Targeting RSK2 by fmk attenuates cell viability and induces apoptosis in FLT3-ITD– but not BCR-ABL–expressing Ba/F3 cells and human leukemia cells. (A) Left: Immunoblotting results show stable expression of BCR-ABL and FLT3-ITD in Ba/F3 cells. Right: A total of 3μM Fmk treatment inhibits RSK2 in Ba/F3-expressing BCR-ABL or FLT3-ITD. (B) A total of 3μM Fmk treatment inhibits RSK2 in BCR-ABL–positive K562 (top) and FLT3-ITD positive Molm14 and Mv(4;11) human leukemia cells (bottom). (C) Treatment with increasing concentrations of fmk resulted in decreased cell viability (left) and increased apoptosis (right) in FLT3-ITD–positive Ba/F3 cells in the absence of IL3 (-IL3), but not in cells expressing BCR-ABL (-IL3), control Ba/F3 cells cultured in the presence of IL3 (+IL3), or FLT3-ITD–positive cells in the presence of IL3 (+IL3). (D-F) Targeting RSK2 by fmk does not affect cell viability nor induce apoptosis in K562 cells expressing BCR-ABL (D), but results in decreased cell viability and increased apoptosis in FLT3-ITD–expressing Molm14 (E) and Mv(4;11) cells (F). (G-H) RNAi-mediated targeted down-regulation of RSK2 (G) significantly induced apoptosis (H; left) and attenuated cell viability (H; right) in FLT3-ITD–expressing Molm14 and Mv(4;11) human leukemia cell lines, but not in K562 cells expressing BCR-ABL. Cells were transiently infected with lentivirus harboring an empty pLKO.1 vector or shRNA specific to RSK2 for 24 hours. The apoptotic population was characterized as the fraction of annexin V–positive cells of total treated cells. The relative cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide–based assay. P value was determined by 2-tailed Student t test. ns indicates not significant.* P < .05; **P < .01.