Abstract 2066: Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer
Authors: Garner Ian M. , Su Zhuyan , Hu Sawyer , Wu Yue , McNeish Iain A. , Fuchter Matthew J. , Brown Robert 
Published: July 2021

Abstract
Defective DNA damage response (DDR) is a hallmark of high grade serous ovarian cancer (HGSOC). Genetic and epigenomic aberrations in DDR pathways including homologous recombination (BRCA1/BRCA2) and non-homologous end joining (PARP-1) make some HGSOC vulnerable to cisplatin and Poly (ADP-ribose) polymerase inhibitors (PARPi). However, mechanisms behind primary and acquired resistance to these agents remain unknown. We hypothesise aberrant epigenomic changes at DDR-associated genes contribute to PARPi resistance and can be reversed using a novel histone methyltransferase inhibitor (HKMT-1-005) which targets two histone methyltransferases (EZH2 and EHMT2) frequently upregulated in HGSOC. MTS assays were used to calculate the IC50 values for HKMT-1-005 and the PARPi Rucaparib in a panel of human HGSOC cell lines (n=8). Isobologram analysis using Combenefit software was used to determine synergy. RNA-seq and ATAC-seq was performed on cell lines derived from the same patient before (PEO1) and after acquired resistance to chemotherapy (PEO4) pre/post treatment with HKMT-1-005. Pathway analysis was used to identify pathways enriched in genes with altered expression and/or chromatin conformation after treatment with HKMT-1-005 and validated with qRT-PCR. Synergy between HKMT-1-005 and Rucaparib and changes in chromatin conformation with HKMT-1-005 treatment were further examined in CRIPSR modified ID8 murine ovarian cell lines with the genotypes: WT, P53-/-, P53-/-BRCA1-/- and P53-/-BRCA2-/-. Isobologram analysis revealed HKMT-1-005 in combination with Rucaparib was synergistic in multiple cell lines. Synergy was greater in PEO1 compared to PEO4. RNA-seq and ATAC-seq identified a significant (FDR &lt;0.05) enrichment of genes associated with DDR pathways which had altered expression and/or chromatin accessibility in PEO1 compared to PEO4, including BRCA1 and BRCA2. qRT-PCR analysis confirmed treatment with HKMT-1-005 reduced expression of BRCA1 and BRCA2 in PEO1 but not PEO4. Synergy between HKMT-1-005 and Rucaparib was greater in P53-/-BRCA1-/- and P53-/-BRCA2-/- compared to WT or P53-/- ID8 mouse cell lines. ATAC-seq identified multiple DDR-associated genes had significantly (FDR &lt;0.05) altered chromatin accessibility in P53-/-BRCA2-/- compared with P53-/- alone. HKMT-1-005 treatment in ID8 cell lines revealed changes in chromatin at multiple DDR-associated genes including BRCA2. HKMT-1-005 is synergistic with the PARP inhibitor Rucaparib and can modulate the expression and chromatin conformation of DDR-associated genes in multiple human and mouse ovarian cell lines. Depletion of BRCA2 may additionally regulate chromatin conformation of genes including those associated with DDR pathways and explain why greater synergy between HKMT-1-005 and Rucaparib was observed in BRCA deficient lines.
Citation Format: Ian M. Garner, Zhuyan Su, Sawyer Hu, Yue Wu, Iain A. McNeish, Matthew J. Fuchter, Robert Brown. Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2066.