Abstract A29: Loss of histone H2B monoubiquitination in ovarian cancer – new therapeutic targeting opportunities based on chromatin relaxation

Abstract

Abstract Post-translational modifications of histones are involved in diverse functions, including gene transcription and the DNA damage response. Targeting elements of the DNA damage response pathway is of increasing interest for the treatment of epithelial ovarian cancer (EOC). Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is present at the coding region of highly expressed genes such as the gene encoding p21, CDKN1A, under conditions of DNA damage. Addition of this histone mark relies on the E3 ubiquitin ligase ring finger protein complex RNF20-RNF40 that we, and others, have previously shown interacts with members of the PAF1 complex necessary for RNA polymerase II-based transcriptional elongation. At 8.5 kDa, ubiquitin constitutes a bulky addition likely sufficient to interfere with condensed chromatin fibres, leading to a more open fibre conformation with greater accessibility for transcription factors and DNA repair proteins. H2Bub1 has been described as a master switch for mammalian gene regulation, with roles in histone cross-talk. Furthermore, H2Bub1 has recently been linked to the cellular DNA damage response, with the protein kinase ATM phosphorylating RNF20-RNF40 to induce H2Bub1 at sites of double strand breaks, leading to a more open chromatin structure for optimal repair. Global H2Bub1 loss has been reported in breast, colon, lung and parathyroid tumors1, and in some of these tumor types has been associated with advancing stage. We have undertaken immunohistochemical analyses of global H2Bub1 and total histone H2B using tissue microarrays of EOC sourced from the Australian Ovarian Cancer Study and the Gynaecological Oncology Biobank at Westmead. Expression of total histone H2B was high and consistent across all tumors, whereas over 70% of tumors showed total loss of global H2Bub1. Contrary to reports that global H2Bub1 levels decreased with advanced stage in breast cancer, we did not observe this for ovarian cancer. This may be related to the fact that cohorts of EOC are skewed towards tumors of advanced stage, as most women present with Stage III-IV disease. The mechanism of H2Bub1 loss in ovarian cancer is currently unknown, but is likely to involve genetic or epigenetic regulation of genes encoding proteins that function as components of E3 ubiquitin ligase machinery. We have previously shown that loss-of-function mutations in the tumor suppressor CDC73, a PAF1 complex member, lead to loss of H2Bub1 in parathyroid cancer1; however, we observed consistent staining of CDC73 in ovarian tumors. We are currently analysing EOCs for expression of RNF20 given that the RNF20 promoter is hypermethylated in breast cancer and TCGA-ovarian reports RNF20 missense mutations with neutral to medium predicted impact scores. This data will be presented. Given the apparent fundamental role of H2Bub1 in determining the accessibility of chromatin for molecules such as DNA repair proteins, H2Bub1 and/or its associated ubiquitin ligase machinery, may be considered as potential therapeutic targets for the treatment of EOC. 1Hahn MA, Dickson K-A, Jackson S, Clarkson A, Gill AJ and Marsh DJ. The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination Human Molecular Genetics 2012; 21(3):559-568. Citation Format: Deborah J. Marsh, Anna deFazio, Adele Clarkson, Catherine Kennedy, Gregory G. Gard, Anthony J. Gill. Loss of histone H2B monoubiquitination in ovarian cancer – new therapeutic targeting opportunities based on chromatin relaxation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A29.