Abstract 2501: Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in diffuse large B-cell lymphoma (DLBCL) pre-clinical models
Authors: Mavis Cory , Gu Juan , Barth Matthew , Khan Sumera , Torka Pallawi , Hernandez-Ilizaliturri Francisco 
Published: August 2019

Background: Upregulation of the anti-apoptotic protein Mcl-1 has been implicated in chemotherapy resistance and poor clinical outcomes in activated B-cell like (ABC)-DLBCL. We hypothesized that singular inhibition of Mcl-1 by AMG-176 and/or dual inhibition of MCL-1 and Bcl-2 by combining AMG-176 with venetoclax could be an effective strategy for inducing cell death in DLBCL pre-clinical models.

Materials and Methods: Using a panel of DLBCL cell lines, we conducted pre-clinical targeting of Mcl-1 with AMG-176. Cells were exposed to AMG-176 as a single agent (0-20uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Subsequently, DOHH-2, OCI-LY1, DHL4, DHL6, TMD8, and VAL cells were exposed to AMG-176 (0-20 uM) in combination with Doxorubicin (0-1uM), Cisplatin (0-10uM), Cytarabine (0-10uM), Vincristine (0-10uM), Ixazomib (0-100nM), Carfilzomib (0-12.5nM), and Venetoclax (0-10uM) for 72 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) as well as changes in cell cycle (PI staining). Bcl-2 family member protein expression was determined by Western blotting. Primary tumor cells isolated from a patient with a composite lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and EBV-driven DLBCL were exposed ex vivo to AMG-176 +/- venetoclax. By immune-phenotype, the primary tumor cells isolated and expanded were ABC-DLBCL.

Results: In vitro, AMG-176 single agent exposure induced cell death in a dose- and time-dependent manner in all DLBCL cell lines tested. AMG-176 IC50 values varied from 0.03 to 11.42 uM for OCI-LY1 and ROS-50 respectively. Western blotting studies confirmed changes in Bcl-2 family members, specifically in Bok, Bcl-XL, Mcl-1 and Bcl-2 following in vitro exposure to AMG-176. The combination of AMG-176 with Doxorubicin, Cisplatin, Cytarabine, Vincristine, Ixazomib, Carfilzomib, or Venetoclax resulted in significant synergistic activity for each combination. The strongest coefficient of synergy was observed when AMG-176 was combined with venetoclax. Similar synergy results were noted in primary tumor cells isolated from the lymphoma patient. Annexin/PI staining with AMG-176 demonstrated caspase dependent apoptosis. No significant changes in cell cycle were observed with AMG-176 exposure.

Conclusion: Our data suggests that AMG-176 exhibits strong synergistic activity with various chemotherapy agents and small molecule inhibitors especially venetoclax. In vivo experiments are planned. Combination therapy targeting Mcl-1 and Bcl-2 may provide an alternative therapeutic approach in DLBCL.

Citation Format: Cory Mavis, Juan Gu, Matthew Barth, Sumera Khan, Pallawi Torka, Francisco Hernandez-Ilizaliturri. Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in diffuse large B-cell lymphoma (DLBCL) pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2501.