PS927 PHOSPHOCHROMATOME ANALYSIS UNCOVERS UNEXPECTED ROLES OF CDK6 IN REGULATING TRANSCRIPTION

Abstract

Background:Cyclin‐dependent kinase 6 (CDK6) together with its close homologue CDK4 enables the progression of cells through the early G1 phase of the cell cycle. CDK4/6‐cyclin D complexes phosphorylate members of the retinoblastoma (Rb) protein family, allowing for the release of E2F transcription factors that induce genes required for cell cycle progression and DNA replication. Alterations of the CDK4/6‐Rb pathway are frequently observed in human malignancies. Recent studies focusing on acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) have uncovered a novel role for CDK6 in regulating transcription. Our group has identified CDK6 as part of chromatin‐bound complexes which drive transcription of disease–relevant genes including the cell cycle inhibitor p16INK4a, the angiogenesis‐promoting factor VEGF‐A (vascular endothelial factor A) and the proto‐oncogene FLT3 (Fms like tyrosine kinase 3).Aims:The aim of this study is to identify novel binding partners of CDK6 on chromatin and to investigate their target gene sets. A better understanding of CDK6 functionality in tumor formation and promotion may help in the design and selection of adequate treatment options.Methods:For further investigation of CDK6́s role in transcription, we transduced murine Cdk6 +/+ and Cdk6 −/− primary bone marrow with the oncogene Bcr/Abl p185. To systematically gain insights on how CDK6 takes influence on chromatin‐binding and activity of transcription factors we performed RNA‐seq paralleled by mass spectrometry‐based phosphoproteomics of chromatin‐bound proteins (Phospho‐Chromatome).Results:RNA‐seq analysis of Cdk6 +/+ and Cdk6 −/− BCR‐ABL transformed cells identified more than 1700 genes that are differentially expressed. Phospho‐Chromatome studies performed in parallel revealed significant changes in the phosphorylation status of the transcription factors ZBTB7A, EBF1 and YY1. In vitro kinase assays verified a direct phosphorylation of ZBTB7A and EBF1 by active CDK6/CyclinD3 complexes.Summary/Conclusion:We show that CDK6 is broadly associated with chromatin where it fulfils different tasks: beside its well‐known role in the E2F‐release it enhances the phosphorylation of various transcription factors and chromatin‐regulating enzymes. This results in pronounced changes in associated gene expression. Therefor our data indicate that the function of CDK6 is not limited to cell cycle progression but regulates a plethora of tumor‐relevant transcriptional programs. Future therapies may consider this unexpected role and even exploit it to improve personalized and cancer‐specific treatment protocols.