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.
Highlights
The balance between cell proliferation and death is vital for normal growth, differentiation, and tissue homeostasis
There was a two-fold decrease in Mcl-1 protein expression in cyclin E-/- compared to wild type (WT) mouse embryonic fibroblasts (MEFs), with minimal reduction in Mcl-1 mRNA levels (Figure 1E, 1F, and 1G)
Based on the experimental evidence presented here and our previous findings [17], we propose a mechanism for cyclin E/Cdk2-dependent Mcl-1 regulation in mediating chemoresistance to Bcl-2 inhibitors
Summary
The balance between cell proliferation and death is vital for normal growth, differentiation, and tissue homeostasis. Cyclin E/cyclindependent kinase (Cdk) 2 plays a crucial role in signaling cascades that govern phosphorylation of a wide range of substrates. These include E2F, Rb, nucleophosmin, and NPAT (nuclear protein, ataxia-telangiectasia), which contain a Cdk2-binding motif and are involved in both physiological and pathological cellular processes [3, 5]. The N-terminal proline, glutamic acid, serine and threonine regions (PEST) domain of Mcl-1 encompass 170 residues that are unique among Bcl-2 family members They contain putative regulatory residues and motifs responsible for Mcl-1 phosphorylation, proteolytic cleavage, and ubiquitination [1, 8]. Regulation of Mcl-1 during mitosis has been reported [14], the role of enhanced cyclin E/Cdk activity in impaired apoptosis and drug resistance has not been defined
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