Glutamine-dependent downregulation of FLT3-ITD is a mechanism of FLT3 inhibitor resistance in FLT3-ITD AML in hypoxia

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O2) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor ICs were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.