Mutation screen of FLT3-ITD identifies AL KD mutations that confer resistance to ponatinib. (A) Numbers of independent ponatinib-resistant Ba/F3/FLT3-ITD subpopulations with amino acid substitution at the indicated residue obtained from a saturation mutagenesis assay (N = 50 clones). (B) Relative proliferation of Ba/F3 populations stably expressing ponatinib-resistant FLT3-ITD mutant isoforms after 48 hours in various concentrations of ponatinib (error bars represent SD of triplicates from the same experiment). Data shown are representative of multiple experiments. (C) Western blot analysis using anti–phospho-FLT3, anti–phospho-STAT5, anti-FLT3, and anti-STAT5 antibody performed on lysates from IL-3–independent Ba/F3 populations expressing the FLT3-ITD mutant isoforms indicated. Cells were exposed to ponatinib at the indicated concentrations for 90 minutes.