Fig. 1

KX2-391 is active against ITD-TKD resistance-causing FLT3 mutations and blocks FLT3 signaling in FLT3-ITD and FLT3-ITD-TKD cells. a Quantification of cellular thermal shift assay was made using western blot Ba/F3 cells expressing FLT3-ITD cells were treated with KX2-391 (1 µM) for 1 h, and temperatures between 43 and 55℃ were defined to perform the test. KX2-391were treated based on 10 different concentrations for 1 h at 51℃. Data were normalized by setting the highest and lowest value in each set to 100% and 0%, respectively. Data were obtained in the presence of the KX2-391 (blue circle) as the positive control and DMSO (red square) as the negative control. b Normalized cell viability of Ba/F3 cells expressing FLT3-ITD TKD mutations after a 48 h exposure to various concentrations of KX2-391 and AC220 (c), measured using CellTiter Glo assays (error bars represent the SD of 3 or more independent experiments). d Viability of FLT3-ITD mutated cells (MV4-11, MOLM-13), and FLT3 nonmutated cells (NB4, SKNO1, TF1) treated with various concentrations of KX2-391 for 48 h, measured by CellTiter Glo assays. e Ba/F3 cells expressing FLT3-ITD TKD mutations were treated with different concentrations of KX2-391 for 24 h and then parental Ba/F + IL3, FLT3-ITD, FLT3-ITD-D835Y, and FLT3-ITD-F691L cells, as well as f MOLM13 and MV4-11 cells were examined by flow cytometry (Annexin V). g Ba/F3 cells expressing FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD/F691L, FLT3-D835V, and FLT3-D835F, as well as FLT3-ITD positive human leukemia cell lines h MOLM13 and MV4-11 were incubated for 12 h with the indicated concentrations of KX2-391 (based on the IC50 values) and subsequently examined by western blotting using antibodies against FLT3/P-FLT3, STAT5/P-STAT5, ERK/P-ERK, AKT/P-AKT and SRC/P-SRC. GAPDH was used as a loading control. All values represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001versus the control