Preclinical studies of gilteritinib, a next-generation FLT3 inhibitor

Lauren Y. Lee, Daniela Hernandez, Trivikram Rajkhowa, Samuel C. Smith, Jayant Ranganathan Raman, Bao Nguyen, Donald Small and Mark Levis

Data supplements

Article Figures & Data


  • Figure 1.

    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.

  • Figure 2.

    Inhibition of c-Kit and effects on erythropoiesis by quizartinib and gilteritinib. (A) TF-1 cells were treated with increasing concentrations of either quizartinib or gilteritinib in RPMI/10% FBS for 1 hour. Immunoprecipitation and immunoblotting were performed to detect the phosphorylation status of c-Kit and total c-Kit. (B) Mononuclear cells isolated from normal donor bone marrow were plated at 105 cells/mL in MethoCult. Increasing concentrations of quizartinib or gilteritinib were added. Counts for colony-forming unit-granulocyte, monocyte (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies were done after 10 to 12 days of incubation (n = 3). P-c-Kit, phosphorylated c-Kit.