Abstract 342: S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations
Authors: Skwarska Anna , Zhang Qi , Herbrich Shelley M. , Baran Natalia , Halilovic Ensar , Ruvolo Peter , Ruvolo Vivian , Morris Erick , Wei Andrew , Moujalled Donia , Andreff Michael , Konopleva Marina 
Published: August 2019

Abstract
Myeloid cell leukemia 1 (Mcl-1) is one of the key anti-apoptotic Bcl-2 family proteins that binds and neutralizes pro-apoptotic BIM, BAX and BAK at the mitochondrial outer membrane, preventing cytochrome c release and caspase activation. Selective upregulation of Mcl-1 functionally contributes to resistance of acute myeloid leukemias (AML) with FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) to chemotherapy (Kasper S. et al. 2012, Blood Cancer J. 2:e60, doi:10.1038/bcj.2012.5). Here we show that a novel Mcl-1 inhibitor S63845 (Kotschy A. et al. 2016, Nature 538, 477-482), has synergistic proapoptotic activity in combination with FLT3-ITD kinase inhibitor midostaurin in pre-clinical models of AML. Our studies demonstrate that S63845 has potent single agent activity in AML cell lines and primary AML samples harboring FLT3-ITD with IC50 values in low nanomolar range. Co-targeting of Mcl-1 and FLT3-ITD with S63845 and midostaurin, respectively, significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage occurring rapidly within 6 hours of treatment. Consistent with markedly reduced cell growth and viability, analysis of drug combinations efficacy using Bliss independence model revealed strong synergistic interactions between S63845 and midostaurin in FLT3-ITD cell lines and primary AML samples. Midostaurin caused de-phosphorylation of FLT3-ITD and its downstream targets such as STAT5, AKT and MAPK. This was accompanied by significant downregulation of MAPK-mediated phosphorylation of Mcl-1 at Thr163 required for Mcl-1 stability. Consequently, midostaurin reduced Mcl-1 protein levels, with no major changes in antiapoptotic Bcl-2 or Bcl-XL. Importantly, midostaurin increased expression of pro-apoptotic Bim, which could in turn bind and negate residual Mcl-1 pro-survival activity. Elevated Bim was sustained upon S63845 co-treatment, suggesting that Bim plays functional role in midostaurin/S63845-mediated lethality. Dynamic BH3 profiling showed that midostaurin primed FLT3-ITD cells to Mcl-1 and Bcl-2 inhibitors and facilitated general apoptosis priming in response to Bim peptide. Importantly, given that Mcl-1 is a major contributing factor to resistance of AML to Bcl-2 selective BH3-mimetic venetoclax, S63845/midostaurin treatment induced cell death in venetoclax-resistant FLT3-ITD mutants. In summary, S63845/midostaurin is highly synergistic in FLT3-ITD mutated AML cells including those resistant to venetoclax. In vivo experiments of tolerability and efficacy are ongoing and will be reported.
Citation Format: Anna Skwarska, Qi Zhang, Shelley M. Herbrich, Natalia Baran, Ensar Halilovic, Peter Ruvolo, Vivian Ruvolo, Erick Morris, Andrew Wei, Donia Moujalled, Michael Andreff, Marina Konopleva. S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations [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 342.