CDK6 but not CDK4 binds the promoter of the FLT3 gene and regulates FLT3 transcription in a kinase-dependent manner. (A-B) Inhibition of FLT3 protein expression with CDK4/6 inhibitor palbociclib at indicated concentrations in a time-dependent manner is depicted. Cells were harvested (A) between 24 and 120 hours or (B) at 48 hours. Cell lysates were subjected to western blot analysis for total FLT3. β-actin was used as loading control. (C) Cells were incubated with increasing concentrations of palbociclib. A time- and dose-dependent decrease in FLT3 phosphorylation at residue Y591 and in STAT5 phosphorylation at residue Y694 was detected by immunoblotting. (D) Palbociclib inhibits FLT3-dependent signaling in a dose-dependent manner. MOLM-14 cells were incubated with palbociclib at indicated concentrations for 4 days. Total cell lysates were immunoblotted with the indicated antibodies: total FLT3, total STAT5, phospho-STAT5, and total MYC. (E) PIM1 gene expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) in FLT3-mutant (MOLM-14, MV4-11, and PL-21) and FLT3-WT (THP-1 and NOMO-1) cell lines after palbociclib (1 µM) administration for 72 hours. Relative PIM1 expression was normalized to the housekeeping gene RPLP0. (F) Effects of individual CDK4 and CDK6 suppression on FLT3 protein levels. (G) FLT3 gene expression was analyzed by quantitative RT-PCR in indicated cell lines after palbociclib (1 µM) administration for 72 hours. Relative FLT3 expression levels were normalized to RPLP0 mRNA. (H-I) Chromatin immunoprecipitation (ChIP) experiments were performed in (H) a murine HPC7 hematopoietic progenitor cell line and in (I) indicated human AML cells. Protein-DNA complexes were immunoprecipitated by using (H) home-made sera against Cdk6 or (I) by using a commercial anti-CDK6 antibody and were analyzed by quantitative PCR (qPCR) for their presence on the FLT3 promoter region. EGR1, p16INK4a, and VEGF-A promoter regions served as positive controls. Bar graphs depict fold enrichment over a negative region as described in the supplemental Data. *P < .05; **P < .01; ***P < .001; ****P < .0001. shRNA, short hairpin RNA.
