THRX-165724 and SU14813 are potent inhibitors of Flt3 autophosphorylation and induce apoptosis in AML cells with Flt3 ITD. (A) Chemical structure of THRX-165724 and SU14813. (B) Inhibition of Flt3 autophosphorylation with THRX-165724 and SU14813. MV4-11 cells were incubated with increasing concentrations of THRX-165724 and SU14813 for 30 minutes. THP-1 cells were incubated with increasing concentrations of THRX-165724 and Flt3 wild-type autophosphorylation was stimulated with FL (50 ng/mL for 5 minutes). Flt3 phosphorylation status was determined by Flt3 immunoprecipitation followed by SDS-PAGE and Western blot with an anti-phosphotyrosine antibody. (C) MTT assay. MV4-11, MOLM13, THP-1, and U937 cells were incubated with increasing concentrations of THRX-165724 and SU14813 for 72 hours. Cell viability and proliferation was assessed using the MTT assay. (D) Induction of apoptosis. MV4-11, MOLM13, THP-1, and U937 cells were incubated for 72 hours with THRX-165724 (300 nM) or SU14813 (100 nM). Apoptosis was assessed by staining with Annexin V-PE and 7-AAD. The percentage of cells in the upper right quadrant denote cells that stain positive for Annexin V and 7-AAD. The cells in the lower right quadrant stain positive for Annexin V only.