Cyclin dependent kinase inhibitors have significant therapeutic potential. We find that the endogenous cdk inhibitor p57 Kip2 is significantly upregulated by three small molecule cdk inhibitors, BMS-387032/SNS-032 (hereafter referred to a SNS-032), flavopiridol or roscovitine. Several lines of evidence demonstrate that p57 Kip2 upregulation by these compounds is transcriptional and is mediated by E2F1. Infection with an adenovirus expressing E2F1, but not a control virus, leads to induction of the p57 Kip2 mRNA and protein. Likewise, an estrogen receptor- E2F1 fusion protein activates endogenous p57 Kip2 transcript in the presence of cycloheximide upon treatment with hydroxytamoxifen demonstrating that activation of p57 by E2F1 is direct. Luciferase constructs driven by the p57 Kip2 promoter verify that upregulation of p57 mRNA by SNS-032 is transcriptional. RNAi experiments show that upregulation of endogenous p57 or a p57 promoter-driven luciferase reporter by SNS-032 is dependent on E2F1 expression. Activation of p57 by E2F1 appears to represent a feedback loop since transfection of a p57 expression vector into cells significantly represses E2F1-driven transcription and significantly decreases the fraction of cells in S phase. Surprisingly, we find that p57-mediated inhibition of E2F1 is retained in cells lacking Rb function due to the expression of the E7 oncoprotein and appears mediated by an interaction between E2F1 and p57. The results presented demonstrate that small molecule cdk inhibitors regulate p57 Kip2 via E2F1 and that p57 Kip2 provides an unanticipated negative feedback to restrain E2F1 activity through a physical interaction.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA