Antimyeloma Effect of the Simultaneous Inhibition of MCL-1 (with S63845) and BCL-2 (with Venetoclax) in the Presence of the Microenvironment
Authors: Algarín Esperanza M , Díaz-Tejedor Andrea , Mogollón Pedro , Hernández-García Susana , Corchete Luis , San-Segundo Laura , Martín-Sánchez Montserrat , González-Méndez Lorena , Schoumacher Marie , Banquet Sébastien , Kraus-Berthier Laurence , Kloos Ioana , Halilovic Ensar , Maacke Heiko , Gutierrez Norma C. , Mateos Maria-Victoria , Paíno Teresa , Garayoa Mercedes , Ocio Enrique M. 
Published: October 2019

Abstract
Background: Venetoclax is a BCL-2 inhibitor particularly effective in patients with multiple myeloma (MM) harboring the t(11;14). However, resistance to venetoclax has been linked to MCL-1 overexpression. On the other hand, it is wellknown that MM cells depend on MCL-1 rather than BCL-2 for survival, and this dependence has recently been reported to be enhanced by the tumor-associated microenvironment. Therefore, the combination of venetoclax with the potent MCL-1 inhibitor S63845 arises as a promising and novel approach for the treatment of MM.
Aims: To evaluate the efficacy and mechanism of action of S63845 alone and in combination with venetoclax in absence and presence of the bone marrow tumor microenvironment in preclinical in vitro, ex vivo and in vivo models of MM.
Methods: S63845 was provided by an agreement with Servier and Novartis. In vitro activity of S63845 and venetoclax alone and in combination was evaluated by bioluminescence on a MM cell line expressing luciferase (MM.1S-luc) in absence and presence of mesenchymal stromal cells isolated from bone marrow aspirates of MM patients (pMSCs). MM.1S cells cultured in absence or presence of pMSCs were analyzed for MCL-1 and BCL-2 protein levels by Western blot. Interactions between these anti-apoptotic proteins with the pro-apoptotic protein BIM were assessed by immunoprecipitation assays. The efficacy of S63845 and venetoclax alone and in combination was also evaluated ex vivo in MM cells and normal lymphocytes from MM patients. Finally, a disseminated MM model in BRG mice was used for in vivo studies.
Results: S63845 and venetoclax showed a strong antimyeloma dose-dependent effect on MM.1S-luc cells co-cultured with pMSCs. However, whereas the presence of tumor-associated MSCs increased the IC50 value of venetoclax in MM.1S-luc cells from 6.2 to 9.8 mM, it reduced that of S63845 from 94.1 to 81 nM, suggesting a mild sensitization to this drug in the context of the microenvironment. Neither S63845 nor venetoclax affected pMSC viability even at high concentrations by MTT assay. The co-culture with the BM stromal microenvironment increased MCL-1 expression on untreated MM.1S cells in two out of four experiments performed with MSCs from different MM patients, whereas it surprisingly induced a decrease on BCL-2 levels in all of them. Treatment with S63845 completely blocked MCL-1 binding to BIM, both in the absence or presence of pMSCs but did not induce the compensatory increase of BCL-2/BIM complexes observed in MM.1S cells in monoculture. Venetoclax also completely blocked the binding of BCL-2 to BIM in MM.1S alone or in co-culture, and induced a similar compensatory increase of MCL-1/BIM complexes in both situations. Importantly, the double combination S63845 + venetoclax was significantly superior to both drugs in monotherapy in killing MM.1S-luc cells co-cultured in the presence of the stromal microenvironment. BIM immunoprecipitation assays showed that the double combination was able to counteract the compensatory upregulation of MCL-1 bound to BIM observed on MM.1S cells treated with venetoclax and to entirely disrupt BCL-2/BIM complexes, both in the absence and presence of pMSCs. Furthermore, S63845 + venetoclax increased the percentage of apoptotic MM plasma cells from three MM patients with respect to single treatments with moderate toxicity detected on normal lymphocytes, suggesting the existence of a therapeutic window for the double combination. Finally, the combination of S63845 + venetoclax clearly delayed tumor growth as compared with the agents in monotherapy in a disseminated model of MM with statistically significant differences from day 19 of treatment. This in vivo effect translated into a significatively improved survival for mice treated with the double combination (median 60 days) vs control mice (median 32 days; log-rank test P=0.045).
Conclusion: Our preclinical data demonstrate the potent activity of the combination of venetoclax with S63845 in MM even in presence of the stromal associated-tumor microenvironment, and provides the rationale for the clinical development of this combination in relapsed or refractory MM patients.
This project was supported by Novartis Pharmaceuticals and by the Spanish , ISCIII-FIS PI15/00067 and PI15/02156, GRS 1604/A/17 and CRMRTC de Castilla y León. Predoctoral grant to EMA by Consejería de Educación de Castilla y León.

Disclosures
Schoumacher: Servier: Employment. Banquet:Servier: Employment. Kraus-Berthier:servier: Employment. Kloos:Servier: Employment; Novartis: Other: Partnership. Halilovic:Novartis: Employment, Equity Ownership. Maacke:Novartis: Employment. Mateos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:AbbVie: Consultancy; Novartis: Consultancy, Honoraria; BMS: Consultancy; Seattle Genetics: Consultancy; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pharmamar: Consultancy; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding.