scholarly journals TP53 Mutations and RNA Binding Protein Musashi 2 Drive Resistance to PRMT5-Targeted Therapy in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 788-788
Author(s):  
Ingrid Tatiana Erazo ◽  
Chiara M Evans ◽  
Daniel Zakheim ◽  
Karen L Chu ◽  
Olena Barbash ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Research Funding ◽  
Rna Binding ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Genome Wide ◽  
Crispr Screen ◽  
Privately Held

Abstract PRMT5 catalyzes the arginine symmetric demethylation of histone and non-histone proteins. PRMT5 is overexpressed in lymphoma and plays a key role in lymphomagenesis through the repression of several tumor suppressors, including P53, while promoting the expression of lymphoma-drivers, such as c-MYC. Currently, three PRMT5 inhibitors are being clinically evaluated in B-cell lymphoma patients, however, the mechanism of action of these molecules remain poorly understood. Thus, to identify the mechanism of action of the potent selective PRMT5 inhibitor, GSK3203591 (GSK-591), we performed a genome-wide CRISPR/Cas9 KO screen in the human mantle cell line, Z-138 and a validation CRISPR /Cas9 screen in the DLBCL cell line, OCI-LY19. Our genome-wide CRISPR screen identified 316 sensitizing genes and 89 genes involved in resistance. The top sensitizing gene was TP53, which validated our screens, as it is a well characterized target of PRMT5. Furthermore, we found that TP53 deletion and the hot spot TP53 R248Wmutation are biomarkers of resistance to GSK-591. RNA-binding protein MUSASHI 2 (MSI2) was identified as the top-ranked driver of resistance to GSK-591. MSI2 plays a key role in hematopoietic stem cells activation, myeloid leukemia and CLL, however, its function in lymphoma remains unknown. MSI2 is overexpressed in DLBCL patients (n=96). Relapsed MCL and DLBCL primary samples express high levels of both PRMT5 and MSI2. Furthermore, MSI2 depletion decreased cell viability and sensitized lymphoma cells to PRMT5 inhibitor in vitro and vivo. In contrast, overexpression of MSI2 reverted sensitivity to GSK-591 in lymphoma cells. Consistent with our genetic studies, inhibition of MSI2 using Ro 08-2750 (Ro) (Minuesa et al. Nat Commun. 2019), conferred sensitivity to GSK-591, and the combination therapy was synergistic inducing cytotoxicity. In order to identify specific downstream targets of MSI2 that may contribute to resistance to PRMT5 inhibition in lymphoma, we performed MSI2-HyperTRIBE, a recently discovered technology that allows mapping the MSI2 targeting network (Nguyen et al. Nat Commun. 2020). Using MSI2-HyperTRIBE, we were able to identify MSI2 targets in lymphoma cells. Moreover, we found that Ro treatment significantly blocked MSI2 binding activity, while GSK-591 had no effect on MSI2 targets. To uncover the mechanism involved in the synergy of PRMT5 and MSI2 inhibitors, we performed RNA-sequencing of Z-138 cells treated with GSK-591, Ro or the combination. Gene set enrichment analysis (GSEA) demonstrated a loss in the c-MYC pathway upon drug combination.MSI2 RNA-IP assays showed that MSI2 binds c-MYC and this interaction is disrupted by the combination of GSK-591 and Ro. We observed that the combination of MSI2 and PRMT5 inhibitors does not affect c-MYC mRNA levels but, rather, controls translation. Interestingly, in the human B cell line P493-6 that express a conditional, tetracycline-regulated c-MYC, the depletion of c-MYC significantly increased the anti-proliferative activity of GSK-591 and Ro alone or in combination. To further investigate whether there are other functionally relevant downstream targets of MSI2 promoting resistance to PRMT5 inhibition, we overlapped the differentially expressed genes upon treatment with GSK-591 and Ro (RNA-Seq), the Ro-direct targets in lymphoma (MSI2-HyperTRIBE) and the resistance genes to PRMT5 inhibition (whole-genome CRISPR screen) and we identified the anti-apoptotic protein BCL-2 as a common target of the PRMT5-MSI2 axis. We established first that the combination of GSK-591 and Ro blocked the binding of MSI2 to BCL-2 mRNA. We also found that the drug combination significantly decreased BCL-2 mRNA and protein abundance. Furthermore, Z-138 and OCI-LY19 BCL-2 KO cells are more sensitive to GSK-591 and Ro alone or in combination. Using venetoclax, we found that BCL-2 inhibition enhanced GSK-591 activity in vitro and in vivo. Thus, these data confirmed that BCL-2 is a key driver of resistance to PRMT5 inhibition. Overall, our study uncovered a novel oncogenic axis, PRMT5/MSI2/c-MYC/BCL-2 that drives resistance to PRMT5-targeted therapy in lymphoma. We demonstrated that TP53 LOF and MSI2 expression could be used as biomarkers for patient stratification. Moreover, we proposed two novel drug combination strategies, with venetoclax or a MSI2 inhibitor, to be considered in further clinical studies with PRMT5 inhibitors. Disclosures Barbash: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Batlevi: Viatris: Current holder of individual stocks in a privately-held company; Juno/Celgene: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Bayer: Research Funding; ADC Therapeutics: Consultancy; TG Therapeutics: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Kite Pharma: Consultancy; Life Sciences: Consultancy; Seattle Genetics: Consultancy; Karyopharm: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; GLG Pharma: Consultancy; Dava Oncology: Honoraria; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Younes: AZ: Current Employment, Other: Senior Vice President, Global Head of Haematology (Early and Late Stage) Oncology R&D at AstraZeneca.

Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5087-5087 ◽  
Author(s):  
Takashi Tokunaga ◽  
Akihiro Tomita ◽  
Kazuyuki Shimada ◽  
Junji Hiraga ◽  
Takumi Sugimoto ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Newly Diagnosed ◽  
Rt Pcr ◽  
Lymphoma Cells ◽  
Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Apoptotic and Non-Apoptotic Cellular Pathways Executed by Bortezomib (BTZ) in Rituximab-Resistant Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4967-4967
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
Karen E Thudium ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mitotic Catastrophe ◽  
M Cell ◽  
Lymphoma Cells

Abstract Abstract 4967 Rituximab-chemotherapy relapsed/refractory (r/r) B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. A better understanding of the mechanism(s)-of-action of BTZ and other proteasome inhibitors (PI) is likely to aid in the identification of biomarkers that can be used to determine clinical responsiveness and/or help in the rational development of novel PI-based therapeutic combinations (e.g. incorporating biologics, small molecules and/or chemotherapy) in r/r B-cell lymphoma. Previously we demonstrated that rituximab resistance was associated with increased proteasome activity leading to a de-regulation in the apoptotic threshold of lymphoma cells to multiple chemotherapy agents. Pharmacological and genetic (e.g. siRNA silencing of BAK/BAX) inhibition of apoptosis partially affected BTZ activity in rituximab-resistant (RSCL) but not in rituximab-sensitive cell lines (RSCL) suggesting the existence of alternative pathways of cell death associated with PI exposure. To this end we evaluated the contribution of cellular senescence, cell cycle inhibition, or mitotic catastrophe to the anti-tumor activity of BTZ as a single agent or in combination with chemotherapeutic agents in RSCL, RRCL and in primary tumor cells. Lymphoma cells were exposed to BTZ (10-25nM) for 24–48 hrs. Cell senescence was determined by SA-β-gal staining using a senescence assay kit and inverted phase-contrast microscopy was performed. Changes in cell cycle were analyzed by the FACScan DNA method and changes in cell cycle regulatory proteins (i.e. cdc2, cyclinA/B, p21, CDK2/4/6) were analyzed by Western blotting. Mitotic index was determined by Wright-Giemsa stain and positive cells were counted under a Nikon microscope. Mitotic catastrophe was determined by confocal microscopy by staining with α-tubulin antibody. Finally, changes in ATP content was determined by the Cell Titer Glo assay. Baseline differences were observed between RSCL and RRCL in terms of cell morphology, proliferation rate and senescence. RRCL (Raji2R and Raji4RH) were considerably larger in size, had a slower proliferation rate and an exhibited a 3-fold increase the number of cells in senescence than RSCL. In vitro exposure of RSCL and RRCL to BTZ attenuated the number of cells in senescence by 50–75%. Cell cycle analysis demonstrated that RRCL had more cells in S phase when compared to RSCL. In vitro exposure to BTZ-induced G2/M arrest in RRCL, but not in RSCL. Overexpression of G2/M cell cycle regulatory proteins cyclin B and cdc2 were observed in RRCL and in tumor cells isolated from r/r B-cell lymphoma patients. Mitotic catastrophe with multi-nucleated cells were only detected in RRCLs exposed to BTZ. In vitro and ex vivo exposure of RSCL and RRCL to BTZ potentiated the cytotoxic effects of paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL and primary tumor cells isolated from r/r lymphoma patients in a dose-dependent manner. Our results suggested that BTZ activates several death pathways in B-cell lymphoma pre-clinical models. In addition to apoptosis, BTZ is capable in triggering mitotic catastrophe in rituximab-chemotherapy lymphoma cells with decreased levels of pro-apoptotic proteins. Moreover, sensitization of RRCL to drug therapy involves interplay between cellular senescence attenuation, G2/M cell cycle regulation, and mitotic catastrophe. Hence, proteasome inhibition may provide a novel therapeutic approach for treating apoptosis-resistant B-cell lymphoma. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute. Disclosures: Hernandez-Ilizaliturri: Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy. Czuczman:Millennium: Honoraria, Research Funding.

CD20 Protein Immunohistochemistry-Positive/Flow Cytometry-Negative Diffuse Large B-Cell Lymphoma—Analyses of the Molecular Mechanisms and Rituximab Effectiveness

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2659-2659
Author(s):  
Takashi Tokunaga ◽  
Akihiro Tomita ◽  
Junji Hiraga ◽  
Takumi Sugimoto ◽  
Kazuyuki Shimada ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Positive Control ◽  
Cell Populations ◽  
Rt Pcr ◽  
Lymphoma Cells

Abstract Abstract 2659 Background: Rituximab is a key molecular targeting drug for CD20(+) B-cell lymphoma, and CD20 protein expression is essential in the treatment mechanism of rituximab. We previously reported that the change to the CD20(−) phenotypic after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009; Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009). Recently, we encountered newly diagnosed patients with diffuse large B-cell lymphomas (DLBCL) which are CD20(+) on immunohistochemistry (IHC) but CD20(−) on flow cytometry (FCM). For these patients, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance have been clarified, and the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims: To confirm the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype using primary DLBCL cells, and to confirm the effectiveness of rituximab in vitro. Methods: Genomic DNA, mRNA, and total protein were extracted from primary DLBCL cells showing the CD20 IHC(+)/FCM(−) phenotype, and subjected to DNA sequencing, quantitative RT-PCR, and immunoblotting. L26 and B1 anti-CD20 antibodies were used in IHC and FCM analyses, respectively. Primary DLBCL cells were transplanted into NOD-SCID mice, and the engrafted lymphoma cells were used for in vitro CDC assay using rituximab. Results and Discussion: Ten patients with primary DLBCL had CD79a(+)/L26(+) results on IHC and CD19(+)/CD20(−) results on FCM. Quantitative RT-PCR indicated that CD20 mRNA expression that was significantly lower (almost 10 times) than that of positive control primary DLBCL cells. Immunoblotting analysis showed that CD20 protein expression was relatively lower than that of positive control cells, and no differences in length could be detected. No genetic mutations in the coding sequence of CD20 gene were detected in all samples examined in DNA sequencing analysis, suggesting that CD20 partial deletion is not the main reason for this phenotype. Interestingly, FCM analyses using fluorescent-labeled rituximab indicated that rituximab could partially recognize FCM CD20-B1(−)/CD19(+) B-cells, suggesting that the binding sensitivity of rituximab is much stronger than that of the B1 antibody. Lymphoma cells showing CD20 L26-IHC(+)/B1-FCM(−) phenotype proliferated in NOD-SCID mice and were partially killed by rituximab on in vitro CDC assay. These data suggest that rituximab is effective even for B-lymphoma cells with low CD20 expression if FCM using rituximab can be made to recognize those cell populations. Conclusion: Lower expression of CD20 mRNA may be one of the critical reasons for the CD20 L26-IHC(+)/B1-FCM(−) phenotype. FCM analysis using rituximab may be helpful to detect cell populations that are sensitive to rituximab treatment. Thus, rituximab therapy may be recommended if we can confirm the existence of cell populations recognized by rituximab on FCM. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo.: Research Funding; Otsuka Pharma.: Research Funding.

Selective Inhibition of Activated B-Cell Lymphoma Cells by a Novel Small Molecule Inhibitor of NF-κB

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2498-2498
Author(s):  
John T ◽  
Tomoko Hayashi ◽  
Raymond P. Wu ◽  
Howard Cottam ◽  
Dennis A. Carson
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Nuclear Translocation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Constitutive Activation ◽  
Structure Activity

Abstract Abstract 2498 Diffuse large B-cell lymphomas (DLBCL) account for approximately 40% of lymphomas in adults, with the activated B-cell lymphoma (ABC) subtype being the least curable. ABC lymphoma cells display the phenotype of activated B-cells, which is induced by constitutive activation of the transcription factor Nuclear Factor-kappa B (NF-κB). NF-κB activation in ABC lymphoma can result from several different mutations or abnormal expression of proteins upstream of NF-κB nuclear translocation. No matter the mechanism of NF-κB constitutive activation, the resulting gene expression pattern confers an anti-apoptotic and pro-survival advantage to B-cells, hence driving the oncogenesis and supporting the survival of ABC lymphomas. Therefore, inhibiting NF-κB activity is an attractive strategy for the treatment of ABC lymphomas. Using a cell line with a reporter for NF-κB transcriptional activity in a high throughput screen of 180,000 compounds we discovered 12 compounds that inhibited NF-κB activation, suggesting these compounds negatively regulate NF-κB. Since ABC lines are generally more dependent on NF-κB signalling for cell survival than are the other types of B-cell lymphoma, the hits from the primary screen were tested in a secondary screen for the ability to selectively inhibit the growth of ABC lymphoma cells. Of the 12 hits, two compounds belonging to the same quinolone chemotype were found to selectively inhibit the growth of an ABC line compared to a non-ABC line and normal human peripheral blood mononuclear cells (PBMCs). Three more structurally related quinolones were obtained to investigate a possible limited structure activity relationship for this class of molecules, designated here as Quinolone Inhibitors of NF-κB (QINs). QIN 1 was significantly more potent (Figure A and B) and selective (Figure B and C) relative to other QINs. The limited structure activity relationship suggests that two structural regions of the chemotype may be important for potency and for ABC selectivity, hence providing impetus to further investigate QINs as possible ABC lymphoma drugs. QIN1 is more potent in inhibiting the growth of ABC lines than a known inhibitor of NF-κB activity, a commercially available selective IKK inhibitor (CAS 507475-17-4). Active IKK causes degradation of IκBα, the natural inhibitor of NF-κB, hence IKK inhibitors prevent NF-κB activation by preventing degradation of IκBα. In addition to being more potent, QIN1 retains similar ABC selectivity as the IKK inhibitor (Figure C). QIN1 and the IKK inhibitor both cause apoptosis at a similar rate and both cause G1 arrest in ABC lines at equi-toxic concentrations, initially suggesting similar mechanisms of action. Subsequently, QIN1was found to inhibit NF-κB in several different assays using the IKK inhibitor as a positive control. First, QIN1 inhibited the activation of a transcription based NF-κB reporter cell line in response to LPS, an NF-κB activator. Supporting these results, QIN1 inhibited cytokine release from human PBMCs stimulated with LPS. In addition, QIN1 prevented degradation of IκBα in response to NF-κB activating stimuli, further demonstrating the ability of QIN1 to inhibit NF-κB activity. Finally, QIN1 inhibited nuclear translocation of active NF-κB, thus preventing pro-survival gene expression. The QINs studied here all contain an alpha-beta unsaturated ketone, an electrophilic chemical moiety known to interact with cysteine thiols. Compounds containing similar electrophilic groups are known to bind a cysteine thiol in IKK, preventing its activity and hence inhibiting NF-κB activation. This suggests that this electrophilic moiety in QINs maybe responsible for the NF-κB inhibition observed in these cell lines. The presence of this electrophilic group is not necessarily detrimental to normal cells as we have shown with QIN1 in vitro (Figure A-C) and in vivo (large daily doses of QIN1 cause no observable toxicities in mice). Overall, our data indicate QIN1 inhibits IKK, a kinase immediately upstream of NF-κB nuclear translocation, allowing the majority of ABC lymphomas to be treated irrespective of the upstream mechanism of pathway activation. The selectivity for inhibition of ABC lymphoma cells by QIN1 and our in vitro data showing synergy with an inhibitor of B-cell receptor signalling, which also promotes ABC survival, provides the impetus for further preclinical development of QINs. Disclosures: No relevant conflicts of interest to declare.

The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Engraftment and Antitumor Responses Against Mantle Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 704-704
Author(s):  
Marco Ruella ◽  
Saad S Kenderian ◽  
Olga Shestova ◽  
Joseph A. Fraietta ◽  
Sohail Qayyum ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
University Of Pennsylvania ◽  
Tumor Control

Abstract Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL. Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival ( p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B). Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Maus:Novartis: Consultancy, Patents & Royalties, Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Patents & Royalties, Research Funding. Mato:Genentech: Consultancy; Pronai Pharmaceuticals: Research Funding; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy. Schuster:Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Hoffman-LaRoche: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Kalos:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Gill:Novartis: Patents & Royalties, Research Funding. Wasik:Janseen and Novartis: Research Funding.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

scholarly journals Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 26-32 ◽  
Author(s):  
T Miki ◽  
N Kawamata ◽  
S Hirosawa ◽  
N Aoki
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Cdna Probe ◽  
B Cell Lymphoma ◽  
Nucleotide Sequencing ◽  
Sequencing Analysis

Abstract Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Kruppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5′ region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus- immortalized B-lymphoblastoid cell lines.

A Fully Human Anti-CD40 Antagonistic Antibody, CHIR-12.12, Inhibit the Proliferation of Human B Cell Non-Hodgkin’s Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3279-3279 ◽  
Author(s):  
Wen-Kai Weng ◽  
Xia Tong ◽  
Mohammad Luqman ◽  
Ronald Levy
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Expression Level ◽  
Primary Lymphoma ◽  
Immunoglobulin Gene ◽  
Dependent Manner ◽  
Lymphoma Cells

Abstract Immunotherapy using anti-tumor antibodies has become a feasible alternative for treating patients with lymphoma. These anti-tumor antibodies may target a specific receptor to disrupt proliferative signaling or mediate their anti-tumor effect by antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated killing. The CD40 antigen is a good target for such anti-tumor antibodies for several reasons: CD40 is expressed on the vast majority of the non-Hodgkin’s B cell lymphomas and it has been proposed that the CD40/CD40L interaction provides a critical survival or proliferative signal for B cell lymphoma, especially the low-grade follicular lymphoma. In addition, B lymphoma cell lines become less sensitive to chemotherapy-induced apoptosis after CD40 cross-linking in an in vitro study. Therefore, an anti-CD40 antagonist that disrupts the CD40/CD40L interaction and mediates effector mechanism could have a therapeutic advantage. In this report, we describe a fully human anti-CD40 antagonistic IgG1 monoclonal antibody, CHIR-12.12 that was generated from mice with a human immunoglobulin gene loci (XenoMouse®mice, Abgenix Inc.). We first compared the antigen expression level of CD40 to the level of CD20, the target for rituximab, on primary lymphoma cells. While the expression level of CD40 was similar between different samples of primary follicular lymphoma cells, it was 10 fold less than the level of CD20. The expression of CD40 and CD20 on chronic lymphocytic leukemia/small lymphocytic lymphoma cells (CLL/SLL) was more variable. However, the level of CD20 was still significantly higher than the level of CD40 in all samples tested (2.4 to 13 fold). While CHIR-12.12 binds to primary lymphoma cells similarly to several other anti-CD40 antibodies, CHIR-12.12 did not induce proliferation of these primary tumore cells. By contrast, an agonist anti-CD40 antibody induced proliferation of these lymphoma cells up to 6-fold over baseline. To study the ability of CHIR-12.12 to interrupt the CD40-CD40L interaction, we cultured lymphoma cells with CD40L-transfected feeder cells in the presence of control IgG1, CHIR-12.12 or rituximab. In this system, the lymphoma cells proliferate in response to CD40-CD40L interaction. The addition of rituximab did not influence the proliferation. However, CHIR-12.12 inhibited the proliferation of follicular lymphoma and of CLL/SLL cells in a dose-dependent manner. The inhibition was observed with antibody concentration at 1 μg/ml and reached maximum of 90% inhibition at 10 μg/ml. We then evaluated the ability of CHIR-12.12 to elicit complement-mediated killing or ADCC. In vitro, rituximab induced complement-mediated cytotoxicity, while CHIR-12.12 did not. However, both CHIR-12.12 and rituximab induced effective ADCC of primary follicular lymphoma cells using purified NK cells from a healthy donor. Even though the level of CD40 is 10-fold less than the level of CD20 on the cell surface of these tumor cells, CHIR-12.12 induced the same degree of ADCC killing as did rituximab. Thus, this novel antagonist CHIR-12.12 antibody both blocks tumor-stimulatory CD40/CD40L interaction and mediates ADCC in the presence of a low number of target antigen. Our results support further development of this antibody to treat patients with B cell lymphoma.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

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