Abstract A15: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation.
Authors: Wilson Andrew J. , Saskowski Jeanette , Khabele Dineo 
Published: May 2016

Background: The lack of treatment options for chemoresistant ovarian cancer, the deadliest gynecologic malignancy, is a central clinical problem. Approximately 50% of high-grade serous ovarian tumors with defects in DNA repair pathways including germline BRCA1 and 2 mutations (the so-called BRCAness phenotype), and are sensitive to DNA damaging agents such as cisplatin and poly ADP ribose polymerase inhibitors (PARPi). In contrast, non-BRCAness tumors such as those exhibiting CCNE1 (cyclin E) amplification are chemoresistant, show upregulated DNA repair processes and cell cycle progression via activation of the CDK2/cyclin E/E2F1 axis, and have poor clinical outcomes. Targeting the cyclin E axis with CDK inhibitors has been unsuccessful in part due to chemoresistance through upregulation of oncogenic E2F1. An area of urgent need is to develop new treatments for these intractable subtypes of non-BRCAness ovarian cancer. To that end, our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs, including PARPi, through reduction of expression of DNA repair genes.

Aims: The objective of this study was to (i) determine expression patterns of cyclin E axis components in publically available databases of high-grade serous ovarian tumors and established cell lines; and (ii) test an epigenetic drug strategy using HDACi to convert non-BRCAness ovarian cancer cells to a BRCAness phenotype as a means of sensitizing chemoresistant cells to PARPi.

Methods: RNA-seq V2 RSEM expression levels of cyclin E, CDK2, BRCA1 and E2F1 in 265 ovarian tumors were extracted from The Cancer Genome Atlas (TCGA) via cbioportal.org. mRNA expression of these genes in ovarian cancer cell lines was extracted from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Preliminary western blot studies indicated that of the FDA-approved HDACi (vorinostat, romidepsin, panobinostat), panobinostat was most effective at reducing protein expression of cyclin E axis components (cyclin E, BRCA1 and E2F1). We therefore treated cultured cyclin E overexpressing ovarian cancer cells via CCNE1 amplification (OVCAR-3) or high-level gain (OVCAR-4) with panobinostat alone and in combination with the PARPi, olaparib. The following endpoints were examined: cell viability in sulforhodamine B (SRB) assays, with drug interactions tested for synergism by isobologram analysis; DNA damage by immunofluorescence analysis of the double-strand DNA break marker, pH2AX(ser139); apoptosis by immunofluorescence and western blot analysis of cleaved PARP and cleaved caspase-3. Two-tailed Student's t tests were used for measuring differences between groups in drug treatment experiments.

Results: There was significant correlation (Spearman) between cyclin E expression and that of CDK2, E2F1 and BRCA1 in ovarian tumors and in serous-like ovarian cancer cell lines. These results are consistent with the relationship between cyclin E overexpression and enhanced E2F1-dependent transcription of established E2F1 targets, including cyclin E itself, and DNA repair genes such as BRCA1. In SRB assays, the combination of panobinostat and olaparib synergized in both OVCAR-3 and OVCAR-4 cells. When combined, the drugs also induced co-operative effects on expression of pH2AX(ser139) and apoptosis markers. Enhanced cytotoxicity of the combination was accompanied by a panobinostat-induced reduction in expression of cyclin E, E2F1 and BRCA1. A key role for E2F1 downregulation in mediating this BRCAness shift was suggested by the observation that E2F1 knockdown with siRNA synergized with panobinostat in reducing BRCA1 expression and inducing apoptosis.

Conclusions: The development of a new PARPi combination therapy with panobinostat has immediate prospects for rapid translation to the clinic and great potential for improving clinical outcomes for non-BRCAness, chemoresistant ovarian cancer.

Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation. [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 A15.