INVESTIGATING EPIGENETIC MECHANISMS OF CHEMORESISTANCE IN OVARIAN CANCER: THE ROLE OF H3K27ME3

Histone Post-Translational Modifications (PTMs) are key epigenetic players in physiology and disease, generating a complex combinatorial code that regulates gene expression, nuclear functions and chromatin organization. Aberrant levels of histone PTMs are recognized as general hallmarks of cancer and their profiling in tumour can both allow the identification of epigenetic markers for patient stratification and suggest novel epigenetic pathways to be targeted for therapy. With the aim to investigate epigenetic aberrations occurring in Epithelial Ovarian Cancer (EOC), we performed a Mass Spectrometry (MS)-based epi-proteomic profiling of histone PTMs in a cohort of 35 EOC patients stratified based on their survival outcomes. This analysis revealed reduced H3K27me3 level in patients displaying short-term survival (< 2 years after diagnosis) and associated with an increased tumour resistance to chemotherapy, compared to Long-Term Survivors (LTS) (> 10 years after diagnosis), with an improved sensitivity to the platinum-based treatment. By epi-proteomic profiling, we also confirmed a similar loss of this histone mark in an independent clinical cohort of chemoresistant tumours, compared to chemosensitive ones. To explore the molecular mechanisms linking H3K27me3 loss to the onset of chemoresistance in EOC, we generated in vitro platinum-resistant cell line models by exposing the platinum-sensitive A2780 cells to increasing concentrations of Carboplatin (CBP). The induction of platinum-resistance correlates with a decrease of H3K27me3, consistent with the findings from the clinical cohorts. Using a multi-omics approach, we investigated the downstream effects of H3K27me3 decrease in chemoresistant patients by integrating H3K27me3 ChIP-sequencing and RNA-sequencing data from A2780 cells before and after the induction of platinum-resistance. Functional enrichment analysis of genes whose transcriptional upregulation was concurrent with a loss of H3K27me3 in chemoresistant cells highlighted a significant association with the presence of H3K27me3-H3K4me3 chromatin bivalent domains and the maintenance of an undifferentiated state, suggesting a link between the onset of chemoresistance and the emergence in the cells of stem-like features. With the goal to test if an H3K27me3 increase could counteract the onset of chemoresistance, we pharmacologically elevated H3K27me3 signal, through treatment of platinum-resistant cells with the KDM6 demethylases inhibitor GSK-J4; this resulted in downregulated transcriptional pathways associated with stemness and invasive tumour features and decreased colony and sphere formation, two processed associated with the emergence of stem-like cell populations. As a next step, we plan to evaluate the in vivo efficacy of GSK-J4 in mouse models implanted with patient-derived tumours exhibiting different responses to the chemotherapy. Collectively, our findings suggest that H3K27me3 is lost during the acquisition of chemoresistance, and such loss is associated with the upregulation of a set of genes promoting the maintenance of a stem-like state of the cells, potentially contributing to EOC recurrence and to poor patient survival. Moreover, the inhibitory effect of the epi-drug GSK-J4 on more undifferentiated cancer cells could represent a novel therapeutic avenue to target chemoresistant EOC cells with enhanced stem-like features.