CDK6 controls platinum sensitivity via the regulation of FOXO3/ATR: a new actionable pathway for ovarian cancer patients

High Grade Epithelial Ovarian Cancers (HGEOCs) consist in a heterogeneous group of tumors. Late diagnosis and drug-resistant recurrences make HGEOCs the most aggressive among the gynecological malignancies. The central role played by CDKs (Cyclin-Dependent Kinase) in several cellular mechanisms, such as control of cell cycle progression, DNA repair, transcription and apoptosis, make them attractive targets to overcome drug-resistance in HGEOCs. Using the RNA interference technology (targeting 23 members of the CDKs family) we performed a functional genomic screening by which we have identified CDK6 as the CDK most significantly involved in platinum sensitivity. The effect of CDK6 silencing on the sensitivity to both carboplatinum (CBDCA) and cisplatin (CDDP) was confirmed in a panel of EOC cell lines using multiple shRNAs. Next, the use of CDK6 dominant negative CDK6D163N and constitutively active CDK6R31C mutants demonstrated that CDK6 kinase activity is necessary to protect from platinum-induced death. Accordingly, an orally active CDK4/CDK6 inhibitor, PD 0332991, was able to sensitize EOC cells to CBDCA or CDDP treatment both in vitro and in vivo, in a CDK6-dependent and CDK4-independent manner. To understand the molecular mechanism whereby CDK6 regulates platinum-induced cell death in HGEOC we focused on CDK6 specific phosphorylation targets demonstrating that the transcription factor FOXO3 is a relevant downstream target of CDK6. FOXO3a downregulation sensitizes HGEOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. Functional and biochemical analyses showed that upon platinum exposure, Cyclin D3-CDK6 complex binds and phosphorylates FOXO3 on Serine 325, preventing FOXO3 degradation and promoting FOXO3 nuclear translocation. We showed that CDK6/FOXO3 axis is necessary to control the DNA damage response in HGEOC cells through the regulation of ATR/CHK1 pathway, and using a chromatin immunoprecipitation approach we demonstrated that FOXO3 binds the ATR promoter confirming the role of FOXO3 and CDK6 as upstream mediators of ATR transcription upon DNA damage. Accordingly, the silencing of both CDK6 or FOXO3 induced ATR downregulation, decreased CHK1 phosphorylation and increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC) mechanism by which cells undergo to death when the ATR/CHK1 pathway is inhibited. In accord with these findings, high CDK6 and FOXO3 expression levels predict poor survival of EOC patients further increasing the translational relevance of this work. Therefore, CDK6 represents an actionable target that could be exploited to improve platinum efficacy in HGEOC patients.