The type I FLT3 inhibitor XL999 overrides secondary FLT3-ITD mutations that arise under gilteritinib and quizartinib treatment in acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (FLT3-ITD) is a well-characterized genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although FLT3 inhibitors, such as gilteritinib and quizartinib, initially show clinical efficacy, resistance frequently emerges because of secondary mutations. Through a kinase inhibitor library screen, we identified XL999, which exhibits potent antileukemic activity across diverse FLT3-mutant AML cell lines and primary patient samples. Mechanistically, biochemical and molecular docking analyses revealed that XL999 directly binds to FLT3-ITD, potentially independent of the common F691 resistance site, thereby suppressing downstream signaling pathways to induce cell cycle arrest and apoptosis. In preclinical mouse models harboring refractory FLT3-ITD-D835Y or ITD-F691L mutations, oral administration of XL999 effectively reduced the leukemic burden across the peripheral blood, spleen, and bone marrow, significantly prolonging survival compared with gilteritinib and quizartinib, with no overt systemic toxicity observed in short-term mouse tolerability studies. Furthermore, XL999 demonstrated robust efficacy in patient-derived xenograft models, complemented by a favorable oral bioavailability profile. Together, these findings suggest that XL999 may represent an orally bioavailable FLT3 inhibitor with the potential to overcome clinically relevant secondary resistance mutations in FLT3-ITD-positive AML, supporting its further investigation as a therapeutic candidate.