Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERa)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERa66 and upregulation of the 36-kDa variant of ER (ERa36). We identified that NF-kB, HIF1a, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-kB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-kB activation, combining didox with tamoxifen treatment cooperatively reverses ER-a alterations and inhibits NF-kB activation. Finally, inhibition of RRM2 by didox reversed tamoxifenresistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.

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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.

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