Abstract B052: FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models

Abstract

20-25% of acute myeloid leukemia (AML) patients harbor FLT3 internal tandem duplication (ITD) mutations, which confer poorer 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 prosurvival proteins BCL-XL and MCL-1. Venetoclax, a potent, selective inhibitor of the prosurvival protein BCL-2, 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 and targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib, a potent FLT3 inhibitor, in FLT3-ITD+ AML models. In vitro, quizartinib reduced expression of BCL-XL and MCL-1, but not BCL-2, in FLT3-ITD+ cells (Molm13 and MV4;11). However, quizartinib had no effect on BCL-XL or MCL-1 expression in FLT3 WT cells (HL60 and OCI-AML3). Combination treatment with venetoclax led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to single agents while FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, cotreatment of quizartinib with either BCL-XL or MCL-1 specific inhibitors had no combination effect in FLT3-ITD+ cells. Additionally, quizartinib and venetoclax were assessed in vivo utilizing MV4;11 and Molm13 orthotopic xenograft models. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg), or the combination of both drugs for 21 continuous days. Quizartinib treatment significantly increased survival compared to the vehicle group in both xenograft models (56 days vs. 26 days and 32 days vs. 14 days for MV4;11 and Molm13 xenografts, respectively). However, venetoclax only marginally improved survival (34 days vs. 26 days) 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 (91 days vs. 56 days and 40 days vs. 32 days in MV4;11 and Molm13 xenografts, respectively). These data underscore the dependency of these models on FLT3-ITD for survival and its regulation of survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD could be a predictive biomarker of venetoclax resistance. To determine mechanisms of venetoclax resistance mediated by FLT3-ITD, mutant 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 compared to single agents. This observation suggests that multiple pathways may contribute to venetoclax resistance rather than a single pathway dependency to confer the prosurvival effects of FLT3-ITD. Additional mechanistic studies are under way to define the role of each pathway in regulating survival. 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. Citation Format: Raghuveer Mali, Elisabeth A. Lasater, Kelly Doyle, Ritu Malla, Erwin Boghaert, Andrew Souers, Joel Leverson, Deepak Sampath. FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B052.

Synergistic effect of BCL2 and FLT3 co-inhibition in acute myeloid leukemia

“…Furthermore, combination of quizartinib with venetoclax was superior to combination with navitoclax plus AMG 176, suggesting that quizartinib elicits anti-tumor activity outside of its reduction of MCL1 levels. In in vitro studies, they also did not see demonstrable synergy using A1331852 (BCL-X L specific) with quizartinib [14]. Unlike our observations with gilteritinib, no synergism was observed in FLT3-WT using quizartinib, highlighting that there are likely differences in how these two drugs elicit their effects.…”

“…Therefore, when venetoclax is added to a FLT3 inhibitor, they suggest there is synergy because both MCL1 and BCL2 expressions are reduced, increasing the dampening of the pro-survival activity of the AML blasts. In their work with the combination of quizartinib and venetoclax, Mali et al explored a similar mechanism in vivo in an MV4-11 xenograft model using inhibitors with different selectivities for each of the BCL2 family members in combination: venetoclax (BCL2), navitoclax (BCL2 and BCL-X L ), and AMG 176 (MCL1) [14]. Their results indicated that the improvement in survival was highest when quizartinib was combined with a BCL2, and not MCL1, inhibitor.…”

“…This work stemmed from the observation that FLT3 inhibitors result in downmodulation of the expression of myeloid cell leukemia 1 (MCL1), a known mechanism of resistance to BCL2 inhibitors, and could improve activity of BCL2 inhibitors. Mali et al also conducted a series of studies using the second-generation FLT3 inhibitor quizartinib [which additionally inhibits KIT and platelet-derived growth factor receptors (PDGFR)] [ 13 ] in combination with venetoclax [ 14 ]. Unlike gilteritinib, quizartinib is a type II inhibitor, interacting only with the inactive conformation and therefore is not effective against a FLT3-tyrosine kinase domain (TKD) mutation; quizartinib was denied FDA approval in June 2019.…”

Targeting sphingolipid metabolism as an approach for combination therapies in haematological malignancies

“…Furthermore, the findings that clinically used small molecule inhibitors of FLT3 39 , BCR-ABL 17 and Bcl-2 67 increase ceramide levels warrant further investigation. In particular, the combining of two separate drugs such as the FLT3 inhibitor, Quizartinib and the Bcl-2 inhibitor Venetoclax which have demonstrated synergistic cell death in AML, could be partially attributed to the increases in ceramide levels that are produced by each drug 39 , 67 , 97 . Thus this could provide an opportunity for targeted combinational therapies under the premise of synergistic increases in ceramide levels.…”

B-cell lymphoma-2 inhibition and resistance in acute myeloid leukemia

“…Sequencing studies were performed to assess the combination of venetoclax and the small molecule FLT3 inhibitor quizartinib in specific FLT3 ITD mutated xenograft models. The combination induced durable tumor regression for up to 3 mo after cessation of treatment[ 83 ]. However, Chyla et al[ 84 ] noted that FLT3-ITD or PTPN11 mutations may confer intrinsic and acquired resistance to venetoclax[ 84 ].…”