HIGH EXPRESSION OF BCL‐2 AND BCL‐XL IN DIFFUSE LARGE B‐CELL LYMPHOMA CONFER POOR PROGNOSIS BUT MAY BE REVERSIBLE BY COMBINED INHIBITION WITH BET INHIBITORS AND BH3 MIMETICS.

Introduction: Double-hit lymphoma (DHL) and double-expressor (DE) Diffuse Large B cell Lymphoma (DLBCL) are both characterised by dysregulation of MYC and BCL-2, and associated with worse survival following treatment with current standard of care (R-CHOP). Novel approaches capable of improving response for these sub-groups are therefore urgently needed. Surprisingly, venetoclax, a BH3-mimetic that potently inhibits BCL-2, has limited activity in this disease. We therefore sought to understand the expression and potential prognostic relevance of all anti-apoptotic BCL-2 family members, not just BCL-2, in DLBCL and how these might be overcome with new therapeutic approaches. To this end we investigated a novel BET inhibitor (BETi), PLX2853, and its combination with the BH3-mimetics, venetoclax (BCL-2) and WEHI-539 (BCL-xL). Methods: We analysed the relationship between survival and expression of BCL-2 family mRNA using the Whole Genome-DASL array from 928 patients from the REMoDL-B trial. E-myc lymphoma cell lines (LCLs) and DLBCL cell lines (CL) (4 GCB, 3 ABC) were utilised to dissect potential mechanisms of cell death following exposure to BETi and BH3-mimetics. Techniques employed included qPCR, western blot, immunoprecipitation, retroviral transduction, flow cytometry and in-vivo mouse studies. Results: We identified differential mRNA expression patterns of anti-apoptotic BCL-2 family members in DLBCL subtypes. These had prognostic relevance: specifically, across sub-types high BCL-2 conferred poor response to R-CHOP, whilst in GCB DLBCL high BCL-xL was associated with worse survival. In Eμ-myc LCLs, a novel BETi developed by structure-based design, PLX2853, induced cell death at lower concentrations than other agents of its class. Cell death was mediated by upregulation of the pro-apoptotic protein, BIM, associated with downregulation of the suppressive miR-17~92. When Eμ-myc LCLs were transduced to overexpress BCL-2 or BCL-xL, reflecting the situation in DLBCL they became resistant to PLX2853. Similar resistance to BETi was seen in human DLBCL CLs. E-myc LCLs overexpressing BCL-2 or BCL-xL could be resensitised to BETi through combination with venetoclax or WEHI-539, respectively, and this was associated with priming of anti-apoptotic proteins with BIM. In GCB DLBCL CLs, BIM upregulation in response to PLX2853 was identified. The combination of PLX2853 and venetoclax produced synergistic increases in cell death, resulting in reduced tumour growth and enhanced survival in NSG mice bearing DLBCL xenografts. Conclusion: We identified a varied landscape of expression of anti-apoptotic BCL-2 proteins in DLBCL and highlighted the contribution of high BCL-xL expression to treatment resistance in DLBCL. High expression of anti-apoptotic BCL-2 family members also provides resistance to BETi. However, this could be overcome by use of specific BH3-mimetics targeting the relevant pro-survival BCL-2 member, involving mitochondrial priming. Keywords: “double-hit” lymphomas; BCL2; venetoclax. Disclosures: Cummin, T: Research Funding: Plexxikon. Barrans, S: Other Remuneration: HTG. Burton, C: Consultant Advisory Role: Roche, Takeda, BMS, Celgene; Honoraria: Roche, Takeda, BMS; Other Remuneration: Roche. Davies, A: Consultant Advisory Role: Roche, Kite, Celgene, Acerta, MorphoSys, BioInvent; Honoraria: Roche, Celgene, Kite, Janssen; Research Funding: Roche, Acerta, Celgene, Gilead, Karyopharma, GSK; Other Remuneration: Roche, Celgene. Cragg, M: Consultant Advisory Role: Bioinvent International, Roche, Boehringer Ingelheim, Baxalta and GLG; Research Funding: Bioinvent, Roche, Iteos, Gilead and GSK. Johnson, P: Consultant Advisory Role: Janssen; Honoraria: Bristol-Myers Squibb, Takeda, Novartis, Celgene, Janssen, Epizyme; Research Funding: Janssen, Epizyme.