Abstract
FLT3-ITD mutations occur in 20–30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.
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Acknowledgements
This work was supported by the National Cancer Institute (NCI) at the National Institutes of health (NIH) through grant R21CA20593601 (MWD), the V Foundation for Cancer Research Translational Research Grant T2017–008 (MWD), NIH grant R01CA178397 (MWD and TOH), and the American Society of Hematology Research Training Award for Fellows (ABP). DY is supported by the International Award from the Lady Tata Memorial Trust. AME is supported by NIH grant 1K22CA216008. We thank the Metabolic Phenotyping Core Facility at the University of Utah for assistance with Seahorse experiments. This work was funded in part by the University of Utah Flow Cytometry Core Facility and the NCI through award 5P30CA042014–24 awarded to the Huntsman Cancer Institute and the National Center for Research Resources of the NIH award 1S10RR026802–01.
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ABP conceptualized the project, designed and performed experiments, analyzed data, interpreted results, and wrote the paper. ADP assisted ABP with phospho-flow cytometry studies and genotyping in cell lines; DY and AME provided technical support to ABP with Seahorse assays and virus production; OA assisted ABP with figure design; TWK and JAS determined FLT3-ITD variant allele frequency in patient samples. SKT, TJK, and PJS provided patient samples. TOH and MWD conceptualized the project, supervised ABP with experimental design, result interpretation, and paper writing. All authors were involved in project discussion and data interpretation.
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MWD reports research funding from and is a paid advisory board member and/or consultant for the following companies: Pfizer Inc, TRM Blueprint, Fusion Pharma, Takeda, Ascentage Pharma, Humana, Adelphi, Medscape, Novartis, Incyte and Sangamo. Inc.
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Patel, A.B., Pomicter, A.D., Yan, D. et al. Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms. Leukemia 34, 2981–2991 (2020). https://doi.org/10.1038/s41375-020-0858-1
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DOI: https://doi.org/10.1038/s41375-020-0858-1
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