Inhibition of the MAP2K7-JNK pathway in T-ALL. (A) The inhibition of MELK and the MAP2K7-JNK pathway in T-ALL cell lines by OTSSP167 (50 nM, 48 hours) was analyzed by immunoblots. (B) Kinase activity was measured by incubating purified MAP2K7, dead-JNK2 (substrate), ATP (100 mM), and vehicle or OTSSP167 and measuring ADP consumption using ADP-Glo kit (n=3). (C) KOPT-K1 cells were treated with 400 mM sorbitol for 4 hours, and OTSSP167 was added for the last 3 hours. Inhibition of MAP2K7 was evaluated by detecting a reduction in phosphorylated JNK and ATF2 proteins. (D) OTSSP167 inhibited constitutive MAP2K7-JNK2 activation in JURKAT and P12-Ichikawa cells at 100 and 50 nM, respectively. (E) Cell viability of KOPT-K1 cells treated with OTSSP167 or MELK-8a. (F) Heatmap of the cell signaling pathways inhibited by OTSSP167 in T-ALL cell lines by RPPA. (G) Immunoblot analysis of inhibition of mTOR and NOTCH1 pathways by OTSSP167 in T-ALL cell lines.