FLT3-ITD Activation Mediates Resistance to the BCL-2 Selective Antagonist, Venetoclax, in FLT3-ITD Mutant AML Models

Abstract

FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~20-25% of adult AML cases and are associated with worse prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the pro-survival proteins BCL-XL and MCL-1. Venetoclax (ABT-199/GDC-0199), a potent, selective inhibitor of the pro-survival protein BCL-2, recently demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors; therefore targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib (AC220), a potent FLT3 inhibitor, in FLT3-ITD+ AML models.Sensitivity to venetoclax was assessed in FLT3-ITD+ (Molm13 and MV4;11) and FLT3 WT (HL60 and OCI-AML3) cells in vitro . Dose dependent growth inhibition and induction of apoptosis was seen in MV4;11, Molm13 and HL60 cells following 48hr venetoclax treatment, with the MV4;11 cells being the most sensitive to venetoclax. Twenty-four hour quizartinib treatment reduced expression of BCL-XL and MCL-1 protein, but not BCL-2, in FLT3-ITD+ cells, while quizartinib had no effect on BCL-XL, MCL-1 or BCL-2 expression in FLT3 WT cells, confirming FLT3-ITD mediated regulation of BCL-XL and MCL-1. To interrogate combined FLT3-ITD and BCL-2 inhibition, cells were treated for 48hrs with venetoclax, quizartinib or the combination. Combination treatment led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to either single agent. FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. To determine the dependence of FLT3-ITD+ cells on BCL-XL and MCL-1, cells were treated with selective BCL-XL (A1331852) or MCL-1 (A1210477) inhibitors in combination with venetoclax. Treatment with either of the selective inhibitors in combination with venetoclax reduced the growth of cells compared to single agents alone, suggesting that all three pro-survival proteins may contribute to the survival of FLT3-ITD+ cells. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, co-treatment of quizartinib with either BCL-XL or MCL-1 inhibitor had no combination effect in FLT3-ITD+ cells. To elucidate the signaling pathway(s) regulated by FLT3-ITD that confer resistance to venetoclax, cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth when compared to each agent alone, indicating that multiple signaling pathways can contribute to venetoclax resistance rather than a single pathway dependence for driving the pro-survival effects of FLT3-ITD. This data underscores the reliance of these models on FLT3-ITD for survival and regulation of multiple survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD itself could be a predictive biomarker of venetoclax resistance.Further, the combination of quizartinib and venetoclax was assessed in vivo utilizing MV4;11 and Molm13 xenograft models grown orthotopically. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg) or the combination for 21 continuous days. Quizartinib treatment significantly increased survival compared to vehicle in both models [56 days vs. 26 days (p<0.0001) and 32 days vs. 14 days (p<0.0001) for MV4;11 and Molm13, respectively]. However, venetoclax alone only marginally improved survival [34 days vs. 26 days (p<0.0001)] in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib alone [91 days vs. 56 days (p<0.0001) and 40 days vs. 32 days (p<0.0001) in MV4;11 and Molm13, respectively]. Monotherapy and combination treatments were tolerated based on minimal changes in animal body weight throughout the duration of the study.Taken together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. [Display omitted] DisclosuresMali:Genentech: Employment. Lasater:Genentech: Employment. Doyle:Abbvie: Employment. Malla:Genentech: Employment. Boghaert:Abbvie: Employment. Souers:Abbvie: Employment.