Primary Diffuse Large B-cell Lymphoma Cells Research Articles

Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

Abstract P04: Role of glucocorticoid receptor in normal and malignant germinal center B cells

Abstract We have reported that super-enhancers (SEs) are specifically hypermutated by Activation-Induced Deaminase (AID)-induced Aberrant Somatic Hypermutation in >90% of diffuse large B cell lymphoma (DLBCL) cases (Bal et al. Nature 2022). Analysis of 122 primary DLBCL biopsies and cell lines identified more than 80 SEs that are recurrently hypermutated, with an average of 12 hypermutated SE/case. Hypermutated SEs are predominantly linked to genes encoding regulators of B cell development and well-known lymphoma oncogenes, including BCL6, BCL2 and CXCR4. We showed that specific hotspot mutations prevent binding and transcriptional downregulation by transcriptional repressors leading to target gene dysregulation. Among these, hypermutation of the SEs linked to BCL2 and CXCR4 abrogates the binding of the glucocorticoid receptor (GR)/transcription factor encoded by NR3C1. As a result, BCL2 and CXCR4 escape GR-mediated transcriptional repression leading to their de-regulated expression in germinal center (GC) B cells. Together with the observation that the NR3C1 gene is genetically inactivated in a small fraction of DLBCL, and the relevance of glucocorticoid-based therapy in DLBCL, these results prompted a comprehensive analysis of the role of GR in normal and malignant B cells. We report that GR is detectable in the nucleus of the great majority of GC B cells, suggesting that it is active as a transcription factor. Similarly, DLBCL primary cases (including GCB-, ABC- and unclassified DLBCL) display GR nuclear expression. Analysis of a conditional GC-specific Nr3c1 knock-out (KO) mouse model showed that GC B cells form normally in the absence of Nr3c1. However, the transcriptional profile of Nr3c1-KO GC B cells was significantly altered compared to wild-type cells. By integrating the GR binding profile obtained in normal human GC B cells with the transcripts differentially expressed in Nr3c1-KO GC B cells, we identified a set of about 2,000 genes that are directly modulated by GR during the GC reaction. Pathway enrichment analysis showed that GR controls several signaling pathways, suggesting a modulatory role on the BCR and CXCR4 pathways. In addition, GR targets display a significant overlap with the BCL6 transcriptional network, suggesting a cooperative role in GC B cell development. Notably, GR was shown to regulate pathways involved in plasma cell differentiation, while repressing targets associated with memory B cell development. In DLBCL cases, several NR3C1 binding sites are recurrently targeted by mutations, suggesting that genetic impairment of GR activity is heterogeneous and extends beyond CXCR4 and BCL2. Together, these data support a role for the glucocorticoid pathway in GC physiology. Recurrent inactivation of the NR3C1 gene or part of its transcriptional regulatory program may contribute to DLBCL pathogenesis, with implications for presently unknown specific roles of glucocorticoids in the therapeutic regimens for DLBCL. Citation Format: Clarissa Corinaldesi, Antony B Holmes, Elodie Bal, Laura Pasqualucci, Katia Basso, Riccardo Dalla-Favera. Role of glucocorticoid receptor in normal and malignant germinal center B cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P04.

Lymphoma-Derived IL-10 Is a Key Immunomodulatory Factor at the Tumor Microenvironment of Activated B-Cell Diffuse Large B-Cell Lymphoma and Influences In Vivo Responses to Immunotherapy

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease to R-CHOP immunochemotherapy. These higher risk patients often exhibit gene expression profiles that resemble a late germinal center-derived activated B-cell (ABC) phenotype, where diverse specific mutations in genes along BCR and TLR signaling pathways converge into constitutive activation of the NF-κB pathway. Identification of factors that contribute to the survival of malignant ABC-DLBCL cells is crucial for designing specific interventions. In this setting, previous in vitro studies in DLBCL cell lines have evidenced that IL-10 is a direct target of NF-κB signaling that sustains lymphoma cell survival by an auto-stimulatory loop via STAT3 signaling. However, the action of IL-10 could be more complex within the lymphoma microenvironment, as IL-10 can have both stimulatory and inhibitory effects depending on the cellular source of IL-10, the timing of its secretion, and the type of immune cells that receives signals via IL-10 receptor (IL-10R). To explore in vivo the relevance of lymphoma-derived IL-10, we have generated a novel quintuple transgenic mouse model that we call pBIC10, after crossing our previous ABC-DLBCL-like pBIC mice carrying constitutive NF-κB signaling (Pascual et al., Blood 2019) to mice with a floxed Il10 gene for conditional knock-out of IL-10 specifically in malignant ABC-DLBCL cells. As expected, increased intracellular IL-10 signaling was detected in primary murine DLBCL cells from pBIC mice but not in IL-10 deficient pBIC10 mice, which promoted lymphoma cell survival ex vivo as revealed by perturbation studies of the IL-10/JAK1/STAT3 autocrine loop with blocking monoclonal antibodies or selected inhibitors. However, in vivo genetic deletion of lymphoma-derived IL-10 did not offer improved overall survival but, on the contrary, notably accelerated DLBCL progression in pBIC10 mice (Figure 1A, left). A comprehensive integration of multiparametric flow cytometry and transcriptomic analyses using bulk and single-cell RNA-seq of lymphoma cells and the tumor microenvironment coupled with BCR- and TCR-seq, identified lymphoma-derived IL-10 as a key immunomodulator of the DLBCL microenvironment, including unexpected protective paracrine functions in the progression of DLBCL. By comparing IL-10-proficient (pBIC) and -deficient (pBIC10) murine DLBCL models, we observed a multifaceted role of IL-10 produced by DLBCL cells, enabling immune-effector processes (facilitating immune chemotaxis, antigen presentation, and IFNγ responses) that yielded higher percentages of long-lived stem-like CD8 +TCF-1 +PD-1 lo/int T cells and dendritic cells, while restraining PD-1/LAG3-driven T-cell exhaustion and reducing immunosuppression by CD4 +CD25 +FOXP3 + regulatory T (Treg) cells. Thus, depletion of lymphoma-derived IL-10 decreased T-cell cytotoxicity and increased Treg-driven immunosuppression, which collectively accelerated lymphoma development. Consequently, T-cell targeted immune-checkpoint therapy (with anti-PD-1 antibodies) was effective only in IL-10-proficient DLBCL but not in IL-10-deficient pBIC10 mice (Figure 1A, right). Further comparative studies between pBIC and pBIC10 mice showed that tumor vascularization and expression of B-cell calcium channels, which have been previously associated with sensitivity to rituximab, were counteracted by lymphoma-derived IL-10. Accordingly, anti-CD20-based targeting of lymphoma cells (with rituximab surrogate antibodies) showed best in vivo responses in IL-10-deficient DLBCL mice with respect to pBIC mice (Figure 1A, right). Consistent with this preclinical data, analyses of transcriptional IL-10-associated hallmarks to stratify DLBCL patients (Schmitz et al., NEJM 2018) identified better clinical responses to R-CHOP in patients with pBIC10-like DLBCL phenotype (Figure 1B). Collectively, our study provides new functional and mechanistic insights into the role of lymphoma-derived IL-10 in the pathogenesis of DLBCL and the restraint of an immunosuppressed tumor microenvironment, which holds predictive potential as a biomarker for immunotherapy responses in DLBCL.

Blocking the CD47-Sirpa Axis Enhances Tafasitamab-Mediated Phagocytosis

Introduction Around 40% of diffuse large B cell lymphoma (DLBCL) patients have refractory disease or experience relapse, thus highlighting the need for novel, more effective therapies (Coiffier et al). The immunotherapy Tafasitamab (MOR208) is an Fc-modified, humanized, monoclonal anti-CD19 antibody approved in combination with lenalidomide as a second line of therapy for R/R DLBCL. Tafasitamab mediates its therapeutic effect directly or via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Macrophages represent a key component of the DLBCL microenvironment and are critical effectors of antibody therapies (Uchida et al). However, ADCP is reportedly counteracted by increased expression of the immune checkpoint molecule CD47 on lymphoma targets (Chao et al). In addition, the inhibitory receptor of CD47, signal regulatory protein alpha (SIRPα) is increased on macrophages from patients with follicular lymphoma who relapse or progress after frontline lenalidomide and rituximab (Marques-Piubelli et al). Aim of the study The current study investigates the impact of the CD47-SIRPα axis on tafasitamab-mediated phagocytosis and the potential of concomitant treatment with an anti-CD47 blocking antibody to improve its efficacy. Results We show that both, CD47 and its inhibitory receptor SIRPa were elevated in lymph node biopsies from DLBCL patients. CD47 expression was evaluated in 6 DLBCL patients and 5 healthy controls and SIRPα was evaluated in 9 patients and 7 controls. In fact, increased expression of SIRPa on macrophages correlated with decreased ADCP activity of tafasitamab, and CRISPR-mediated CD47 overexpression on lymphoma targets impaired tafasitamab-mediated phagocytosis, in vitro. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination therapy. Importantly, the combination enhanced tafasitamab-mediated phagocytosis of primary DLBCL cells using primary, patient-derived lymphoma-associated macrophages (LAMs) as effectors, in an autologous setting. Conclusion Our results show increased expression of CD47-SIRPa axis in primary DLBCL samples and indicate that CD47 blockade can enhance the phagocytic potential of tumor-targeting immunotherapies such as tafasitamab. Our findings suggest that there is merit in exploring the clinical utility of combining tafasitamab with an anti-CD47 blocking antibody in lymphoma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DSF/Cu induces antitumor effect against diffuse large B-cell lymphoma through suppressing NF-κB/BCL6 pathways

BackgroundThe B-cell lymphoma 6 (BCL6) oncogene is required for the survival of diffuse large B-cell lymphoma (DLBCL), which is incurable using conventional chemotherapy. Thus, it is imperative to improve the survival of patients with DLBCL. Disulfide (DSF) has been shown to have anticancer effects, but its effect on DLBCL remains unclear.MethodsFour DLBCL cell lines (OCI-LY1, OCI-LY7, OCI-LY10 and U2932) and primary DLBCL cells from eight newly diagnosed DLBCL patients were pretreated with DSF alone or in combination with Cu. Cell morphology was observed under microscope. Flow cytometry was performed to evaluate the cell apoptosis, cell cycle, the mitochondrial membrane potential and the intracellular accumulation of reactive oxygen species (ROS). The protein expression was respectively measured by flow cytometry and western blotting.ResultsDSF or DSF/Cu exhibited a marked inhibitory effect on the growth of DLBCL cells, accompanied by cell cycle arrest at the G0/G1 phase. Meanwhile, DSF or DSF/Cu significantly induced DLBCL cells apoptosis. Further study revealed that DSF or DSF/Cu promoted apoptosis by inhibiting NF-κB signaling pathway. Interestingly, DSF/Cu significantly reduced BCL6 and AIP levels. In addition, DSF significantly up-regulate p53 protein in OCI-LY7 and OCI-LY10 while down-regulate p53 protein in OCI-LY1 and U2932.ConclusionThese results provided evidence for the anti-lymphoma effects of DSF on DLBCL and suggested that DSF has therapeutic potential to DLBCL.

PB2103: CAN WE ACCURATELY PREDICT SURVIVAL IN DIFFUSE LARGE B CELL LYMPHOMA IN THE RITUXIMAB ERA? RETROSPECTIVE COMPARISON OF IPI, AAIPI, R-IPI AND NCCN-IPI

Background: In diffuse large B cell lymphoma (DLBCL), the value of the International Prognostic Index (IPI) and age-adjusted IPI (aaIPI) changed with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) as standard first line. The revised (R-IPI) and enhanced (NCCN-IPI) indices have emerged to improve prognostic ability. An analysis from Surrogate Endpoints for Aggressive Lymphoma (SEAL) database suggests NCCN-IPI better discriminates prognosis. Aims: In light of data scarcity and future changes in second line therapy for relapsed/refactory patients, we aimed to analyse the suitability of 4 survival prognostic indices in a DLBCL cohort. Methods: Single centre analysis of 169 patients diagnosed with de novo DLBCL from 2008 to 2016 and treated with R-CHOP. Primary central nervous system DLBCL or upfront stem cell transplant were exclusion criteria. IPI, aaIPI, R-IPI and NCCN-IPI scores at diagnosis were calculated. Analysis for 5-year overall survival (5y-OS) and progression free survival (5y-PFS) were performed using Kaplan-Meier curves and compared using the log-rank test. For each prognostic index, C-index test from receiver operating characteristic curves was calculated and area under the curve comparisons between prognostic indices were made using DeLong’s test. Results: Median age was 61 years (interval 18-82), 55% of patients had >60 years and 54% were male. At diagnosis, 23% had an ECOG performance status ≥2, 68% had Ann-Arbor stage III-IV, 63% had increased lactic dehydrogenase levels and 77% had extranodal disease (24% >1 site). Median follow-up was 71 months and 5y-OS and 5y-PFS were 74% and 67%, respectively. For the whole cohort, C-index for 5y-OS was 0.701, 0.629 and 0.751 for IPI, R-IPI and NCCN-IPI, respectively, and R-IPI was outperformed by IPI and NCCN-IPI (p=0.007 and p<0.001, respectively). For 5y-PFS C-index was 0.673, 0.695 and 0.689 for IPI, R-IPI and NCCN-IPI, with no statistically significant differences between indices. NCCN-IPI managed to discriminate the intermediate low (IL) and intermediate high (IH) risk groups for both 5y-OS (IL 89% versus (vs) IH 64%, p=0.001) and 5y-PFS (IL 77% vs IH 60%, p=0.029), contrary to IPI (5y-OS IL 69% vs IH 74%, p=0.552; 5y-PFS IL 63% vs HI 64%, p=0.925). The cohort was divided into age groups so that R-IPI and NCCN-IPI could be compared with aaIPI. For patients with ≤60 years, C-index for 5y-OS was 0.722, 0.585 and 0.724 for aaIPI, R-IPI and NCCN-IPI, respectively, and R-IPI was outperformed by NCCN-IPI but not by aaIPI (p=0.023 and p=0.077, respectively). For 5y-PFS C-index was 0.705, 0.622 and 0.625 for aaIPI, R-IPI and NCCN-IPI, with no statistically significant differences between indices. IL and IH risk groups could not be differentiated neither by aaIPI (5y-OS IL 85% vs IH 82%, p=0.725; 5y-PFS 76% vs 69%, p=0.556) nor NCCN-IPI (5y-OS IL 85% vs IH 70%, p=0.162; 5y-PFS IL 70% vs IH 70%, p=0.905), although in the last index there were no high-risk patients. Summary/Conclusion: NCCN-IPI seems the most robust prognostic tool for OS with frontline R-CHOP. Our results are in accordance with the SEAL analysis, although our sample was significantly smaller and SEAL included other immunochemotherapy regimens. However, improvements are needed for PFS prognostication, especially in younger patients within intermediate risk groups, and all indices performed similarly. Accurate prediction of PFS will ultimately allow for early benchmarking of patients for advanced or more aggressive therapies, and future research will be needed from bigger cohorts.

The Effect of Erythropoiesis-Stimulating Agent on the Management of Chemotherapy-Induced Anemia and Tumor Growth in Diffuse Large B-Cell Lymphoma Patients

Bacground : Erythropoiesis stimulating agents (ESA) increase hemoglobin levels, reduce red blood cell (RBC) transfusion requirements and improve the quality of life (QoL) in patients with chemotherapy-associated anemia (CAA). The purpose of this study is to investigate the efficacy and safety of ESA for management of anemia caused by chemotherapy in patients with diffuse large B-cell lymphoma (DLBCL). And this study estimated quality of life as measured by Functional Assessment of Cancer Therapy Scales for anemia (FACT-An) additionally. Until now, several studies suggested that ESA can affect the progression of tumors. This study conducted experiments to confirm whether the results of previous studies were also applied to lymphoma cells.Patients and Methods : This prospective, multicenter, phase II study enrolled 50 Histologically confirmed DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristin, and prednisolone (R-CHOP) chemotherapy. This study enrolled patients who were shown hemoglobin < 10.0 g/dL at least 3 cycles after starting R-CHOP, were planning to receive at least 4 times of darbepoetin, could read Quality of life as measured by FACT-An and agreed with informed consent. However, this study excluded patients who combined iron deficiency anemia, received erythropoietin at least one months before starting darbepoetin, serious pre-existing medical condition, and untreated primary or metastatic CNS(central nervous system) malignancy. This study was registered in clinicaltrial.gov, and more information can be found at NCT02890602. All patients received darbepoietin alfa at fixed dose of 360 mg once every 3 weeks and if the hemoglobin level exceeds 12 g/dL, administration of darbepoietin was stopped temporarily. In addition, DLBCL cell lines or primary DLBCL cells were tested in vitro to investigate if there were any adverse effects of ESAs on the cancer cell growth.Results : Fifty patients were enrolled between Sep 2012 and Oct 2016. The median age was 64.5 years (range: 34-79 years). The baseline clinical features were as follows: female sex, 68.0%; ECOG >1, 75.5%; stage III/IV, 80.0% and International Prognostic Index score (IPI) > 2, 53.3%, and mean serum ferritin level, 648.13±982.52. The baseline mean hemoglobin level was 9.24 g/dL and overall mean change in hemoglobin level was 1.54±1.3 g/dL at 16 weeks. 46% of total enrolled patients achieved an increase in Hb levels of at least 2 g/dL after 16 weeks of darbepoietin administration and median time to response was 8 weeks. The change of OoL was not significant from baseline to finish of ESA. Most adverse events were hematologic complications which were attributed to the primary disease or received chemotherapy. Only one patient had a thrombotic event which was Common Terminology Criteria for Adverse Events (CTCAE) grade I. Transfusion necessity was determined by clinical judgment and the transfusion rates were 18%. In in vitro study using DLBCL cells, erythropoietin or darbepoietin did not alter cancer cell viability nor rituximab-mediated cytotoxicity under the conditions we have tested.Conclusions : Our study has demonstrated that ESA is effective and safe for patients suffering from anemia after RCHOP chemotherapy. The use of ESA in patients with DLBCL did not deteriorate the quality of life of the patients during chemotherapy. Finally, our research have also shown that the use of ESA does not affect the growth of lymphoma cells. (EPOMA ClinicalTrials.gov number, NCT02890602) DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Polo-like kinase 4 induces mitotic defects and DNA damage in diffuse large B-cell lymphoma

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, has recently been shown to play key roles in tumorigenesis. Blocking PLK4 expression by interference or targeted drugs exhibits attractive potential in improving the efficacy of chemotherapy. Nevertheless, the role of PLK4 in diffuse large B-cell lymphoma (DLBCL) is still undefined. In this study, we discover that PLK4 is a potential target for the treatment of DLBCL, and demonstrate the efficacy of a PLK4 inhibitor when used in combination with doxorubicin. Pharmaceutical inhibition of PLK4 with CFI-400945 inhibited DLBCL cell proliferation and induced apoptotic cell death. The anti-tumor effects were accompanied by mitotic defects, including polyploidy and cytokinesis failure. Activation of p53 and Hippo/YAP tumor suppressor signaling pathway was identified as the potential mechanisms driving CFI-400945 activity. Moreover, CFI-400945 treatment resulted in activation of DNA damage response. Combining CFI-400945 with doxorubicin markedly delayed tumor progression in DLBCL xenografts. Finally, PLK4 was increased in primary DLBCL tissues and cell lines. High levels of PLK4 expression were associated with poor survival in the patients receiving CHOP-based treatment, implicating PLK4 as a predictive biomarker of DLBCL chemosensitivity. These results provide the therapeutic potential of CFI-400945 both as monotherapy or in combination with doxorubicin for the treatment of DLBCL.

DSP107, a Novel Bi-Functional Fusion Protein That Combines Inhibition of CD47 with Targeted Activation of 4-1BB to Trigger Innate and Adaptive Anticancer Immune Responses

The mainstay of treatment for Diffuse Large B cell Lymphoma (DLBCL) is conventional chemotherapy combined with anti-CD20 monoclonal antibody rituximab (RTX). However, a subset of patients is refractory to treatment and between 20 to 50% of patients will, after experiencing an initial complete response (CR), develop resistance to treatment and relapse with poor prognosis. Therefore, additional therapeutic options are urgently needed. In this respect, combination of RTX treatment with CD47 monoclonal antibodies has yielded high objective response rates in patients with relapsed/refractory DLBCL in recent phase I trials. Interestingly, although CD47-targeting specifically activates the innate immune system, treatment with CD47 antibodies augments antigen-presentation in the context of MHC by macrophages and dendritic cells, thereby, triggering cross-priming of T cells in murine models. This T cell activation was pivotal in vivo efficacy in these murine models. Thus, a clear rationale exists for the development of novel therapeutics that exploit CD47 checkpoint inhibition while simultaneously stimulating anticancer T cell immunity. Here, we report on such an immunotherapeutic, termed Dual Signaling Protein 107 (DSP107), comprising a computationally-designed fusion of human soluble SIRPα and 4-1BBL. DSP107 was designed to bind to CD47 on cancer cells and block the CD47/SIRPα inhibitory signal delivered to phagocytes. Further, DSP107 was designed to bind to 4-1BB, a costimulatory receptor upregulated upon TCR/MHC interaction and a validated surrogate marker for the tumor-reactive subset of T cells in tumor tissue. Since 4-1BB activation by soluble 4-1BBL requires cross-linking, DSP107 will trigger 4-1BB signaling only after binding to CD47. This CD47-mediated surface immobilization of DSP107 enables delivery of the 4-1BBL-4-1BB costimulatory signal to tumor localized T cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site (Figure 1). Treatment with DSP107 alone or in combination with RTX triggered significant phagocytosis of a panel of DLBCL cancer cell lines as well as primary patient-derived DLBCL cells by macrophages and neutrophils within 3 hours. Further, after longer term incubation of 24h an ~85% reduction in remaining tumor cells was detected upon combined DSP107 and RTX treatment compared to medium control, whereas an increase in apoptosis was detected in the remaining cells. The pro-phagocytic activity of DSP107 was equal to both CD47 antibody as well as SIRPα:Fc. Simultaneously, binding of DSP107 to CD47 enabled 4-1BB costimulatory signaling by reporter cell line HT1080.4-1BB only on CD47-coated plates. Further, in co-cultures of HT1080.4-1BB with CHO.wt and CHO cells ectopically expressing human CD47, 41BB activation was only observed after binding of DSP107 to human CD47. This activation of 4-1BB costimulatory signaling triggered prominent T cell proliferation in mixed cultures of isolated peripheral blood T cells with cancer cells and augmented T cell cytotoxicity in vitro in a concentration and Effector to Target ratio dependent manner. Finally, injection of peripheral blood mononuclear cells (PBMCs) in mice with established SUDHL6 xenografts and simultaneous treatment with DSP107 triggered a strong reduction in tumor size compared to treatment with PBMCs alone. In conclusion, DSP107 clearly inhibits the CD47/SIRPα inhibitory axis and augments phagocytic removal of cancer cells by innate immune cells. Moreover, binding of DSP107 to CD47 enables the 4-1BBL-mediated costimulation of antitumor T cell cytotoxicity. Thus, DSP107 activates both innate and adaptive anticancer immunity and may be of use for the treatment of DLBCL alone or in combination with RTX. Disclosures Cendrowicz: Kahr Medical: Research Funding. Jacob:Kahr Medical: Current Employment. Greenwald:Kahr Medical: Current Employment. Tamir:Kahr Medical: Current Employment. Huls:Kahr Medical: Research Funding. Foley-Comer:Kahr Medical: Current Employment. Pereg:Kahr Medical: Current Employment. Chajut:Kahr Medical: Current Employment. Peled:Kahr Medical: Consultancy. Bremer:Kahr Medical: Consultancy, Research Funding.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting theUPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

Targeting PIN-1 Attenuates GCB DLBCL Cell Proliferation Through Inhibition of PI3K/AKT Signaling.

IntroductionDiffuse large B cell lymphoma (DLBCL) is a highly heterogeneous type of non-Hodgkin lymphoma with many molecular subtypes that can be distinguished by gene expression profiling (GEP). However, the pathogenesis of DLBCL is still unclear.Materials and MethodsThe expression levels of the prolyl isomerase PIN-1 and other related proteins were determined in 73 primary DLBCL patient samples and cell lines by Western blotting (WB) and immunohistochemical (IHC) staining. Cell cycle and apoptosis were evaluated by flow cytometry. Lymphoma cell viability was detected by CCK-8 proliferation assay.ResultsHigh levels of PIN-1 expression were detected in 55% of germinal center B cell (GCB) DLBCL patient samples, whereas such abnormal expression levels were found in only 11% of non-GCB DLBCL patient samples. PIN-1 expression was positively associated with activation of the oncogenic phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in both GCB DLBCL cell lines and primary patient samples. Depletion of PIN-1 was cytotoxic to GCB DLBCL model cell lines because it led to inhibition of the PI3K/AKT signaling pathway, revealing a GCB DLBCL subgroup that is dependent on this pathway. A PI3K inhibitor was selectively toxic to GCB DLBCL lines expressing high levels of PIN-1.ConclusionOur study used PIN-1 to identify a new subgroup of GCB DLBCL associated with the PI3K/AKT signaling pathway, and our findings reveal that inhibition of PI3K is a promising therapeutic approach for GCB DLBCL.

Ironomycin Induces Diffuse Large B-Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy. Although more than half of these patients may achieve long-term remission, the majority of the remaining patients succumb to DLBCL.These patients relapse after conventional Rituximab (R)-based chemotherapy regimen, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) in association with drug resistance. Iron is essential for many fundamental cellular functions, including proliferation and DNA synthesis. Accumulating evidence reveals that abnormal iron metabolism plays an important role in carcinogenesis and in the progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify if iron metabolism pathway is deregulated in DLBCL and could be exploited to develop novel therapeutic strategies. A list of 63 genes related to iron metabolism in cancer was defined. Using Maxstat R function, we showed that 12 genes out of the 63 investigated have a prognostic value in two independent cohorts of DLBCL patients treated by R-CHOP (Melnick cohort, n=69 and Lenz cohort, n=233). Based on these prognostic genes, we created a gene expression profile (GEP)-based risk score as the sum of the beta coefficients weighted by ±1 according to the patient signal above or below the probe set Maxstat value as previously reported (Herviou L et al. Clinical epigenetics 2018). The iron score was significantly associated with high-risk DLBCL in 3 independent cohorts of patients. GSEA analysis revealed that high-risk DLBCL patients defined by iron score are significantly enriched in genes involved in MYC amplification and MYC targets (p&lt; 0.01). These data demonstrated that high iron score allows to identify DLBCL patients with a poor outcome and that could benefit from targeted therapy. We analyzed the therapeutic interest of Ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Ironomycin inhibits DLBCL cell proliferation in a panel of 16 DLBCL cell lines, at nanomolar concentrations (Median IC50: 30nM; range: 7.2 - 91.5 nM) compared to other iron chelators. Ironomycin induces significant cell growth inhibition, apoptosis of DLBCL cells and DNA double strand break accumulation. Furthermore, apoptosis induced by Ironomycin was not reversed by Iron supplementation. Caspase activation and apoptosis induction mediated by Ironomycin could be partially reversed by pan-caspase inhibitor QVD (p&lt;0.001). Ferroptosis inhibitor, Ferrostatin-1, also partially reversed Ironomycin-induced apoptosis (p&lt;0.001). According to ferroptosis induction, Ironomycin induced ROS (CM-H2DCFDA staining), lipid peroxidation (BODIPY-C11 staining) and autophagy (LC3B puncta formation), at 48H, in DLBCL cells. We also identified a delayed progression of replication forks upon Ironomycin treatment in DLBCL cell lines (p&lt;0.0001). Ironomycin induces significant phosphorylation of RPA2 that is a marker of DNA-replication stress and DNA damage response. Interestingly, we identified a significant correlation between basal replication stress monitored by ATR and CHK2 phosphorylation and sensitivity to Ironomycin in DLBCL cell lines. With major importance, we validated the therapeutic interest of Ironomycin in primary DLBCL cells of patients (n=5) without major toxicity for non-tumor cells from the microenvironment. Ironomycin significantly reduces the median number of viable primary DLBCL cells by 69.3% and 77.8%, at respectively 50 and 100nM concentrations (p&lt;0.01 and p&lt;0.001). Furthermore, Ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment (p&lt;0.001). We tested the therapeutic interest to combine Ironomycin with conventional chemotherapy used in DLBCL including cyclophosphamide, doxorubicin and etoposide. Interestingly, we identified a significant synergistic effect when Ironomycin is combined with Doxorubicin. Altogether, these data demonstrated that a subgroup of high-risk DLBCL patients could be identified with the iron score and could benefit from Iron metabolism inhibitor treatment. Disclosures Cartron: Roche, Celgene: Consultancy; Sanofi, Gilead, Janssen, Roche, Celgene: Honoraria. Moreaux:Diag2Tec: Other: Co-founder of Diag2Tec company.

The IL‐6 signaling complex is a critical driver, negative prognostic factor, and therapeutic target in diffuse large B‐cell lymphoma

Interleukin‐6 (IL‐6) is a growth factor for normal B cells and plasma cell‐derived malignancies. Here, we show that the IL‐6 signaling pathway is also active in a subset of diffuse large B‐cell lymphoma (DLBCL) patients with particularly poor prognosis. Primary DLBCL cells and DLBCL cell lines expressing IL‐6R engraft and form orthotopic lymphomas in humanized mice that ectopically produce human IL‐6, and in mice reconstituted with a human immune system. We show that a subset of DLBCL cases have evolved mechanisms that ensure constitutive activation of the IL‐6 signaling pathway, i.e., the expression of both chains of the IL‐6R, the expression of the cytokine itself, and the mutational inactivation of a negative regulator of IL‐6 signaling, SOCS1. IL‐6 signaling promotes MYC‐driven lymphomagenesis in a genetically engineered model, and treatment with the IL‐6R‐specific antibody tocilizumab reduces growth of primary DLBCL cells and of DLBCL cell lines in various therapeutic settings. The combined results uncover the IL‐6 signaling pathway as a driver and negative prognosticator in aggressive DLBCL that can be targeted with a safe and well‐tolerated biologic.

Retraction: In Vitro and In Vivo Interactions between the HDAC6 Inhibitor Ricolinostat (ACY1215) and the Irreversible Proteasome Inhibitor Carfilzomib in Non-Hodgkin Lymphoma Cells

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2–M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib–ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. Mol Cancer Ther; 13(12); 2886–97. ©2014 AACR .

Targeting ubiquitin-activating enzyme induces ER stress–mediated apoptosis in B-cell lymphoma cells

AbstractAlterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)–like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243–induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

LMO2 Facilitates Synthetic Lethality after PARP Inhibition (PARPi) in Diffuse Large B-Cell Lymphoma (DLBCL)

Novel therapies for DLBCL are needed to improve patients' outcomes. LIM domain only 2 (LMO2) protein is ubiquitously expressed and plays important roles in endothelial and hematopoietic cell development. LMO2 protein expression is upregulated in germinal center B (GCB) cells, the cell of origin of DLBCL. 73% of GCB and 45% of ABC DLBCLs express LMO2 protein at levels of reactive GCB cells. Although the function of LMO2 in B cells and DLBCL is unknown, expression of LMO2 serves as one of the best prognostic markers of longer survival following R-CHOP therapy. Additionally, LMO2 expression in DLBCL cells results in genomic instability. These observations suggest that LMO2 may decrease DNA repair efficiency. Indeed, here we demonstrate that primary DLBCL tumors and cell lines expressing high levels of LMO2 protein (LMO2HIGH) are defective in DNA double-strand break (DSB) repair via the homologous recombination (HR) pathway. We found that LMO2HIGH DLBCL cells and LMO2LOW DLBCL cells expressing a GFP-LMO2 fusion protein via a doxycycline-inducible system have a defective accumulation of the HR proteins BRCA1 and RAD51 to damaged chromosomes as visualized by immunofluorescence (IF) and Western blot assays. Furthermore, LMO2HIGH DLBCL exposed to ionizing radiation showed decreased levels of phosphorylated (S4 and S8) replication protein A (RPA32 subunit), a marker of DSB end-resection activity and an essential step for HR-dependent DSB repair. Consequently, LMO2HIGH DLBCL showed decreased HR activity as assessed via a DR-GFP reporter system and scoring the frequency of HR-dependent sister chromatid exchanges. Also, in LMO2HIGH vs LMO2LOW DLBCLs, we observed higher levels of ionizing radiation-induced foci (IRIF) for 53BP1 and RIF1 - non-homologous end-joining (NHEJ) core factors playing a critical role in defining DSB repair pathway choice. Similarly, we found a higher accumulation of 53BP1 and RIF1 chromatin-enriched fraction after DNA damage in LMO2HIGH than LMO2LOW DLBCLs. Furthermore, we show that LMO2 forms a complex with 53BP1 but not BRCA1 after DNA damage, as demonstrated by Co-IP, GST pull-down assays and spatial co-localization in IRIFs. This suggests that LMO2 functionally interacts with 53BP1 during DSB repair. Indeed, knockdown of 53BP1 in LMO2HIGH cells increased the levels of BRCA1 and RAD51 IRIF to values similar to LMO2LOW cells without affecting LMO2 levels, revealing that LMO2 depends on 53BP1 to inhibit HR activity.Since LMO2HIGH DLBCL cells exhibit a defective HR-pathway, we next explored the therapeutic potential of PARPi in DLBCL. We found that in LMO2HIGH but not LMO2LOW DLBCL cell lines the PARPi olaparib induced a significant decrease in cell proliferation and colony formation and an increase in cell death via apoptosis. The sensitivity to olaparib directly correlated with LMO2 protein levels. The proliferation defect in LMO2HIGH DLBCL cells was due to the increased DNA damage caused by exposure to PARPi, as observed by an increase in γH2AX foci. Induction of LMO2 expression in LMO2LOW DLBCL cell lines led to sensitivity to olaparib, demonstrating that the proliferation defect induced by olaparib was dependent on LMO2 expression. Silencing of LMO2 via shRNA or CRISP/Cas9 in LMO2HIGH cell lines rescued the proliferation defect induced by olaparib. The proliferation deficiency induced by olaparib was synergistic with doxorubicin in LMO2HIGH but not in LMO2LOW DLBCL cell lines and untreated patient-derived primary DLBCL tumors.We also examined the in vivo efficacy of olaparib in DLBCL mice models. Olaparib treatment prolonged survival of mice harboring LMO2HIGH but not LMO2LOW DLBCL xenograft tumors. Olaparib in combination with RCHOP significantly prolonged survival of mice harboring LMO2HIGH DLBCL tumors compared to cohorts treated with either olaparib or RCHOP alone. Further, analysis of tumors excised from OCI-LY1 bearing animals treated with olaparib for 3 days revealed increased cell death and a higher sensitivity to PARPi in OCI-LY1 LMO2 cells compared to the OCI-LY1 LMO2 negative-GFP control cells, indicating in vivo survival advantage for LMO2LOW cells upon olaparib treatment.In summary, high expression of LMO2 results in HR-dysfunction phenocopying the BRCA1/2 mutations observed in breast and ovarian tumors. In LMO2HIGH DLBCL, PARPi-induced killing is synergistic with doxorubicin, thus providing a clear path for therapeutic development of PARPi in DLBCL. DisclosuresLossos:Affimed: Research Funding.

Selective Targeting Cyclin-Dependent Kinase-9 (CDK9) Downmodulates c-MYC and Induces Apoptosis in Diffuse Large B-Cell Lymphoma (DLBCL) Cells

DLBCL accounts for >10,000 deaths in the U.S. annually. Despite efficacy of upfront chemo-immunotherapy, up to 50% of patients die within 5 years. While inhibitors of B-cell receptor signaling transformed the therapeutic paradigm in indolent lymphomas, they show poor activity in DLBCL, which thus represents an unmet clinical need.MYC oncogene, a transcription factor which regulates cell proliferation and growth, is a key driver in lymphomagenesis. Myc overexpression is associated with chemoresistance in NHL, while "double-hit" lymphomas characterized by MYC and BCL2 rearrangement are most aggressive. Myc proteins have extraordinarily short half-life. To regulate transcription, Myc recruits pTEFb, an elongation factor whose catalytic core comprises CDK9/cyclin T complexes. Hence, Myc expression and function may be susceptible to CDK9 inhibition. Non-specific inhibitors of multiple CDKs have shown promise in B-cell malignancies, where their pro-apoptotic effect has been attributed to a reduction in transcription and downmodulation of short lived pro-survival proteins, e.g. MCL1. However, such inhibitors lack a therapeutic window or a refined mechanism of action. Here we selectively targeted CDK9 in a pre-clinical study in DLBCL and demonstrate that this therapeutic approach disrupts Myc oncogenic function.Experiments were conducted in activated B-cell(ABC)-type (OCI-LY3, U2932, NuDUL-1) and germinal center(GC)-type (OCI-LY18/19, SUDHL4/6/10/16, VAL) DLBCL cell lines and in primary lymph node-derived B-cells from patients with refractory DLBCL. Tumor xenografts were established in NSG mice. We used AZ5576, a novel selective CDK9 inhibitor (Astra Zeneca), pan-CDK inhibitors (flavopiridol, dinaciclib) and genetic knockdown of CDK9.Treatment with AZ5576 promoted rapid apoptosis induction (within 2 hours) in DLBCL cell lines and primary tumor cells, as evident by PARP cleavage. Quantification of Annexin V+ cells after 24 hour drug exposure rendered an IC50 of 300-500 nM in DLBCL cell lines independent of cell of origin and 100 nM in primary DLBCL cells. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis. Contrary to pan-CDK inhibitors, AZ-5576 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect.CDK9 inhibition resulted in rapid (within 1 hour) and dose-dependent downregulation of Myc and MCL1 mRNA and protein in DLBCL cell lines and primary cells. Cell cycle profiling demonstrated that treatment with AZ-5576 led to S phase reduction, consistent with loss of Myc, while pan-CDK inhibitors downmodulated S/G2/M, reflecting inhibition of multiple CDKs. Cyclohexamide chase experiments demonstrated enhanced degradation of Myc in DLBCL cells treated with AZ-5576. Myc protein degradation is tightly regulated: while phosphorylation at Ser62 enhances Myc stability, MycT58 is primed for degradation. CDK9 inhibition decreased pMycS62 prior to apoptosis induction in DLBCL cell lines. Raji cells, which carry a stabilizing MycT58A mutation, were less sensitive to AZ-5576. This suggests that CDK9 may govern Myc protein turnover, thus regulating its expression through multiple mechanisms.Overexpression of Myc was associated with enhanced sensitivity to CDK9 inhibition. Treatment with 300 nM AZ-5576 for 24 h induced apoptosis of 35-70% of Myc-high cells (e.g., U-2932, VAL), compared with 10-20% of Myc-low cells (e.g., OCI-LY3, SU-DHL10). CDK9 inhibition did not disrupt Myc-CDK9 complex formation. Despite this, it abrogated Myc transcriptional activity in a functional reporter assay. Meanwhile, sensitivity to AZ-5576 did not correlate with expression of MCL1, BCL2 or BCLX.Our findings were confirmed in a mouse xenograft model. VAL cells were inoculated subcutaneously and treatment with AZ-5576 (30 mg/kg orally twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression (Figure), prolonged survival, decreased cell proliferation (Ki-67), enhanced apoptosis (Annexin V), and reduction in Myc total and p-S62 levels and MCL1.Thus, our data suggests that targeting CDK9 is a promising therapeutic strategy poised to disrupt Myc oncogenic activity in DLBCL and provides rationale for clinical development of AZ-5576. [Display omitted] DisclosuresChen:Seattle Genetics: Consultancy. Drew:Astra Zeneca: Employment. Danilov:ImmunoGen: Consultancy; Pharmacyclics: Consultancy; Astra Zeneca: Research Funding; Prime Oncology: Honoraria; Dava Oncology: Honoraria; GIlead Sciences: Research Funding; Takeda: Research Funding.

The Bcl-2 BH4 Antagonist Bda-366 Induces Apoptosis in CLL and DLBCL Cells through a Bcl-2 Independent Mechanism

Overexpression of the antiapoptotic protein Bcl-2 is considered primarily responsible for the increased apoptosis resistance and prolonged survival of the malignant B cells in chronic lymphocytic leukemia (CLL), follicular lymphoma and subsets of diffuse large B cell lymphoma (DLBCL). The importance of Bcl-2 overexpression is corroborated by the significant clinical activity of the recently approved Bcl-2 BH3 antagonist venetoclax. Venetoclax induces apoptosis by sequestering Bcl-2 from the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, resulting in consequent Bax/Bak activation, mitochondrial outer membrane permeabilization and cytochrome c release. Although venetoclax is extremely toxic against CLL cells in vitro, resistance can arise in vivo because of overexpression of other anti-apoptotic Bcl-2 family members, such as Bcl-xL and Mcl-1.In the present study we investigated the activity of BDA-366, another small molecule Bcl-2 inhibitor that was recently reported to selectively induce apoptosis in Bcl-2-overexpressing lung cancer cells through a distinct mechanism (Han B et al, Cancer Cell. 2015;27:852-63). This drug was designed to selectively bind the BH4 domain of Bcl-2 and was reported to trigger apoptosis by inducing a conformational change in Bcl-2 that converts it into a pro-apoptotic protein. To investigate whether BDA-366 is also selectively toxic for CLL cells, we analyzed the viability of CLL (n=31) and normal peripheral blood mononuclear cells (PBMCs)(n=6) after 48 hours in culture with increasing concentrations of BDA-366. Annexin V/PI analysis showed that BDA-366 is significantly more toxic against CLL (LD50=1.11±0.46 mM) than normal PBMCs (LD50=2.03±0.31 mM, P<0.001). However, considerable variability was noted in the sensitivity of the different CLL samples, with approximatelly 50% of the cases demonstrating LD50 values in the nanomolar range and the remaining cases displaying sensitity similar to normal PBMC. To investigate the reasons for this variability, we correlated LD50 values with IGHV mutation status, ZAP-70 expression, cytogenetic abnormalities, and expression of various Bcl-2 family members, including Bcl-2, Bcl-xL, Mcl-1, BimEL, BimL, Bax, Bak and Hrk. The only positive finding from this analysis was the inverse correlation between LD50 and expression of BimL (n=23, r=-0.446, p=0.033), whereas no correlation was observed with Bcl-2 expression.Variable sensitivity to BDA-366 and absence of correlation with Bcl-2 expression was also observed in the analysis of a series of DLBCL and CLL cell lines. These cell lines showed LD50 values ranging from nanomolar (Pfeiffer, 0.19 µM; OCI-LY-18, 0.32 µM; OCI-LY-1, 0.33 µM; TOLEDO, 0.41 µM; OCI-Ly10, 0.50 µM; HT, 0.56 µM; Ri-1, 0.58 µM; and SU-DHL-6, 0.81 µM) to micromolar range (MEC1, 1.5 µM; HBL-1, 4.0 µM; Karpas, 4.34 µM; BJAB, 4.8 µM; and SU-DHL-4, 6.31 µM). Although most of the sensitive lines expressed Bcl-2, some had very low or even undetectable Bcl-2 expression (e.g., Pfeiffer and HT), suggesting that BDA-366 can induce apoptosis independently of Bcl-2. In support of this possibility, we observed that BDA-366 was equally effective against Bcl-2-negative HT cells and HT cells stably transfected with Bcl-2. In addition, transient overexpression of Bcl-2 in the DLBCL cell line BJAB and the CLL cell line MEC1 resulted in greater resistance to BDA-366, further suggesting that BDA-366 does not induce apoptosis by converting Bcl-2 into a proapoptotic protein.To determine whether mechanisms that induce resistance to venetoclax can also induce resistance to BDA-366, we investigated viability of BDA-366-treated CLL cells following stimulation with immobilized anti-IgM. Pre-treatment of CLL cells with immobilized anti-IgM resulted in increased resistance to both BDA-366 and venetoclax. Interestingly, the combination of BDA-366 and venetoclax was significantly more toxic than each agent alone (Figure 1), suggesting that combining these drugs could be a potential approach to overcome resistance induced by BCR signals.In conclusion, these data show that BDA-366 is selectively toxic at low nanomolar concentrations for a large proportion of primary CLL samples and DLBCL cell lines. The primary mechanism of action is still unclear, but the lack of a correlation with Bcl-2 expression suggests that it differs from the one initially proposed. [Display omitted] DisclosuresEfremov:Gilead: Honoraria.

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5+ CD4+ T cells, in DLBCL. Data showed that compared to CXCR5- CD4+ T cells, CXCR5+ CD4+ T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5+ CD4+ T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5- CD4+ T cells, while the level of IL-10 secretion was significant elevated in the CXCR5+ compartment compared to the CXCR5- compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5+ CD4+ T cell coculture compromised the CXCR5+ CD4+ T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5+ compartment also contained significantly lower frequencies of cytotoxic CD4+ T cells than the CXCR5- compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4+ T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10.