Momelotinib potently inhibits FLT3^ITD and its resistant variants from the activation loop. (A) Sigmoidal curve showing the viability of BaF3 cells expressing FLT3-WT, Jak2V617F, and FLT3^ITD treated with dimethyl sulfoxide or increasing concentrations of momelotinib for 72 hours. IC₅₀ values for each cell line is indicated in parentheses. (B) Sigmoidal curve showing the potent inhibition of human FLT3^ITD-mutant AML cells (MV4-11 and MOLM13), whereas proliferation of K562 cells was not significantly affected. IC₅₀ values for each cell line is indicated in parentheses. pFLT3 and pSTAT5 levels determined by western blotting using total cell extracts of MV4-11 cells (C) and MOLM13 cells (D) treated with increasing concentrations of momelotinib for 2 hours. Dose-response sigmoidal curve showing the proliferation of BaF3 cells expressing FLT3^ITD and its quizartinib-resistant variants at different concentrations of momelotinib (E), quizartinib (F), and gilteritinib (G) (left panels). Fold differences in the IC₅₀ values for each FLT3^ITD variant normalized to FLT3^ITD are presented as a bar graph with a logarithmic scale (right panels). Note that momelotinib efficiently inhibited the activation loop quizartinib-resistant mutants, as well as compound mutant FLT3^{ITD/F691L/Y842H}, which is fully resistant to gilteritinib (brown bar). (H) Immunoblot analysis showing inhibition of the kinase activity of FLT3^ITD and resistant variants treated with different concentrations of momelotinib. Total cell extracts from the cells treated with momelotinib for 2 hours were probed with anti-pFLT3, anti-pSTAT5, anti-FLT3, and anti-STAT5 antibodies. Representative cell proliferation data (± standard deviation) are shown from 2 independent experiments. Error bars represent standard error of the mean. *P < .05, **P < .01, ***P < .001. ns, not significant.