Effects of KW-2449 on human leukemia cells with FLT3 mutation. (A) Chemical structure of KW-2449. (B) MOLM-13 cells, which express FLT3/ITD, were treated with KW-2449 at the indicated concentrations for 24 hours. For analysis of FLT3 and its phosphorylated form (P-FLT3), the blots of immunoprecipitated FLT3 SDS-PAGE samples were analyzed by antiphospho-tyrosine (P-Tyr) antibody and anti-FLT3 antibody. For detection of STAT5 and its phosphorylated form (P-STAT5), total cell lysate SDS-PAGE samples were analyzed by anti-P-STAT5 antibody and anti-STAT5 antibody as primary antibodies. Phosphorylation levels of FLT3 and STAT5 were decreased by KW-2449 in a dose-dependent manner. (C) MOLM-13 cells were treated with various concentrations of KW-2449 for 48 hours, and cell-cycle distribution was analyzed as described in “Growth inhibition profile and cell-cycle analysis.” (D) MOLM-13 cells were treated with various concentrations of KW-2449 for 48 hours, and apoptosis induction was analyzed by 7-amino-actinomycin D/annexin V staining. (E) Inhibitory effects of hAGP on growth inhibitory activity against MOLM-13 cells were compared between KW-2449 and potent FLT3 inhibitors, PKC-412 and CEP-701, as described in “Effects of hAGP on growth inhibitory activity by FLT3 inhibitors.” Growth inhibitory activity of KW-2449 was not affected by the presence of hAGP.