TTT-3002 is a kinase inhibitor of FLT3 signaling. (A) Proliferation of leukemia cell lines at 48 hours following treatment with TTT-3002, measured by MTT assay. Error bars represent average ± SD. Data are representative of 3 independent experiments. (B) STAT5 signaling is rescued by interleukin-3 (IL-3) stimulation. Expression of pFLT3 and pSTAT5 by western blotting of Ba/F3-FLT3/ITD cell lysates following treatment with TTT-3002 ± IL-3 (10 ng/mL). (C) Ba/F3-FLT3/ITD cell proliferation is rescued by IL-3 stimulation. Ba/F3-FLT3/ITD cells were plated in quadruplicate with TTT-3002 (0 to 10 nM) with and without IL-3 (10 ng/mL), and proliferation was measured at 48 hours by MTT assay. Error bars represent average of 3 independent experiments ± SD. (D) FLT3 phosphorylation is inhibited by TTT-3002 in FLT3/ITD, FLT3/PM, and FLT3/WT cell lines. Expression of FLT3 and pFLT3 was measured by western blotting following 1 hour of treatment with TTT-3002. Data from 3 independent experiments are represented as a fraction of pFLT3/FLT3 relative to DMSO control ± SD. IC₅₀ is indicated on the graph. (E) FLT3/ITD (Ba/F3-FLT3/ITD), FLT3/PM (Ba/F3-FLT3/D835Y), and overexpressed FLT3/WT (SEM-K2) cells were treated with TTT-3002 for 1 hour, and expression of pFLT3, FLT3, pSTAT5, STAT5, pAKT, AKT, pMAPK, and MAPK were measured by western blotting. Data are representative of 3 independent experiments.