Anti-leukemic activity of gilteritinib. (A) IC₅₀ values for different FLT3 receptor subtypes. Cells expressing the indicated subtype were incubated with ASP2215 for 1 hour at increasing concentrations in RPMI/10% fetal bovine serum (FBS), analyzed by immunoprecipitation/immunoblotting for phospho- and total FLT3, and followed by densitometry. SEMK2 cells express wild-type FLT3, Molm14 cells express a 21-bp ITD mutation, and TF/ITD and Ba/F3 ITD cells express a transfected FLT3 construct containing an 18-bp ITD (see supplemental Methods). Ba/F3 cell lines with the indicated mutant FLT3 isoforms were generated by transfecting the murine lymphocyte Ba/F3 line with constructs expressing the FLT3 receptor (non-ITD–containing, except for the F691L variant) containing the indicated single point mutation. Each IC₅₀ value was calculated from multiple experiments. (B) Gilteritinib inhibits FLT3 phosphorylation in vivo. Plasma samples collected at trough time points from 3 patients enrolled in a gilteritinib study were incubated with Molm14 cells for 1 hour. Immunoprecipitation and immunoblotting were used to detect phospho-FLT3 and total FLT3 at these time points. (C) FLT3 TKI sensitivity ex vivo in primary patient samples is dependent on FLT3 mutation type. (AML1) A comparison of the cytotoxic effects of gilteritinib against 2 samples from patientAML1: 1 collected at diagnosis and the other at relapse. (AML2, AML3) Patients with relapsed AML and a high FLT/ITD allelic burden, respectively. (AML4) A patient with both FLT/ITD and TKD (D835Y) mutations, which confer resistance to type II inhibitors sorafenib and quizartinib when compared with counterparts lacking a FLT3/TKD mutation. (AML5) A patient with a high FLT3-ITD allelic burden who developed a FLT3/TKD (D835I) mutation after being treated with quizartinib. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) data in were performed with the patient’s blasts after progression on quizartinib and indicate that gilteritinib was the most potent of all 5 FLT3 TKIs against this patient’s blasts. The immunoblot shows the level of phospho-FLT3 expression in blasts after a 1-hour treatment with 20 nM of each FLT3 TKI in RPMI/10% FBS. The patient then enrolled on a crenolanib study and was refractory and was noted to have a new F691L gatekeeper mutation. (AML5 refractory) The MTT assay was repeated with the patient’s blasts after treatment with crenolanib and shows the resistance conferred by the F691L mutation. Supplemental Table 1 provides the mutation profiles of these patients.