Abstract A77: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma
Authors: Watson Zachary L. , Yamamoto Tomomi M. , Hughes Connor J. , Wheeler Lindsay J. , Post Miriam D. , Behbakht Kian , Bitler Benjamin G. 
Published: September 2020

Abstract
The purpose of this study is to examine the contribution of epigenetics to PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC). To identify epigenetic mechanisms of PARPi resistance in HGSOC we utilized unbiased exploratory techniques, including mass spectrometry profiling of histone modifications and RNA-sequencing. Our data show that, compared to sensitive cells and tumors, PARPi-resistant HGSOC cell lines and in vivo patient-derived ascites display a global increase of histone H3 lysine 9 dimethylation (H3K9me2) accompanied by overexpression of histone methyltransferases EHMT1 and EHMT2. EHMT1/2 catalyze H3K9me2 and have canonical roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and have previously been implicated in erlotinib resistance in non-small cell lung cancer. The role of EHMT1/2 in PARPi response has not been evaluated in HGSOC. Using immunohistochemistry, we stained a tissue microarray of over 100 primary ovarian tumors for H3K9me2. High H3K9me2 correlates with significantly worse overall survival. We then used genetic and pharmacologic methods to disrupt EHMT1/2 and examined PARPi sensitivity, apoptosis, DNA repair, and cell cycle. EHMT1/2 disruption reduced H3K9me2 and sensitizes HGSOC cells to PARPi. EHMT1/2 disruption does not increase PARPi-induced apoptosis, suggesting a cytostatic effect, rather than cytotoxic. Functional assays of DNA repair show that disruption of EHMT1/2 ablates both homologous recombination and nonhomologous end joining. Consistently, immunofluorescent staining of phosphorylated histone gamma-H2AX shows increases in DNA damage following EHMT1/2 inhibition. Propidium iodide staining and flow cytometry analysis of cell cycle shows PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of Cyclin A, Cyclin B1, and M-phase inducer CDC25C. We also observed reduced phosphorylation of CDC25C and mitotic markers MPM2 and histone H3 serine 28. These data suggest EHMT1/2 increases DNA damage repair and regulates cell cycle progression, both of which contribute to PARPi resistance. In summary, this study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi and suggests a potential mechanism through DNA damage and cell cycle dysregulation. Identification of factors promoting PARPi resistance, such as EHMT1/2 and H3K9me2, will facilitate development of novel prognostic and therapeutic strategies to successfully treat resistant HGSOC.
Citation Format: Zachary L. Watson, Tomomi M. Yamamoto, Connor J. Hughes, Lindsay J. Wheeler, Miriam D. Post, Kian Behbakht, Benjamin G. Bitler. Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A77.