Plasma inhibitory assay shows ponatinib is active against FLT3-ITD and FLT3-ITD/F691 mutants at clinically achievable plasma concentrations. (A) Western blot analysis for phosphotyrosine and total FLT3 performed after immunoprecipitation using anti-FLT3 antibody on lysates prepared from Molm14 cells expressing the FLT3-ITD mutant isoforms indicated. Cells were exposed for 120 minutes to healthy control and steady-state plasma obtained from a patient treated with 45 mg of ponatinib per day. (B) Quantitation of FLT3 autophosphorylation after immunoprecipitation from Molm14 cells expressing the indicated FLT3 mutant isoforms. (C) Western blot analysis using anti–phospho-S6, anti–phospho-STAT5, anti-S6, and anti-STAT5 antibody performed on whole-cell lysates prepared from Molm14 cells expressing the FLT3-ITD mutant isoforms indicated. Cells were exposed for 120 minutes to normal control and steady-state plasma obtained from a patient treated with 45 mg ponatinib per day.