Abstract A02: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients.
Authors: Dall'Acqua Alessandra , Monica Schiappacassi , Baldassarre Gustavo 
Published: May 2016

Epithelial Ovarian Cancers (EOCs) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOCs the most lethal gynecological malignancy. The central role played by CDKs (Cyclin-Dependent Kinase) in several cellular mechanisms, such as control of cell cycle progression, DNA repair and transcription and the association of their activity in different apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOCs.

We explored the possibility that the activity of one or more CDK was necessary to overcome the platinum-induced cell death in EOC cells. To this aim we used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the CDK most significantly involved in the regulation of platinum sensitivity in EOC cells since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death.

The effect of CDK6 silencing on the sensitivity to both carboplatinum and cisplatin was confirmed in a larger 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 was necessary to protect from platinum-induced death. Accordingly, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to CBDCA or CDDP treatment both in vitro and in vivo, in a CDK6-dependent, CDK4-independent manner.

To understand the molecular mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one principally involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Accordingly, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death.

Mechanistically, we showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway since, after platinum treatment, the knock-down of CDK6 or FOXO3a induced the downregulation of ATR and the decrease of CHK1 phosphorylation. Accordingly, we identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR was a FOXO3a transcriptional target, in platinum treated EOC cells.

Inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA.

Overall we demonstrated that in EOC platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select those patients that may benefit from a combination therapy based on platinum and CDK6 inhibition, further increasing the translational relevance of our work.

Citation Format: Alessandra Dall'Acqua, Schiappacassi Monica, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A02.