Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms

Ami B Patel, Anthony D Pomicter, Dongqing Yan, Anna M Eiring, Orlando Antelope, Jonathan A Schumacher, Todd W Kelley, Srinivas K Tantravahi, Tibor J Kovacsovics, Paul J Shami, Thomas O'Hare, Michael W Deininger, Ami B Patel, Anthony D Pomicter, Dongqing Yan, Anna M Eiring, Orlando Antelope, Jonathan A Schumacher, Todd W Kelley, Srinivas K Tantravahi, Tibor J Kovacsovics, Paul J Shami, Thomas O'Hare, Michael W Deininger

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.

Conflict of interest statement

Competing Interests

Michael W. Deininger 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.

Figures

Figure 1.. Culture in HS-5 CM protects…
Figure 1.. Culture in HS-5 CM protects FLT3-ITD+ AML cells from quizartinib.
A. Apoptosis at 48 hours following treatment of various FLT3-ITD+ AML cell lines and the K562 cell line (negative control) with quizartinib 10 nM. Treated cells were cultured in medium conditions indicated by the blue, red and black bars. (n=3) B. Bar graphs comparing the IC50 values of quizartinib in FLT3-ITD+ AML cell lines cultured in RM or CM for 72 hours. (n=3) C. Primary CD34+ cells from AML patients with or without FLT3-ITD mutations were cultured in RM or CM and treated with graded concentrations of quizartinib in cell proliferation experiments (n=3)
Figure 2.. CM activates STAT3 and STAT5…
Figure 2.. CM activates STAT3 and STAT5 in AML cells and knockdown of STAT5 is associated with impaired cell growth in FLT3-ITD+ AML.
A. Immunoblots demonstrate STAT3 and STAT5 activation in AML cell lines cultured in RM and CM. B and C. Patterns of STAT3 and STAT5 activation in CD34+ cells from patients with FLT3-ITD+ and FLT3-ITD− AML. D. MOLM-13 cells containing an empty vector or shRNA constructs targeting STAT3, STAT5 or both STAT3 and STAT5 were cultured in RM or CM and treated with graded concentrations of quizartinib. Cell proliferation was measured at 72 hours. (n=3)
Figure 3.. The combination of dasatinib and…
Figure 3.. The combination of dasatinib and quizartinib decreases STAT5 activation in CM and overcomes stroma-based resistance to FLT3 TKI.
A. MOLM-13 cells cultured in CM were treated with quizartinib 10 nM alone or in combination with fixed doses (0.1 μM) of various inhibitors. Following 4 hours of culture, cells were harvested and STAT5 phosphorylation was assessed by flow cytometry. (n=3) B. Cell proliferation assay in MOLM-13 cells cultured in CM and treated with graded doses of quizartinib alone or in combination with fixed doses (0.1 μM) of inhibitors for 72 hours. (n=3) C and D. STAT5, Lyn and Src activation in MOLM-13 cells following culture in RM or CM and treatment with quizartinib ± dasatinib for 4 hours.
Figure 4.. The combination of dasatinib and…
Figure 4.. The combination of dasatinib and quizartinib suppresses stroma-enhanced glycolysis in a STAT5-independent manner in FLT3-ITD+ AML.
A. Glycolysis and glycolytic capacity were measured in MOLM-13 cells cultured in RM or CM and treated with quizartinib ± dasatinib for 24 hours. (n=3) B. Glycolysis and glycolytic capacity were measured in MOLM-13 cells expressing a doxycycline-inducible shRNA against STAT5. Cells were cultured in CM ± doxycycline (DOX) and treated with quizartinib and/or dasatinib for 24 hours. (n=3)

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