Dissecting the role of G9a/GLP histone methyltransferases in Platinum-resistant ovarian cancer

Epithelial Ovarian Cancer (EOC) is the most aggressive gynecological malignancy, mainly due to the advanced stage at diagnosis and the development of drug-resistant recurrences. Epigenetic modifications, such as histone methylation/acetylation, are emerging as key regulators of tumor growth and response to therapies. To verify if they are also involved in the onset of drug re-sistance in EOC, we performed a high-throughput Epigenetic Modifiers (EMs)-based screening using four different models of EOC isogenic Platinum-Resistant (PT-Res) cells. The screening identified G9a/GLP histone methyltransferases (HKMTs) inhibitors (G9a/GLPi) as the most ac-tive compounds in killing all tested models. Based on these results and the current literature pro-posing a role for G9a in EOC progression, the aim of this thesis was to dissect the role of these HKMTs in the development of EOC PT-resistance. Using biochemical and cell biology approaches we characterized the activity of the G9a/GLPi UNC0631 in inducing cell death alone or in combination with cisplatin (PT), demonstrating that UNC0631 was able to improve PT efficacy and increase PT-induced DNA damage both in parental but especially in PT-Res cells. Using OVSAHO parental and PT-Res cells as a study model we observed an altered expression of cell-cell junction markers (i.e. β-catenin, occludin and p120-catenin) modified by PT and then re-verted by UNC0631, indicating a role of G9a/GLP in cell-cell junction organization. Accordingly, PT-Res clones displayed greater ability to form spheres in extracellular matrix respect to their pa-rental counterpart and UNC0631 treatment decreased their sphere forming activity. In search of the possible molecular mechanism(s) explaining UNC0631 activity we verified that G9a expression and localization is regulated by PT and is altered in PT-Res EOC cells. According-ly, PT treatment increased H3K9me2 and these effects were completely reverted by UNC0631 treatment. Then Gene Expression Profile (GEP) in OVSAHO parental and PT-Res cells treated with PT and/or UNC0631, combined with Gene Set Enrichment Analyses (GSEA), reveled that Focal Adhesion and Canonical Wnt pathways were the most significantly enriched in PT-Res re-spect parental cells in untreated condition. Moreover, genes belonging to Tight Junction, Canoni-cal Wnt and Focal Adhesion pathways were all enriched in PT-Res cells treated with PT and re-verted by UNC0631. qRT-PCR analysis confirmed that two genes of the Wnt pathway, namely FBXW11 and BTRC, were modified by PT in a G9a/GLP-dependent manner in all tested PT-Res isogenic models. Chromatin immunoprecipitation analyses demonstrated that G9a bound the dis-tal region of BTRC promoter, more efficiently in PT-Res respect to the parental cells and, accord-ingly, that BTRC promoter had a higher degree of H3K9me2 in PT-Res cells. Finally, to prove the role of G9a/GLP in mediating the effects of UNC0631 we generated OVSAHO PT-Res G9a/GLPKO. When compared with than PT-Res wild type cells PT-Res G9a/GLPKO cells were more sensitive to PT, had increased PT-induce DNA damage, and the partial recovery of properly organized cell-cell junctions. Collectively, our data suggest that G9a/GLP inhibition could represent a good strategy to over-come PT-resistance in EOC. Mechanistically the data support a role for G9a/GLP HKMTs in the regulation of Wnt/β-Catenin pathway that could explain the acquisition of a mesenchymal stem-like phenotype observed in PT-Res cells that, in the end, could affect the response to anticancer drugs.