Inhibition of PIM kinase activity suppresses growth of AML cells with high CD25 expression by down-regulating STAT5 activity. (A) Differential response of patient leukemia blasts subpopulations to the inhibition of PIM kinase activity. Primary leukemia blasts from 4 patients were sorted into high-CD25 expression or low-CD25 expression subpopulations and treated with the indicated concentrations of PIM inhibitor. IC₅₀ values were specified. (B) Verification of CD25 mRNA levels in responder cell lines. Quantitative reverse-transcription polymerase chain reaction assays measuring CD25 mRNA levels in the indicated cell lines were implemented. Relative CD25 expression was calculated by normalizing the CD25 mRNA expression value of each cell line to that of the PLB985 cell line. (C) Level of STAT5 tyrosine phosphorylation in a panel of responders and nonresponders. Responders of PIM kinase inhibition exhibited a high level of STAT5 tyrosine phosphorylation (Y694/699), with the exception of NOMO1. In some nonresponders, the expression of total STAT5 was not obvious. (D) Effect of STAT5 knockdown on the growth of KG1, EOL1, NB4, and SKM1 cell lines. Sustained knockdown of endogenous STAT5A/B was achieved by continuous exposure to Accell siRNAs (see supplemental Methods) over the course of specified experiments. Culture volume was diluted 1:1 every day to avoid overconfluency and ensure the optimal growth of cells. (E) Proliferative response to PIM kinase inhibition on expression of constitutive STAT5B in a nonresponder cell line. Introduction of STAT5B C715F drastically upregulated CD25 levels in a nonresponder ML2 cell line (i). (F) Regulation of CD25 expression in KG1 cells. Knockdown of STAT5 in KG1 cells abrogated CD25 mRNA (i) and protein expression (ii). (G) The effect of PIM kinase inhibition on CD25 mRNA expression. PIM inhibitor dose-dependently downregulates CD25 expression at the mRNA level (i) and the protein level (ii). (H) Modulation of STAT5 DNA binding capacity in the presence of a PIM inhibitor. Nuclear extracts were made from either dimethylsulfoxide or compound C-treated cells and incubated with biotin-labeled double-stranded oligonucleotides derived from a CD25 promoter region that contains STAT5 DNA binding sequences (Biotin-GAS_c+n). “Cold- GAS_c+n” denotes unlabeled oligonucleotide that serves as a control for binding specificity. (I) Interaction between PIM kinase family and STAT5 transcription factors. Immunoprecipitation of PIM kinases was carried out in radioimmunoprecipitation assay buffer, and the presence of STAT5 was detected by western blot. (J) Modulation of STAT5B pS731 by PIM kinase inhibition. Treatment with PIM inhibitor significantly downregulated S731 phosphorylation on STAT5B in a dose-dependent (i) and time-dependent (ii) manner. (K) Direct phosphorylation of STAT5B S731 by PIM kinases. Biochemical reaction was carried out in the presence of recombinant STAT5B and PIM kinases. Protein input is illustrated by Coomassie staining (lower).