KW-2249 and its metabolite inhibit FLT3. (A) Molm14 cells in culture medium were incubated for 1 hour in increasing concentrations of KW-2449 (top blots) and M1 (bottom blots). The cells were lysed, and FLT3 was immunoprecipitated from the extracts and resolved by SDS-PAGE. After probing the blots with antiphosphotyrosine (4G10), the blots were stripped and reprobed with anti-FLT3 to confirm equal lane loading. The blots were analyzed by densitometry, and the IC₅₀ values were calculated using linear regression analysis. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Extracts from the experiment in panel A were resolved directly with SDS-PAGE and the blots were probed with anti–phospho-STAT5, then stripped and reprobed with anti-STAT5 to confirm equal lane loading. Densitometry was performed as in panel A. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Molm14 cells were incubated in culture medium with increasing concentrations of KW-2449 (solid line) or M1 (dashed line) for 48 hours. The MTT assay was then performed, with results plotted as percent untreated control. (D) Primary AML cells isolated from the peripheral blood of patients on the clinical trial prior to beginning treatment with KW-2449 were incubated for 48 hours with increasing concentrations of KW-2449. After 48 hours, the MTT assay was performed and the results plotted as percent untreated control. Four of the patients harbored FLT3/ITD mutations (solid lines), and 4 did not (dashed lines).