scholarly journals Mouse Models of Germinal Center Derived B-Cell Lymphomas

2021 ◽  
Vol 12 ◽  
Author(s):  
Stefanie N. Meyer ◽  
Sanjay Koul ◽  
Laura Pasqualucci
Keyword(s):  
B Cell ◽  
Mouse Models ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Genetic Alterations ◽  
Genetically Engineered ◽  
Lessons Learned ◽  
B Cell Lymphomas ◽  
Therapeutic Principles

Over the last decades, the revolution in DNA sequencing has changed the way we understand the genetics and biology of B-cell lymphomas by uncovering a large number of recurrently mutated genes, whose aberrant function is likely to play an important role in the initiation and/or maintenance of these cancers. Dissecting how the involved genes contribute to the physiology and pathology of germinal center (GC) B cells –the origin of most B-cell lymphomas– will be key to advance our ability to diagnose and treat these patients. Genetically engineered mouse models (GEMM) that faithfully recapitulate lymphoma-associated genetic alterations offer a valuable platform to investigate the pathogenic roles of candidate oncogenes and tumor suppressors in vivo, and to pre-clinically develop new therapeutic principles in the context of an intact tumor immune microenvironment. In this review, we provide a summary of state-of-the art GEMMs obtained by accurately modelling the most common genetic alterations found in human GC B cell malignancies, with a focus on Burkitt lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma, and we discuss how lessons learned from these models can help guide the design of novel therapeutic approaches for this disease.

CD40L Modulates TRAIL-Induced Apoptosis in Germinal Center Derived B Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4630-4630
Author(s):  
Marion Travert ◽  
Patricia Ame-Thomas ◽  
Thierry Fest ◽  
Céline Pangault ◽  
Gilbert Semana ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Over Expression ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Follicular lymphoma are characterized by the rearrangement of the bcl-2 gene, present in more than 90% of patients. Over-expression of the bcl-2 protein resulting from this translocation is associated with the inability to eradicate the lymphoma, by inhibiting apoptosis. Despite the median survival ranges from 8 to 15 years, leading to the designation of indolent lymphoma, patients with advanced-stage follicular lymphoma are not cured with current therapeutic options. Numerous reports have shown that Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in a wide variety of transformed cell lines of diverse lineage, but does not appear to kill normal cells, even though TRAIL mRNA is expressed at significant levels in most normal tissues. As cell death induced by TRAIL occurs almost exclusively in tumor cells, it suggests that this drug is safe to use as an antitumor therapy. We therefore investigated the efficiency of this cytokine to induce apoptosis in germinal center derived B cell lymphoma, despite bcl-2 over-expression. Our study was also designed to evaluate the role of CD40L, one of the main differentiation signal involved in B cell maturation during the germinal center reaction, on the regulation of TRAIL-induced apoptosis. This study was performed on three germinal center derived tumor cell lines (BL2, VAL and RL), and on normal and tumor primary cells obtained from human tonsils and lymph nodes. Our data show that normal B lymphocytes obtained from tonsil biopsies are resistant to TRAIL-mediated apoptosis, when B lymphoma cells issued from lymph node of numerous patients are significantly sensitive to the cytokine. When we treat these lymphoma cells with trimeric huCD40L, we partly rescue these cells from spontaneous apoptosis which naturally occurs after few days of culture, and reverse by 50% TRAIL-mediated apoptosis when cells were co-treated with huCD40L for 16 hours. Similar results were reproduced on some germinal center derived cell lines. BL2 was indeed found highly sensitive to TRAIL-induced apoptosis following a 24 hour exposure. On the opposite, VAL and RL were almost insensitive. We have demonstrate that apoptosis is exclusively mediated by TRAIL-R1 in BL2. Analysis of signalling pathways revealed that the protection to TRAIL-induced apoptosis by CD40L is due to some specific anti-apoptotic molecules that will be described. Genes encoding these molecules are targets of the NFκB signalling pathway activated by CD40L. Our results suggest that activation of NFκB and induction of anti-apoptotic molecules by CD40L play an important role in the protection of germinal center derived B cell lymphomas against apoptosis. Then, NFκB inhibitors may be wise to use in clinical trials in conjunction with TRAIL against follicular lymphomas.

Profiles Of De Novo CD25-Positive Mature B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4308-4308
Author(s):  
Shin-ichiro Fujiwara ◽  
Raine Tatara ◽  
Kiyoshi Okazuka ◽  
Iekuni Oh ◽  
Ken Ohmine ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
High Expression ◽  
B Cell Lymphomas ◽  
Cns Involvement ◽  
Cd25 Expression

Abstract Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as >20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P<0.001,<0.001, =0.019, and <0.001, respectively). BL and FL, which were derived from germinal center B-cells, did not express CD25. These results indicate that pre- or post- germinal center-derived B-cells, activated by IL-2/IL-2R signaling, may give rise to CD25+ BCL such as CD25+ MCL, CLL, MZL, and DLBCL. The highest median CD25 expression (41.5%) was observed in MCL. CD25 expression was higher in MCL than CD5+ BCL (CLL and CD5+ DLBCL) (median, 41.5 vs. 16.9%, respectively; P<0.001). With a cut-off value of 60% CD25-positivity, patients with CD25-high (>60%) MCL (n=9) were not treated with aggressive chemotherapy regimens such as Hyper-CVAD due to their age and characteristics, compared with those with CD25-low (<60%) MCL (n=12) (11.1 vs. 72.7%, respectively, P=0.021). In patients with CLL, the range of CD25 expression was wide (0.4-90.7%), and 29 patients (60%) showed CD25-positivity (CD25+ CLL). CD25+ CLL showed higher soluble IL-2R (sIL-2R) levels and an inferior overall survival (OS) than CD25- CLL (median sIL-2R, 2,195 vs. 706 U/ml P=0.047; 5-year OS, 62.7 vs. 100%; P=0.037). There was a significant correlation between levels of CD25 and sIL-2R (r=0.53, P=0.0053). It is clinically important to distinguish between DLBCL and BCL involving MYC oncogene rearrangement (BL and BL/DLBCL, MYC+ BCL). The former showed higher CD25 expression than the latter (median, 10.2 vs. 2.1%, respectively, P=0.04). The progression-free survival rate (PFS) after rituximab containing chemotherapy was inferior in patients with CD25+ DLBCL (n=72) than those with CD25- DLBCL (n=110) and MYC+ BCL (5-year PFS, 49 vs. 70.4, 66.3%, respectively). In patients with DLBCL, central nerve system (CNS) involvement was observed in 15 patients (7 at diagnosis and 8 at relapse). CD25+ DLBCL showed a higher frequency of CNS involvement than CD25– DLBCL (13.8 vs. 4.5%, respectively, P=0.049). Regarding MZL, CD25 was highly expressed in nodal MZL, but it showed a low expression in splenic MZL. Regarding the sites of extranodal MZL, CD25 expression was lower in the thyroid than at other sites (median, 5.1 vs. 21.2%, respectively, P=0.37). There were some differences between CD25+ (n=9) and CD25- (n=14) MZL concerning the presence of B symptoms (33.3 vs. 0%, respectively) and advanced stage (66.6 vs. 35.7%, respectively). Conclusion CD25 expression using flow cytometry can potentially provide diagnostic and prognostic implications on BCL patient. The high expression of CD25 in MCL and CLL suggests the possibility of targeted anti-CD25 immunotherapy. These findings may shed light on the role of CD25 expression in B-cell lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus

2010 ◽  
Vol 84 (22) ◽  
pp. 11670-11678 ◽  
Author(s):  
Sarah D. Linnstaedt ◽  
Eva Gottwein ◽  
Rebecca L. Skalsky ◽  
Micah A. Luftig ◽  
Bryan R. Cullen
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
B Cell Lymphoma ◽  
Selective Inhibition ◽  
B Cell Lymphomas ◽  
Cell Infection ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL in vivo.

scholarly journals TP53 Aberrations By FISH in CLL and Complex Karyotype at Transformation Predict for Worse Outcome in Diffuse Large B-Cell Lymphoma - Richter Transformation: A Single Institution Series of 75 DLBCL-RT Cases

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2984-2984 ◽  
Author(s):  
Shiraz Fidai ◽  
Madina Sukhanova ◽  
Brian C.-H. Chiu ◽  
Y. Lynn Lynn Wang ◽  
Wendy Stock ◽  
...  
Keyword(s):  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Genetic Alterations ◽  
Survival Outcome ◽  
Complex Karyotype ◽  
Large B Cell Lymphoma ◽  
Trisomy 12 ◽  
Large B Cell

Abstract Background: Richter transformation (RT), also known as Richter syndrome, is a rare complication of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) that has an aggressive clinical course and unfavorable prognosis. The majority of these transformations occur in the form of diffuse large B-cell lymphoma (DLBCL), and less frequently in the form of prolymphocytic leukemia (PLL), or classical Hodgkin lymphoma (cHL). We examined a series of RT cases with available CLL/SLL data diagnosed at our institution between 2000 and 2018 to identify clinical/biologic characteristics associated with adverse outcome. Design: After searching our pathology archives between the years 2000-2018, we identified a total of 83 RT cases: 75 DLBCL-RT (including 7 PLL-RT) and 8 cHL-RT. For the purposes of this study, we focused only on DLBCL-RT cases. All clinical, demographic, and pathologic data including cytogenetics (karyotype and FISH), immunohistochemistry (IHC), and flow cytometry were collected. Data points included age, gender, CLL/SLL histology (typical vs. atypical), CD5, CD38, ZAP-70 status, CLL karyotype, FISH [chromosomes 11q (ATM), trisomy 12, 13q, 14 (IGH), 17p(P53)]. In addition, time to transformation (months), site of transformation, transformation biopsy characteristics (germinal center vs. non-germinal center, CD5 status, along with MYC and BCL2 IHC status), and karyotype at transformation were collected. For the current analysis , karyotype and FISH data were dichotomized into (complex vs. non-complex) or (abnormality present vs. absent) respectively. Limited CLL treatment data was also collected but details on performance status, stage and IPI at transformation were not available. The clinical end point was overall survival (OS) determined as age at the time of transformation to death due to any cause, censoring patients without an event at last follow-up. Hazard ratios and 95% confidence intervals were derived from Cox proportional hazards regression models. Data analyses were performed using Stata®11. Results: Among DLBCL-RT patients, the median age at transformation was 66 years (range: 43-95). 87% of cases had antecedent CLL with a typical phenotype (CD5+, CD23+, FMC7-), 66% of CLL cases expressed CD38 (flow > 20% positivity) and 77% were positive for ZAP-70 (IHC). There was no correlation between CD38 expression and ZAP-70 (p=0.7). Fifty-three percent of the CLL cases harbored a complex karyotype. The most frequent abnormalities detected by FISH were TP53 aberrations (39%), followed by del13q (37%), Trisomy 12 (33%), IGH breaks (21%) and 11q/ATM deletion (17%). Transformation occurred at median of 59 months (0-352 range) after CLL diagnosis. DLBCL-RT was non-germinal center B-cell phenotype in 85% (n=27) of cases, with variable expression of CD5 (61%), p53 (50%), MYC (83%) and BCL2 (90%). EBER was infrequent at transformation (4%; n=23). Among DLBCL-RT, a total of 29 patients died at a median of 6 months (0-58 months) after RT diagnosis. In univariate analysis, age at transformation, type of CLL (typical vs. atypical), site (nodal vs. extranodal), CLL CD38 or ZAP-70 expression did not impact survival outcome. Only TP53 aberrations by FISH in antecendent CLL (p=0.02) (Figure 1) and the presence of complex karyotype at transformation were associated with adverse survival outcome (p=0.004) (Figure 2). MYC and/or BCL2 genetic alterations at transformation did not impact outcome. In a multivariable Cox model including both CLL TP53 aberrations and complex karyotype at transformation, only complex karyotype at transformation is weakly associated with outcome (p=0.09; hazard ratio 0.14 [0.015-1.36]; 95% CI) although this analysis is limited by the number of available patients with complete data (n=17). Conclusion: DLBCL-RT from an underlying CLL has a poor survival outcome and only TP53 alterations in the CLL and complex karyotype at transformation impacted survival. Figure. Figure. Disclosures Stock: Jazz Pharmaceuticals: Consultancy. Riedell:Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy; Kite Pharmaceuticals: Consultancy, Speakers Bureau. Smith:BMS: Consultancy; Portola: Honoraria.

scholarly journals Epstein Barr virus-positive B-cell lymphoma is highly vulnerable to MDM2 inhibitors in vivo

2021 ◽  
Author(s):  
Xiaoshan Zhang ◽  
Ran Zhang ◽  
Chenghui Ren ◽  
Yi Xu ◽  
Shuhong Wu ◽  
...  
Keyword(s):  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
T Cell Therapy ◽  
Barr Virus ◽  
Epstein Barr

Epstein-Barr virus (EBV)-positive B-cell lymphomas are common in immunocompromised patients and remain an unmet medical need. Here we report that MDM2 inhibitors (MDM2i) navtemadlin and idasanutlin have potent in vivo activity in EBV+ B-cell lymphoma established in immunocompromised mice. Tumor regression was observed in all 5 EBV+ xenograft-associated B-cell lymphomas treated with navtemadlin or idasanutlin. Molecular characterization showed that treatment with MDM2i resulted in activation of p53 pathways and downregulation of cell cycle effectors in human lymphoma cell lines that either were EBV+ or had undetectable expression of BCL6, a transcriptional inhibitor of the TP53 gene. Moreover, treatment with navtemadlin resulted in tumor regression and prevented systemic dissemination of EBV+ lymphoma derived from 2 juvenile patients with posttransplant lymphoproliferative diseases, including one whose tumor was resistant to virus-specific T-cell therapy. These results provide proof-of-concept for targeted therapy of EBV+ lymphoma with MDM2i and the feasibility of using EBV infection or loss of BCL6 expression to identify responders to MDM2i.

scholarly journals Identification of Potent Paracaspase MALT1 Inhibitors for Hematological Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Wu Yin ◽  
Nie Zhe ◽  
Andrew Placzek ◽  
Michael Trzoss ◽  
Goran Krilov ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Synergistic Effects ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Publicly Traded ◽  
Ongoing Work

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), was identified as a translocation protein fused with cIAP2 in mucosa-associated lymphoid tissue (MALT) B cell lymphomas. MALT1, a key mediator of NF-κB signaling and the main driver of a subset of B-cell lymphomas, functions via formation of a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 has been considered as a potential therapeutic target for several non-Hodgkin B cell lymphomas as well as chronic lymphocytic leukemia (CLL). Here, we describe the discovery of novel, potent MALT1 inhibitors that result in antiproliferative effects in non-Hodgkin B-cell lymphoma cells. Results: We have identified novel small molecule MALT1 inhibitors using our proprietary physics-based Free Energy Perturbation (FEP+) modeling technology. Our compounds show potent (sub nM) inhibition of MALT1 enzymatic activity, as well as high binding affinity (sub nM) to MALT1 protein measured by Surface Plasmon Resonance (SPR). BCL10 is a binding partner of MALT1 that is cleaved by MALT1 at the C-terminus. Our inhibitors were efficacious in a target engagement assay showing prevention of BCL10 cleavage in Activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines OCI-LY3 and OCI-LY10, which are Bruton tyrosine kinase (BTK) inhibitor ibrutinib-resistant and -responsive respectively. Our compounds are potent inhibitors of IL10 secretion in both OCI-LY3 and OCI-LY10 cells, which is consistent with the inhibition of NF-κB signaling. We also examined the effect of our MALT1 inhibitors on ABC-DLBCL cell proliferation. Our inhibitors demonstrated potent anti-proliferative effects in both OCI-LY3 and OCI-LY10 cell lines, as well as synergistic effects with ibrutinib in a BTKi sensitive ABC-DLBCL cell panel. Examinations of a protease panel and off-target safety screening panel, as well as in vivo high dose tolerability study showed our compound had excellent selectivity and significant safety margin. Plasma IL10 and tumor BCL10 have been identified as robust PD markers in PK/PD studies in both OCI-LY3 and OCI-LY10 tumor bearing mice. Dose-dependent tumor growth inhibition was observed after 3 weeks of treatment in OCI-LY3 xenograft model, with efficacy also observed in combination with venetoclax. Ongoing work: We are continuing to explore the synergistic effects of our compounds with BTK inhibitors in B-cell lymphoma mouse models. Preliminary data showed potent inhibition of IL-2 secretion in Jurkat cells from our compound treatment. Additional studies are ongoing to elucidate the role of MALT1 inhibition in Treg as well as Teffector cells in vitro and in vivo. Refinement of the current inhibitor series, using co-crystal structures, is in progress in preparation for further development of optimized molecules. Conclusion and Future Plans: We have identified novel potent MALT1 protease small molecule inhibitors that are efficacious in the in vitro B-cell lymphoma cell proliferation assays and in the in vivo B-cell lymphoma xenograft model. Our data suggest that targeting MALT1 may expand therapy options for patients with selected B-cell lymphomas, such as ABC-DLBCL. Our work provided insight into the anti-tumor efficacy of our inhibitors in B-cell lymphomas as single agent, and ongoing work will continue to assess the potential combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphoma. Disclosures Yin: Schrodinger: Current Employment, Current equity holder in publicly-traded company. Zhe:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Placzek:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Trzoss:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Krilov:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feng:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lawrenz:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Pelletier:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lai:Triplet Therapeutics: Current Employment, Current equity holder in private company. Bell:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Calkins:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Grimes:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Tang:Schrodinger: Current Employment, Current equity holder in publicly-traded company. McRobb:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Gerasyuto:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feher:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Mondal:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Jensen:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Wright:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Akinsanya:Schrodinger: Current Employment, Current equity holder in publicly-traded company.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Inactivation of the PRDM1/BLIMP1 Gene in Non-GC-Type Diffuse Large B-Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2614-2614
Author(s):  
Laura Pasqualucci ◽  
Mara Compagno ◽  
Jane Houldsworth ◽  
Stefano Monti ◽  
Adina Grunn ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Genetic Alterations ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Nonsense Mutations ◽  
Hodgkin's Lymphomas

Abstract PRDI-BF1/Blimp1 is a zinc finger transcriptional repressor expressed in post-germinal center (GC) B cells and required for terminal B cell differentiation. The PRDM1/BLIMP 1 locus lies on chromosomal band 6q21–q22.1, a region frequently deleted in B-cell non-Hodgkin’s lymphomas suggesting the existence of one or more tumor suppressor loci (Gaidano et al, Blood80:1781, 1992). To test whether genetic alterations affecting the BLIMP1 gene may be involved in DLBCL pathogenesis, we performed PCR amplification and direct sequencing of the BLIMP1 coding sequences in 136 cases, including 20 cell lines and 116 primary biopsies. Gene expression profiling analysis using the Affymetrix Gene Chip system was used in 93 cases to classify them as germinal center B-cell like (GCB, N=38), activated B-cell like (ABC, N=35) and type III (N=20). Nonsense mutations were found in 7 of 136 cases. Four of these mutations generated premature stop codons, predicting severely truncated proteins of 61 to 244 aminoacids; in three cases, a single bp substitution affecting the exon 2 splice donor site led to insertion of 101 nucleotides from intron 3 and a premature stop codon. One primary tumor case carried a mutation within the intron 3 splice acceptor site, and one cell line displayed a gene rearrangement in one allele with deletion of the second allele. Strikingly, 6 of the 7 nonsense mutations identified segregated with the ABC phenotype (the remaining case was not profiled), suggesting that these alterations may be common and specific in this subtype of DLBCL (6/35, 17%). In addition, missense mutations generating aminoacid substitutions were found in 8 additional cases, and complete lack of Blimp-1 protein expression was found in most ABC-type DLBCL cases. These observations suggest that inactivation of the BLIMP1 gene may occur also by other mechanisms, including the generation of dominant negative mutants, chromosomal deletion or epigenetic silencing, in a large fraction of DLBCL. Functional studies are currently being performed to corroborate these data. Overall, these results suggest that BLIMP1 may act as a tumor suppressor gene, whose loss or inactivation may contribute to lymphomagenesis by blocking post-GC differentiation of B cells toward plasma cells.

Constitutively Activated STAT3 Promotes Cell Proliferation and Survival in the Activated B Cell Subtype of Diffuse Large B Cell Lymphomas.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1621-1621
Author(s):  
Bihui Hilda Ye ◽  
Beibei Belinda Ding ◽  
Jian Jessica Yu ◽  
Raymond Y.-L. Yu ◽  
Lourdes M. Mendez ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Cell Development ◽  
B Cell Development ◽  
Reciprocal Relationship ◽  
B Cell Lymphomas ◽  
Large B Cell

Abstract During B cell development, cell proliferation and survival are regulated by stage-specific transcription factors. Accordingly, distinct oncogenic pathways are employed by B cell lymphomas representing different stages of B cell development. Diffuse large B cell lymphoma (DLBCL) contains at least two main phenotypic subtypes, i.e. the germinal center B cell-like (GCB-DLBCL) and the activated B cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated NF-kappaB and tends to be refractory to chemotherapy. In this study, we investigated the relationship between BCL6 and STAT3 expression/activation in DLBCL and normal GC B cells. Our results demonstrate that BCL6 directly inhibits transcription of the STAT3 gene by binding to two BCL6 sites in its 5′ regulatory region. As a result, high level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Specifically, in tonsillar GCs, STAT3 expression and activation is restricted to a previously uncharacterized subset of BCL6−Blimp-1− B cells in the apical light zone. The location and phenotype of these cells suggest that they are in the process of exiting the BCL6-directed GC program and transitioning to a plasma cell differentiation process governed by Blimp-1. The reciprocal relationship between BCL6 and STAT3 is also conserved in DLBCL such that STAT3 expression and activation is preferentially associated with the BCL6-low, ABC subtype. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB and Mcl-1, and increased expression of the cell cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define STAT3 activation as a second oncogenic pathway operating in ABC-DLBCL and suggest that blocking STAT3 may be potentially therapeutic in treatment of these aggressive lymphomas.

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