Mutation screen of FLT3-ITD and FLT3-ITD/D835 reveals kinase domain mutations that cause resistance to gilteritinib. Numbers of independently derived gilteritinib-resistant Ba/F3/FLT3-ITD subpopulations with amino acid substitution at the indicated residue obtained from a saturation mutagenesis assay for FLT3-ITD (A) and FLT3-ITD/D835V at the indicated gilteritinib concentration (B). (C) Relative resistance of Ba/F3 cells expressing indicated FLT3 TKD mutations in FLT3-ITD (red) and FLT3-ITD/D835V (blue) backgrounds. Resistance is expressed as fold-change compared with FLT3-ITD (ratio of mutant cell line IC₅₀ over IC₅₀ of FLT3-ITD cells). Error bars represent SD of 3 or more independent experiments. *Mutation was only assessed in FLT3-ITD background. Dotted line indicates fold-change in IC₅₀ for FLT3-ITD/F691L cell line. (D) Western blot analysis using anti–phospho-FLT3 and anti-FLT3 antibody performed on lysates from interleukin-3-independent Ba/F3 populations expressing the FLT3-ITD–mutated isoforms indicated. Cells were exposed to gilteritinib at the indicated concentrations for 90 minutes.