Ba/F3 FLT3-ITD-TKD dual mutants show resistance to the FLT3 PTK inhibitor SU5614. (A) Dose-response curves of the inhibitory activity of SU5614 in Ba/F3 FLT3-ITD (▪) and FLT3-ITD-D835Y cells (○) after 72 hours of incubation. Ba/F3 cells expressing different FLT3 constructs were seeded at a density of 4 × 10⁴ cells/mL in the absence or presence of different concentrations of SU5614. Viable cells were counted after 72 hours by trypan blue exclusion. The growth of cells that were incubated without inhibitor was defined as 100%. Values represent means and standard deviations from 3 independent experiments. (B) Dose-response curves of the inhibitory activity of SU5614 in Ba/F3 FLT3-ITD (▪), FLT3-ITD/WT (○), and FLT3-ITD/D835N cells (▾) after 72 hours of incubation. Values represent means and standard deviations from 3 independent experiments. (C) Ba/F3 cells transduced with the FLT3-ITD (▪), FLT3-ITD-D835N (○), or FLT3-ITD-D835Y constructs (▾) were incubated with different concentrations of SU5614 for 24 hours and were analyzed by flow cytometry after staining with annexin V–PE and 7-AAD. Values represent means and standard deviations from 3 independent experiments. (D) MIG FLT3-ITD (GFP+; ○) and MIY FLT3-ITD-D835N (YFP⁺)–expressing Ba/F3 cells (▪) were mixed in a ratio of 10:1, and the percentage of GFP⁺ and YFP⁺ cells was measured every 3 to 4 days for a time period of 12 days by FACS analysis in the presence of 0.2 μM of SU5614.