FLT3 inhibitors have become a cornerstone in the treatment of FLT3-mutated acute myeloid leukemia (AML), however, durable clinical responses are frequently limited by the emergence of acquired resistance. In this study, we established and comprehensively characterized a quizartinib-resistant FLT3-ITD AML model to elucidate the molecular and functional mechanisms underlying therapeutic failure. Prolonged exposure of MV4-11 cells to escalating concentrations of quizartinib resulted in the selection of quizartinib resistant clones (MV4-11QR), displaying an increase in IC 50 and a shift from cytotoxic to predominantly cytostatic responses. Resistant cells maintained MAPK signaling despite FLT3 inhibition. Global proteomic profiling revealed extensive reprogramming, with enrichment of pathways related to energy metabolism, RNA processing, and translational regulation, accompanied by enhanced mitochondrial respiration and glycolytic capacity. Whole-genome sequencing identified acquisition of the FLT3 D835H mutation and clonal expansion of TP53 R248W with loss of the wild-type TP53 allele, indicating strong treatment-driven clonal selection. Functionally, MV4-11QR cells showed broad cross-resistance to clinically relevant agents, including midostaurin, venetoclax, and cytarabine. Importantly, pharmacological targeting of mutant p53 with eprenetapopt or MAPK signaling with trametinib restored sensitivity to quizartinib, inducing synergistic or additive cytotoxic effects and increased apoptosis. Together, these findings define a multilayered resistance program involving genetic, signaling, and metabolic adaptations and support rational combination strategies to overcome FLT3 inhibitor resistance in AML. • Long-term quizartinib exposure selects resistant FLT3-ITD AML clones • MV4-11QR cells maintain MAPK signaling despite FLT3 inhibition • Resistance associates with FLT3D835H acquisition and TP53 clonal dominance • Proteomic rewiring enhances mitochondrial respiration and glycolysis • Targeting mutant p53 or MAPK restores quizartinib sensitivity
Miranda et al. (Wed,) studied this question.