Fig 2: Inhibitory activity of quizartinib and other FLT3 inhibitors on the FLT3 signaling pathway and the viability of AML cells.(A) MV4-11 cells were treated with quizartinib or AC886 at the indicated concentrations for 2 hours. Western blot analysis was performed on cell lysates using antibodies against the indicated proteins. (B–C) MOLM-14 cells were incubated with FLT3 inhibitors at 20 nM or with DMSO control for (B) 1 or (C) 2 hours. Cells were pelleted, washed, and resuspended in medium and incubated for (B) up to 3 hours or (C) up to 24 hours. (B) Western blot analysis was performed on FLT3 immunoprecipitants (FLT3) and whole-cell lysates (ERK). (C) Western blot analysis was performed on cell lysates using antibodies against the indicated proteins. (D) AML cells were incubated with each FLT3 inhibitor for 3 days. Cell viability was assessed using the CellTiter-Glo 2.0 Assay (Promega Corporation, Madison, Wisconsin, USA) and plotted at each concentration. Each data point and bar represent the mean and standard deviation of each concentration group, respectively (n = 3).

Fig 3: BPR1J-340 induces apoptosis in MOLM-13 and MV4;11 cells.Western blotting revealed that BPR1J-340 was able to induce apoptosis in FLT3-ITD-driven FLT3-ITD+ AML cells. MOLM-13 (A) and MV4;11 (B) cells were treated with BPR1J-340 at the indicated concentrations for 24 hours, and the cell lysates were then subjected to Western blot analysis using antibodies against caspase-3 and PARP (poly-ADP-ribose polymerase). (full length caspase-3 (FL-caspase-3), cleavage of caspase-3 (CL-caspase-3), full length poly(ADP-ribose) polymerase (FL-PARP), cleavage of PARP (CL-PARP)).

Fig 4: The effect of BPR1J-340 and SAHA versus drug combination on AML FLT3-ITD+ cells.MOLM-13 or MV4;11 cells were seeded at an initial concentration of 1×105 cell mL-1 for 24 hours and then treated with BPR-1J340 either alone or in combination with SAHA in the indicated concentrations. (A) Viable cells were counted after staining with trypan blue dye at indicated time point (B) At 48-hours after treatment, cells were stained with FITC-labeled annexin V and propidium iodide and percentages of apoptotic cells were analyzed by flow cytometry. (C) The representative flow cytometry histograms of assays of annexin positive apoptotic cells at 48 hours drug treatment (D) MOLM-13 cells were treated with drugs for 20 hours, and then followed by Western blot analysis to assess changes in protein levels with the indicated antibodies. Statistical analysis was performed using Student's t-test. *Indicates significant difference compared to vehicle control (* p<0.05, * * * p<0.01). Data shown are representative of multiple independent experiments.

Fig 5: BPR1J-340 inhibits FLT3-dependent signaling.(A) MV4;11 cells were treated with BPR1J-340 at the indicated concentrations for 1 hour. The phosphorylation status of FLT3 and STAT5 were evaluated by Western blot analysis. (B) HEK 293T cells were transfected with FLT3-WT-, FLT3-ITD-, or FLT3-D835Y-expressing plasmids for 24 hours and then incubated with various concentrations of BPR1J-340 for 1 hour. The FLT3 phosphorylation status in the transfected cells was evaluated by Western blot analysis.