concentrations of all tested immunological checkpoints and their ligands in patients included in this study.The test results obtained are presented in Table 6 and Figure 8.The presented study results confirm the changes observed in the immunophenotype.Here, patients with EBV+ also showed higher serum concentrations of soluble forms of all analyzed molecules in relation to the thigh of EBV-patients in both study populations.In patients with CLL, these changes ranged from 1.30-fold for sPD-1 to 1.54-fold for sCD200, and for patients with CVID, from 1.32-fold for sCD86 to 1.86-fold for sCTLA-4.A detailed analysis of EBV+ patients between both patient populations showed an increase in the concentration of all molecules tested in CLL patients relative to CVID of 1.20-fold for sCD200; 1.50 times for sCD86; 1.73 times for sCD200R; 1.85-fold for sCTLA-4; 2.15 times for sPD-1; and 3.50 for sPD-L1 (Figure 8).The statistical significance observed between the study groups of patients and healthy volunteers are listed in Supplementary Materials Table S5.The presented study results confirm the changes observed in the immunophenotype.Here, patients with EBV+ also showed higher serum concentrations of soluble forms of all analyzed molecules in relation to the thigh of EBV-patients in both study populations.In patients with CLL, these changes ranged from 1.30-fold for sPD-1 to 1.54-fold for sCD200, and for patients with CVID, from 1.32-fold for sCD86 to 1.86-fold for sCTLA-4.A detailed analysis of EBV+ patients between both patient populations showed an increase in the concentration of all molecules tested in CLL patients relative to CVID of 1.20-fold for sCD200; 1.50 times for sCD86; 1.73 times for sCD200R; 1.85-fold for sCTLA-4; 2.15 times for sPD-1; and 3.50 for sPD-L1 (Figure 8).The statistical significance observed between the study groups of patients and healthy volunteers are listed in Supplementary Materials Table S5. ",
"section_name": "Analysis of the Effect of EBV Reactivation on Serum Concentrations of Soluble Forms of Immunological Checkpoints and Their Ligands",
"section_num": "3.3."
},
{
"section_content": "Due to the observed changes, we decided to analyze the effect of EBV reactivation on selected parameters of the immune system of patients with CLL and CVID.The analysis performed is graphically presented in Supplementary Materials Figure S1 for CLL patients and Figure S2 for CVID patients. Due to the size of the sample, only several dozen statistically significant correlations were recorded in particular groups of patients.For CLL EBV+ patients, we observed 36 significant correlations, of which 10 were negative (four moderate and 6 low) and 26 were positive (seven moderate and 19 low) (Supplementary materials Table S6). For CLL EBV-patients, there were 25 significant correlations, of which 11 were negative (two high and 9 moderate) and 14 were positive (one high and 13 moderate) (Supplementary materials Table S7). In CVID EBV+ patients, there were 28 significant correlations, of which 15 were negative (one high, 10 moderate, and four low) and 13 were positive (10 moderate and three low) (Supplementary materials Table S8). In patients with CVID EBV-, there were 29 significant correlations, of which 14 were negative (one high and 13 moderate) and 15 were positive (one high and 14 moderate) (Supplementary materials Table S9).The last stage of the analysis was the assessment of the sensitivity and specificity of the observed changes in the immunophenotype and serological profiles, with particular emphasis on the tested immunological checkpoints and their ligands in the context of EBV reactivation in patients with CLL and CVID. In the first step, we evaluated the results obtained from analyzing serological profiles of antibodies against specific EBV antigens to confirm their diagnostic effectiveness.These analyses confirmed that for both CLL and CVID patients, highly specific markers of EBV reactivation within the studied populations are: anti-EBV EA IgA and IgG; anti-EBV VCA IgA and IgM; and anti-EBV EBNA-1 IgA and IgG (Figure 9A,B).However, in the case of comparing the results obtained between CLL EBV+ and CVID EBV+ patients, we cannot unambiguously indicate the most sensitive marker indicator to distinguish between these two disease entities (Figure 9C).However, such a function may be performed by assessing the number of EBV virus copies in the examined genetic material (Figure 10). The analysis also shows that in the case of patients with CLL EBV+ in relation to CLL EBV-, a highly specific marker may be: the percentage of CD4+ PD-1+ and CD8+ PD-1+; percentage of total lymphocytes positive for PD-L1 (Figure 11A) and CTLA-4 (Figure 12A); the percentage of CD4+ CD86+ and CD19+ CD86+ (Figure 12A); and the percentage of total lymphocytes positive for CD200R and CD200 expression (Figure 13A). In the case of evaluating the serum concentration of soluble forms of the test molecules, almost all, except for sPD-1 and sCTLA-4, may be potential markers associated with EBV reactivation in this group of patients (Figure 14A). We observed similar trends in patients with CVID.Of particular note, the potential biomarker molecules of EBV reactivation status are the percentage of CD8 + PD-1 and CD19+ PD-1+ (Figure 11B); the percentage of all tested lymphocytes positive for PD-L1 (Figure 11B), CTLA-4 and CD86 (Figure 12B), and CD200R (Figure 13B) as well as the percentage of CD4+ CD200+ and CD8+ CD200+ (Figure 13B).Serum concentration analysis of the soluble forms of the tested molecules showed that all except sCD86 could be a potential biomarker molecule (Figure 14B). We recently analyzed the relationship between CLL EBV+ and CVID EBV-patients, which showed a significant advantage of CD4+ PD-1 and CD8+ PD-1 (Figure 11C); CD19+ CTLA-4+ and CD19+ CD86+ (Figure 12C); and all serum concentrations of the molecules tested except for sCD200 (Figure 14C).Detailed results from this analysis are provided in Supplementary Materials Table S10.The analysis also shows that in the case of patients with CLL EBV+ in relation to CLL EBV-, a highly specific marker may be: the percentage of CD4+ PD-1+ and CD8+ PD-1+; percentage of total lymphocytes positive for PD-L1 (Figure 11A) and CTLA-4 (Figure 12A); the percentage of CD4+ CD86+ and CD19+ CD86+ (Figure 12A); and the percentage of total lymphocytes positive for CD200R and CD200 expression (Figure 13A). In the case of evaluating the serum concentration of soluble forms of the test molecules, almost all, except for sPD-1 and sCTLA-4, may be potential markers associated with EBV reactivation in this group of patients (Figure 14A). We observed similar trends in patients with CVID.Of particular note, the potential biomarker molecules of EBV reactivation status are the percentage of CD8 + PD-1 and CD19+ PD-1+ (Figure 11B); the percentage of all tested lymphocytes positive for PD-L1 (Figure 11B), CTLA-4 and CD86 (Figure 12B), and CD200R (Figure 13B) as well as the percentage of CD4+ CD200+ and CD8+ CD200+ (Figure 13B).Serum concentration analysis of the soluble forms of the tested molecules showed that all except sCD86 could be a potential biomarker molecule (Figure 14B). We recently analyzed the relationship between CLL EBV+ and CVID EBV-patients, which showed a significant advantage of CD4+ PD-1 and CD8+ PD-1 (Figure 11C); CD19+ CTLA-4+ and CD19+ CD86+ (Figure 12C); and all serum concentrations of the molecules tested except for sCD200 (Figure 14C). Detailed results from this analysis are provided in Supplementary Materials Table S10. ",
"section_name": "Influence of EBV Reactivation on Correlations of Selected Analyzed Parameters of the Immune System",
"section_num": "3.4."
},
{
"section_content": "The presented study is a correlational analysis of two important aspects including EBV reactivation and the expression of immune checkpoints and their ligands in the context of two diseases, representing PID-CVID and SID-CLL.The observed significant increase in the expression of immune checkpoints and ligands after EBV reactivation highlights the potential role of this virus in immune dysregulation.The fact that similar changes were observed in both CLL and CVID patients indicates the universality of the effect of EBV on the immune system, regardless of the underlying disease.These findings are consistent with previous studies suggesting a link between EBV and immune modulation, especially in CLL patients.In the work by Gamaleldin et al. from 2021 [44], the researchers showed that high expression of PD-1 and PD-L1 together with high EBD-DNA load was associated with a worse prognosis in CLL.Moreover, studies have shown that B cell lymphomas that are associated with EBV infection express PD-L1, which binds to PD-1, inhibiting T cell cytotoxicity.In the same context, CLL T cells show upregulation of inhibitory molecules, such as PD-1 and PD-L2.In addition, researchers have suggested that PD-1 and PD-L1 may be suitable therapeutic targets for patients suffering from aggressive CLL [44].This is also confirmed by research by Malpic et al. [45], which showed that CD30 and PD-1/PD-L1 expression appear as potentially unfavorable but targetable biomarkers in EBV-positive diffuse large B-cell lymphoma, and as indicated by the Ozturk study in the course of classical Hodgkin lymphoma [46].The relationship between EBV reactivation and the PD-1/PD-L1 pathway has been quite well documented in the course of many cancers, including gastric cancer [47,48], breast cancer [49], and nasopharyngeal carcinoma [50], where EBV reactivation was associated with worsening of the tested clinical parameters and a worse prognosis of patients. The studies conducted by our research team on the interaction between CTLA-4 and CD86 in the course of CLL in the context of EBV reactivation, as well as the currently presented studies, show how important this pathway is in inhibiting the anti-cancer response.The disorders described above are closely related to the presence of EBV genetic material in PBMCs in patients with CLL and the production of antibodies against antigens of this virus, which indicates their involvement in the abnormal cellular response in the course of CLL [51].The importance of CTLA-4/CD86 is also confirmed in research by other scientists, e.g., work by Mittal et al. [28], Do et al. [29], and Ma et al. [52].As in the case of PD-1, the CTLA-4 molecule and its participation in the pathogenesis of neoplastic diseases, including EBV reactivation, is quite well documented in the literature, especially in gastric cancer [53,54], breast cancer [55], and epithelial cancers [56]. Studies on the participation of the last CD200R/CD200 pathway analyzed in our work are also scarce.The only study on the relationship between the presence of EBV and the studied pathway in the course of leukemia concerns its immunohistochemical assessment by Christopher Batuello and Emily F Mason from 2023 [57].Researchers have shown that CD200 can help differentiate Epstein-Barr virus-positive (EBV+) classical Hodgkin lymphoma (CHL) from common Reed-Sternberg cell (RS) and EBV+ large B-cell lymphoma (LBCL). Very little information can be found on the impact of EBV reactivation and the relationship between immune checkpoints in the course of CVID and its consequences related to the development of neoplastic diseases.Data from the literature indicate that EBV infection in patients with PID may lead to immune dysregulation and increased risk of malignancies, in addition to the severe course of acute infection.Researchers also emphasize that the recognition of various genetic defects and their impact on immune pathways provide us with fundamental insights into the pathophysiology of EBV infection and related diseases and may lead to the development of better-targeted therapies in the future.The studies so far compare EBV infections to several PIDs, which are presented in Table 7. Comparing CLL and CVID patients with EBV reactivation in this study reveals intriguing differences and similarities in the immune responses of these two patient populations.While both groups show elevated expression of checkpoints and ligands, CLL patients seem to show more pronounced changes.This discrepancy raises questions about potential disease-specific factors that may contribute to changes in immune modulation after EBV reactivation; however, the specific mechanisms require further multifactorial research. The study of serum concentrations of soluble forms of the analyzed molecules suggests the potential utility of these molecules as biomarkers of EBV reactivation.The distinct patterns observed in CLL and CVID patients, particularly in relation to healthy volunteers, highlight the diagnostic potential of these markers.However, the presented research results concern a small population of patients; therefore, it will be necessary to perform further studies in order to determine the sensitivity and specificity of these markers and their ability to differentiate disease entities for a much larger group of patients. The present study raises important questions about the functional implications of the observed changes in immune checkpoints and ligands.Increased expression of these molecules may indicate an elevated state of immune activation or exhaustion, potentially affecting immune surveillance, infection response, and disease progression.Future research should aim to elucidate the functional consequences of these changes and their implications for patient outcomes.The marked changes in immune checkpoints and ligands suggest that targeting these pathways may hold promise as a therapeutic approach in CLL and CVID patients with EBV reactivation.Strategies that modulate these checkpoints have the potential to rebalance the immune system and enhance antiviral responses.However, careful consideration of the broader immune landscape and potential off-target effects is critical when designing such interventions. The studies presented in this publication have some limitations and show only a small part of the changes that occur during the reactivation of the EBV virus.This study's focus on specific molecules and pathways may not capture the full complexity of immune dysregulation after EBV reactivation, and the observed changes may be incomplete due to sample size.Future research should explore broader immunological profiling to gain a fuller understanding of the changes that occur in CLL and CVID patients and to monitor changes in their bodies over a longer period of time. Taken together, the analysis of immune checkpoints and ligands in the context of EBV reactivation in CLL and CVID patients sheds light on the intricate relationship between viral infection and immune dysregulation.The findings provide valuable insights into potential diagnostic markers, therapeutic targets, and broader implications of immune modulation in these patient populations.However, further research is needed to uncover the underlying mechanisms, functional consequences, and clinical applications of these observed changes.The heightened vulnerability of XLP-1 patients to EBV infection is likely a result of diminished NK cytolytic activity and reduced CD8+ T cell killing.This susceptibility stems from EBV's strong attraction to B cells, coupled with the compromised ability of T and NK cells to interact with B cells due to a deficiency in the SLAM receptor-associated pathway. [ 58,59] CD27 deficiency These patients were documented to exhibit symptomatic primary EBV infection or lymphadenopathy during early life, and a subset also experienced persistent EBV viremia.About half of the individuals developed a malignant neoplasm. [60] ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Patients with RASGRP1 deficiency commonly display a clinical profile characterized by recurrent infections, enlarged liver and spleen (hepatosplenomegaly), swollen lymph nodes (lymphadenopathy), EBV-related excessive lymph cell growth, and the emergence of B-cell lymphoma.Additionally, they may also present autoimmune traits like autoimmune hemolytic anemia, thrombocytopenia, and uveitis. [61,62] CD70 deficiency The clinical signs of CD70 deficiency closely mirror those of CD27 deficiency.In both cases, patients universally exhibit EBV viremia, and the majority of them go on to develop EBV-associated lymphoproliferation or B-cell malignancy, alongside conditions like hypogammaglobulinemia and compromised targeted antibody responses.[63,64] Deficiency of the actin regulator-coronin 1A Dysfunctional calcium flux and the buildup of β-actin at the immune synapse lead to heightened T cell apoptosis and a reduction in CD4+ lymphocyte count.In the case of coronin 1A deficiency, individuals experienced profound infections, and five of them went on to develop B-cell lymphoma induced by EBV. [ 65,66] Serine/threonine kinase 4 (STK4) deficiency The aberrations in the immune system result in autoimmunity, EBV viremia, and the recurrence of sinopulmonary and mucocutaneous infections, predominantly associated with herpes viruses.Additionally, patients also face susceptibility to other infections caused by viruses such as molluscum contagiosum, fungi like candidiasis, and bacteria like staphylococci.A deficit in the Fas-mediated apoptotic pathway could elevate the susceptibility to EBV-related lymphomas.Nonetheless, it is plausible that over extended periods of virus transmission, one of the mechanisms by which the immune system regulates EBV within the B cell framework involves the process of Fas-mediated cell elimination. [71] ",
"section_name": "RASGRP1 deficiency",
"section_num": null
},
{
"section_content": "In conclusion, the analysis of EBV reactivation in patients with chronic lymphocytic leukemia (CLL) and common variable immunodeficiency (CVID) revealed substantial alterations in the expression of immunological checkpoints and their ligands across various T and B lymphocyte subpopulations.This investigation underscores a significant increase in the percentages of these checkpoints and ligands in both patient groups compared with EBV-negative individuals.Specifically, the PD-1, PD-L1, CTLA-4, CD86, CD200R, and CD200 pathways exhibited pronounced upregulation in response to EBV reactivation.Notably, the observed changes were more prominent in CLL patients with EBV reactivation compared with those in CVID patients with the same condition.These findings suggest that EBV reactivation plays a crucial role in modifying the immune landscape, potentially contributing to immune dysregulation in both CLL and CVID patients.Furthermore, this study's serological analysis demonstrated elevated serum concentrations of soluble forms ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Data Availability Statement: All necessary information regarding the preparation of this work is available on written request from the corresponding author. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "of the analyzed molecules in EBV-positive patients, further affirming the impact of EBV reactivation on immune system modulation.The diagnostic potential of several markers was assessed, revealing distinct patterns that could aid in differentiating between CLL and CVID patients with EBV reactivation.In essence, this correlation analysis highlights the intricate interplay between EBV reactivation and the expression of immunological checkpoints and their ligands in CLL and CVID patients.These findings offer valuable insights into the potential mechanisms underlying immune dysfunction in these conditions and emphasize the significance of further research to elucidate the precise implications of these alterations for patient prognosis and therapeutic interventions. The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15194786/s1,Table S1.Statistical analysis of the concentration of antibodies against specific EBV antigens in the serum of patients with CLL and CVID, taking into account EBV reactivation in relation to HV.Table S2.Statistical analysis of selected parameters of the morphology and biochemistry of peripheral blood of patients with CLL and CVID, including EBV reactivation in relation to HV.Table S3.Statistical analysis of selected peripheral blood immunophenotype parameters of patients with CLL and CVID, including EBV reactivation in relation to HV.Table S4.Statistical analysis of the percentage of T and B lymphocytes positively expressing the tested immunological checkpoints and their ligands in patients with CLL and CVID, including EBV reclamation against HV.Table S5.Statistical analysis of serum concentrations of tested immunological checkpoints and their ligands in patients with CLL and CVID, including EBV reclamation against HV.Table S6.Spearman rank correlations for CLL EBV+ patients.Table S7.Spearman rank correlations for CLL EBV-patients.Table S8.Spearman rank correlations for CVID EBV+ patients.Table S9.Spearman rank correlations for CVID EBV-patients.Table S10 Informed Consent Statement: Before participating in this study, patients provided informed consent.This ensured that each participant was fully aware of the study's objectives, along with its associated risks and benefits. The authors declare no conflict of interest.The funders had no role in the design of this 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": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "of the analyzed molecules in EBV-positive patients, further affirming the impact of EBV reactivation on immune system modulation.The diagnostic potential of several markers was assessed, revealing distinct patterns that could aid in differentiating between CLL and CVID patients with EBV reactivation.In essence, this correlation analysis highlights the intricate interplay between EBV reactivation and the expression of immunological checkpoints and their ligands in CLL and CVID patients.These findings offer valuable insights into the potential mechanisms underlying immune dysfunction in these conditions and emphasize the significance of further research to elucidate the precise implications of these alterations for patient prognosis and therapeutic interventions. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15194786/s1,Table S1.Statistical analysis of the concentration of antibodies against specific EBV antigens in the serum of patients with CLL and CVID, taking into account EBV reactivation in relation to HV.Table S2.Statistical analysis of selected parameters of the morphology and biochemistry of peripheral blood of patients with CLL and CVID, including EBV reactivation in relation to HV.Table S3.Statistical analysis of selected peripheral blood immunophenotype parameters of patients with CLL and CVID, including EBV reactivation in relation to HV.Table S4.Statistical analysis of the percentage of T and B lymphocytes positively expressing the tested immunological checkpoints and their ligands in patients with CLL and CVID, including EBV reclamation against HV.Table S5.Statistical analysis of serum concentrations of tested immunological checkpoints and their ligands in patients with CLL and CVID, including EBV reclamation against HV.Table S6.Spearman rank correlations for CLL EBV+ patients.Table S7.Spearman rank correlations for CLL EBV-patients.Table S8.Spearman rank correlations for CVID EBV+ patients.Table S9.Spearman rank correlations for CVID EBV-patients.Table S10 Informed Consent Statement: Before participating in this study, patients provided informed consent.This ensured that each participant was fully aware of the study's objectives, along with its associated risks and benefits. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1186/s12886-017-0532-1
|
Notch1 signaling induces epithelial-mesenchymal transition in lens epithelium cells during hypoxia
|
Posterior Capsular Opacification (PCO) is one of the most common complications of cataract surgery which can result in severe visual damage. Epithelial-Mesenchymal Transition (EMT) of lens epithelium cells (LEC) is the pathological basis of PCO. Recent research showed that hypoxia acted as an inducer of EMT through a Notch1/Snail1/E-cadherin pathway. However, it remains unclear whether the Notch1/Snail1/E-cadherin pathway is involved in PCO under hypoxia.The morphology of SRA01/04 cells treating with Cobalt Chloride (CoCl2) was observed and the markers of EMT and Notch1/Snail1/E-cadherin pathway were analyzed by Western blot and Immunocytochemistry assay. Transwell invasion assay and Wound healing assay were used to detected the effect of p3 × FLAG-CMV-7-NICD1 transfection on the SRA01/04 cells.The SRA01/04 cells lost cell polarity and cell junction culturing with CoCl2. The expression of Keratin, Hypoxia-inducible factor-1 alpha (HIF-1α), Notch1, Snail1were upregulated, on the other side, Fibronectin and E-cadherin were downregulated in hypoxia. Furthermore, the overexpression of Notch1 induced the expression of E-cadherin and increased the invasion and migration ability of SRA01/04 cells.These results suggest that Notch1/Snail1/E-cadherin pathway facilitates the EMT through HIF-1α in SRA01/04 cells during hypoxia and promotes LEC motility.
|
[
{
"section_content": "Cataract surgery is the most common ophthalmic surgical procedure in now days.However, Posterior Capsular Opacification (PCO), which can result in severe visual damage, is one of the most common complications of modern cataract surgery [1].Close examination of lens cell populations in human donor capsular bags with implanted intraocular lenses [2][3][4] revealed that the Epithelial-Mesenchymal Transition (EMT) of LEC migration from the anterior capsular membrane is the pathological basis of PCO. The lens metabolizes actively and requires nourishment to maintain its growth and transparency.However, the lens need lower energy demands, lack of aerobic respiration and consume little oxygen comparing to other tissues in the eye [5].The anterior portion of lens absorbs the nutrition including oxygen from the anterior chamber, aqueous humor, while the oxygen supply of the posterior portion of the lens is maintained by the vitreous [6].The oxygen tension in aqueous humor is much higher than that in the vitreous [7][8][9], which creates an oxygen-rich environment in the anterior portion of the lens but an oxygen-poor environment in the posterior part.The lens epithelium is physiologically located only in the oxygen-rich anterior portion of the lens.After cataract surgery, the LECs move to the posterior portion of the lens to a hypoxic microenvironment, where it may undergo EMT. EMT is defined as a transition from epithelial cells to mesenchymal cells [10] and is an essential process not only in development [11], but also in fibrosis [12], tumor metastasis [13,14], and wound healing [15].In this process, epithelial cells down-regulate the expression of cell adhesion molecule (such as E-cadherin), deposit ECM proteins, dissolve cell-cell junctions, lose their apical-basal polarity, and obtain migratory and invasive behavior [16,17].Due to the change in oxygen environment during EMT, hypoxia has gained significant attention in the recent research of EMT [18,19].Variation of the microenvironmental oxygen levels and activation of hypoxic signaling pathway through Hypoxia-Inducible Factor (HIF) are regarded as significant triggers and modulators of EMT [20]. Notch1/Snail1/E-cadherin pathway is recently reported to induce EMT in several diseases [21][22][23][24].The Notch gene was discovered by Morgan T.H.in Drosophila melanogaster nearly a century ago [25], the absence of which causes a 'notched' defective wing in drosophila.Notch signaling influenced several cellular processes, including proliferation, differentiation and apoptosis [26].Notch1 signaling pathway also played an essential role in the growth and differentiation in developing lens [27,28].HIF-1α enhances Notch signaling [29] and interacts with Notch Intracellular domain, NICD [30], the intracellular activating portion of Notch.Although the activation of Notch signaling induces EMT, it remains unclear whether the Notch1/ Snail1/E-cadherin pathway is involved in PCO under hypoxia.Therefore, this study investigated the involvement of Notch1/Snail1/E-cadherin signaling in EMT in LEC during hypoxia. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Cell culture SRA01/04 cells (The human Lens Epithelium Cell line) was cultured in RPMI-1640 with 10% fetal bovine serum, 10 U/l penicillin G, and 100 mg/l streptomycin.Hypoxic mimic condition was generated by the treatment with Cobalt Chloride (CoCl 2 , Sigma).The RPMI-1640 medium containing 150 μM CoCl 2 was sterilized by 0.22 μM filters and preserved at 4 °C.The control groups were cultured in the absence of CoCl 2 . ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "SRA01/04 cells were treated with 150 μM CoCl 2 or not .Cells were fixed in 4% paraformaldehyde, then permeabilized in 0.5% Triton X-100 three times.After blocking with 0.5% bovine serum albumin (BSA) for 2 h, the cells were incubated with each primary antibodies in a wet box at 4 °C overnight.The primary antibodies used were mouse anti-Keratin (1:500) and rabbit anti-Fibronectin (1:500) (Thermo Fisher, USA).On the following day, cells were incubated with secondary antibody diluted 1:500 (Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody, Thermo Fisher, USA) for 2 h at room temperature.Then 5 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA) was used to stain nuclei.Immunostaining was visualized by using a Nikon Eclipse 80i microscope (20 × magnification) and digitally imaged with NIS-Elements F 2.3 software package.Assessment of the staining intensity was based on the ratio of Keratin and Fibronectin to DAPI respectively.The Data presented are representative of five randomly selcected fields and five cells were measured in each field.The immunocytochemistry assay was repeated three times. ",
"section_name": "Immunocytochemistry",
"section_num": null
},
{
"section_content": "The p3 × FLAG-CMV-7-NICD1 plasmid was obtained from Raphael Kopan (Addgene plasmid # 20183).The SRA01/04 cells were transfected with 3 μg specific p3 × FLAG-CMV-7-NICD1 for 24 h, using Lipofectamine™ LTX reagent(Invitrogen) in serum-free RPMI-1640 medium according to the manufacture's instruction.Control SRA01/04 cells were transfected with p3 × FLAG-CMV-7(Sigma).Western blot was used to verify the efficiency of transfection. ",
"section_name": "Plasmid and transfection",
"section_num": null
},
{
"section_content": "Different groups of SRA01/04 cells were harvested and lysed in using cell lysis buffer (Beyotime, China).The lysed proteins were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane.After blocking, the membrane was incubated with primary antibodies (Notch1 1:1000, Snail 1:1000, E-cadherin, Keratin, Fibronectin and GAPDH 1:500), followed by correspongding horseradish peroxidase-conjugated secondary antibodies (1:2000) for 90 min at RT.The protein bands were detected by enhanced chemiluminescence (SuperSignal West Pico.Thermo, USA) and recorded using a DNR Bio-Imaging System (Israel).The ratio of the optical densities between Notch-1, Snail, E-cadherin, Keratin, Fibronectin and GAPDH represent the relative protein expression.The primary antibodies against Notch1 and Snail were from Cell Signaling Technology (USA).The primary antibodies against E-cadherin, Keratin, Fibronectin, GAPDH and the secondary antibody were form Bioss (China).WB was repeated three times and three samples were used in each time. ",
"section_name": "Western blot and antibodies",
"section_num": null
},
{
"section_content": "24-well Transwell chambers (8-μm pore size, Corning) were used in transwell invasion assay.The transwell filters were covered with matrigel (50 μl) on the upper surface of the polycarbonic membrane.At 24 h after transfection, 4 × 10 4 cells were seeded in the upper chamber in RPMI1640 medium without serum.The lower chamber was filled with the same medium with 10% FBS.48 h later, non-invading cells were cleaned.The invading cells were fixed in 4% paraformaldehyde and stained by Crystal Violet.The number of invaded cells was counted in three randomly selected fields at high magnification.Data presented are representative of three individual wells and transwell invasion assay was repeated three times. Wound healing assay SRA01/04 cells were cultured and transfected in a 6-well plate.A 10 μl pipette tip was used to make a straight scratch through the monolayers.In the subsequent period, cells migrated into the wound surface.The scratch width was used to represent the migrating ability of SRA01/04 cells.24 h later, the scratch width was measured in five randomly selected fields.Data presented are repeated three times. ",
"section_name": "Transwell invasion assay",
"section_num": null
},
{
"section_content": "All the statistical analyses were performed with SPSS13.0 using one-way ANOVA followed by S-N-K post-hot test.p < 0.05 is considered significantly difference. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Effect of CoCl 2 treatment on the SRA01/04 cells CoCl 2 is mostly used to mimic hypoxic conditions in cell cultures.we first examined the morphology changes of SRA01/04 cells cultured in CoCl 2 for 12,24 and 48 h (Fig. 1a-c).The most obvious changes happened when cells were cultured in CoCl 2 for 48 h.They had no cell polarity and lost cell junction (Fig. 1c), the characteristics of mesenchymal cells.In addition, 48 h CoCl 2 treatment induced a significant decrease in the expression of Keratin, and an increase in the expression of Fibronectin (p < 0.05) (Fig. 1g-i).So the expression of Keratin and Fibronectin in SRA01/04 cells were also tested in immunocytochemical assay especially on 48 h (Fig. 2).As a result, the red fluorescence (Fibronectin) was most obvious (Fig. 2j,p<0.05)and the green fluorescence (Keratin) weakened distinctly (Fig. 2i, p<0.05) in SRA01/04 cells treated by CoCl 2 for 48 h. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Western Blot analysis showed that the expression of HIF-1α (Fig. 3a &b), Notch1 (Fig. 3a &c), Snail1 (Fig. 3d & e) all increased after 48 h CoCl 2 treatment in SRA01/04 cells, (p < 0.05) while the E-cadherin expression was decreased (Fig. 3d & f, p<0.05) ",
"section_name": "Expression of HIF-1α,Notch1, Snail1 and E-cadherin in SRA01/04 cells following treatment with cobalt chloride",
"section_num": null
},
{
"section_content": "Notch1 was then overexpressed in SRA01/04 cells to examine the effect of Notch1/Snail1/E-cadherin activation.SRA01/04 cells were transfected with p3 × FLAG-CMV-7-NICD1 (Fig. 4 group3), using cells transfected with empty vector p3 × FLAG-CMV-7 (Fig. 4 group1) and non-transfected cells (Fig. 4 group2) as controls.The protein level of Notch1 was confirmed to be upregulated in group 3 (Fig. 4a &b), compared with group1 and group2.Snail1 was correspondingly upregulated (Fig. 4a &c) and E-cadherin was downregulated (Fig. 4a &d) in group3. ",
"section_name": "Overexpression of Notch1 decreased expression of Ecadherin via activation of Snail1",
"section_num": null
},
{
"section_content": "Transwell assay was performed to study the effect of Notch1 on migration ability of SRA01/04 cells.As shown (group 3) and increased expression of Snail1 and decreased expression of E-cadherin compared with negative control either transfected with empty vector p3 × FLAG-CMV-7 (group 1) or non-transfected cells (group 2).e & g Representative images (g) and average number of invaded cells in the lower chamber during transwell assay 24 h after transfection of empty vector (group1), non-transfected (group2) and transfected with Notch1-NICD (group 3).f & h Representative images (h) and average scratch width in wound healing assay following the same transfection group as e & g in Fig. 3g, the number of cells invaded across the polycarbonate membrane significantly increased in group3 (142.3 ± 4.9) compared with group1 (54.6 ± 5.5) and group2 (51.2 ± 5.2) (Fig. 4e P < 0.05).But the difference between group 1 and group 2 was not significant (P > 0.05). Wound healing assay was also used to examine the effect of Notch1 on cell migration.The scratch width, 24 h post injury reflects the cell migration ability.As shown in Fig. 4h, the narrowest width was founded in group3 (16.2 ± 2.1 μm) compared with group1 (48.0 ± 2.6 μm) and group2 (41.2 ± 2.5 μm) (P < 0.05).No significant difference was found between group 1 and group 2 (Fig. 3f, P>0.05). ",
"section_name": "Overexpression of Notch1 enhanced the migration of SRA01/04 cells",
"section_num": null
},
{
"section_content": "Harold Ridley, a British ophthalmologist, made the first intraocular lens implantation to treat cataract in 1949.From then on, clinical treatment of cataract has improved continuously and studies on the pathogenesis of cataract have advanced significantly.However, PCO is still a common complication of cataract surgery with unknown cause, which is affecting the overall results of cataract treatment.The incidence and severity of PCO correlates to the use of surgical techniques, intraocular lens optic edge designs and intraocular lens materials in clinic [31].On a biochemical level, the proliferation, migration and abnormal differentiation of residual lens epithelial cells and fibers in the capsular bag have been involved in the pathogenesis of PCO.Among these factors, EMT was recognized to play an critical role in the development of PCO. Hypoxia has been associated with EMT through potentiating Notch signaling [32][33][34].HIF-1a also played an essential role in the EMT under hypoxia.Since HIF-1a interacts with Notch-NICD, this suggest that HIF-1a may induce EMT through activating the Notch signaling pathway during hypoxia. In this study, we found that EMT occurs in SRA01/04 cells induced by CoCl 2 .CoCl 2 indicating that hypoxia led to profound morphological changes in LEC.The upregulatin of Notch1,Snail1 and HIF-1a in SRA01/04 cells following treatment suggest that both the Notch1/ Snail1/E-cadherin signaling and HIF-1a are involved in EMT induced by hypoxia. Former studies have indicated that Snail expression can be directly induced by the Notch signaling pathway [35,36].However, this process was never examined in LEC.In addition, the biological function of Notch1/ Snail1/E-cadherin in LEC has never been reported either.In this present study, we confirmed that the expression of Snail is upregulated upon over expression of Notch1 in SRA01/04 cells, suggesting a tight correlation between the regulations of the two proteins in LEC.The enhanced migration ability in SRA01/04 cells following Notch1 overexpression is consistent with the transition of LEC from epithelial cells to mesenchymal cells (typically high mobility) and provides strong evidence on the involvement of Notch1 in EMT in LEC. Therefore, we hypothesize that the residue LEC migrated to the posterior capsule after cataract surgery where the lower oxygen level initiates the Notch1 pathway through HIF-1a in LEC, and promotes the transformation of LEC into mesenchymal cells.This notion is also supported by another research from reverse [37].In this research, active oxygen processing intraocular lenses were used to prevent PCO. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, we showed initially that Notch1/Snail1/E-Cadherin pathway may facilitate the EMT and promote cell motility, possibly through HIF-1a in SRA01/04 cells under hypoxia, which may participate in PCO pathogenesis.Further studies are needed to determine a causative effect of HIF-1a and Notch1 in the occurrence of EMT during hypoxia using gene knockdown or knockout both in cell lines and in animal models.The elucidation of the molecular mechanism of EMT through Notch1/Snail1/E-Cadherin pathway may provide new molecular targets for the prevention and treatment of patients with PCO. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "No additional acknowledgments. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All data generated or analysed during this study are included in this published article.More details are available from the corresponding author on reasonable request. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The ethics committee of Shengjing Hospital of China Medical University (Shenyang, China) ruled that no formal ethics approval was required in this case. 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": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "The ethics committee of Shengjing Hospital of China Medical University (Shenyang, China) ruled that no formal ethics approval was required in this case. ",
"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
}
] |
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