PS1351 CRITICAL ANALYSIS OF THE STRINGENT COMPLETE RESPONSE IN MULTIPLE MYELOMA. AN UPDATED OF THE ANALYSIS BASED ON THE PETHEMA/GEM2012MENOS65 PHASE III CLINICAL TRIAL

Abstract

Background: Multiple myeloma (MM) is a heterogenous hematological cancer that remains largely incurable. Apoptosis dysregulation by overexpression of BCL2 family anti-apoptotic proteins (BCL2, MCL1, BCLXL) is involved in MM cell survival and resistance to chemotherapy. This particularity makes MM cells vulnerable to BH3 mimetics that trigger apoptosis by specifically disrupting complexes between anti-apoptotic and pro-apoptotic proteins. BH3 mimetics targeting either BCL2 (Venetoclax) or MCL1 (S63845, AMG176 and AZD5991) are currently tested in clinic for MM treatment. BCL2 targeting was shown to be effective in a minority of patients, mainly t(11;14), while MCL1 targeting is widely effective, particularly at relapse. However, some patients remain resistant to BCL2 and MCL1 BH3 mimetics and could benefit from a dual targeting. Aims: The objective of this study was to highlight the interest of combining two clinically available BH3 mimetics, Venetoclax and S63845, targeting respectively BCL2 and MCL1. We also intended to decipher the mechanism of action of this combination and to identify resistance factors. Methods: For this study, we treated a panel of 25 Human Myeloma cell lines (HMCLs) and a cohort of more than 40 myeloma patient samples with Venetoclax and S63845 to identify those resistant to both agents. This group of resistant HMCLs and patient samples was treated with the combination S63845/Venetoclax at low doses. siRNA assays were performed to identify the role of effectors BAX and BAK and BH3-only proteins BIM and NOXA in combination-induced apoptosis. To understand the mechanism of action of this combination, we also studied the activation of BAX and BAK using specific antibodies. Finally, the role playing by BCLXL in the resistance to the S63845/Venetoclax combination was studied by both silencing and overexpression experiments. Results: One third of our cell line collection as well as our patient cohort was resistant to each BH3 mimetic. The combination was highly effective and synergic at low doses in three fourth of these cells. We demonstrated that the efficiency of the combination was equally dependent on BAX and BAK and that the simultaneous silencing of both effectors completely abrogated the combination effect. Conversely, the response to S63845 was mainly dependent on BAK. Interestingly, we also showed that the combination-induced apoptosis was associated with a hetero-complex formation between BAX and BAK. The BH3-only protein BIM, but not NOXA, was also shown to play a role in the combination response. Finally, BCLXL remained a resistance factor to the S63845/Venetoclax combination in agreement with its role in the resistance to both MCL1 and BCL2 BH3 mimetics alone. Summary/Conclusion: Our work demonstrates the interest of using the combination of two BH3 mimetics targeting both MCL1 and BCL2 in MM cells otherwise resistant to each inhibitor alone. Using low doses, this therapeutic strategy could potentially minimize side effects. We also demonstrated the crucial role of BAX and BAK in this BH3 mimetic combination-induced cell death. This is in agreement with the hetero-complex formation of BAX and BAK observed during myeloma cell apoptosis resulting from the combination treatment.