Cyclin E/Cdk2-dependent phosphorylation of Mcl-1 determines its stability and cellular sensitivity to BH3 mimetics

// Gaurav S. Choudhary 1,6 , Trinh T. Tat 7 , Saurav Misra 2 , Brian T. Hill 4 , Mitchell R. Smith 4 , Alexandru Almasan 1,5 and Suparna Mazumder 3,5 1 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 2 Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 3 Department of Immunology, Lerner Research Institute, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA 4 Department of Hematology and Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA 5 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA 6 Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA 7 Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH, USA Correspondence to: Suparna Mazumder, email: // Alexandru Almasan, email: // Keywords : Mcl-1, cyclin E, Cdk2, dinaciclib ABT-737, ABT-199, CLL Received : February 26, 2015 Accepted : July 01, 2015 Published : July 15, 2015 Abstract Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E -/- mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors.