Abstract
BackgroundEuchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown.ResultsTo identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that 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 specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR.ConclusionsThis study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.
Highlights
Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression
Dimethylated histone H3 lysine 9 (H3K9me2) is globally increased in Poly ADP ribose polymerase inhibitors (PARPi)-resistant high-grade serous ovarian carcinoma (HGSOC) cells and correlates with poorer overall survival To examine the epigenetic landscape of PARPi-resistant cells, we subjected a PARPi-sensitive HGSOC cell line (PEO1, TP53/BRCA2-mutated) to step-wise dose escalation of olaparib to select for resistant cells
In agreement with mass spectrometry, H3K9me1 was decreased in PEO1-OR by 8%, H3K9me2 was increased by 20%, and H3K14ac was increased by 11%
Summary
Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown. ADP ribose polymerase inhibitors (PARPi) were initially developed to treat homologous recombination (HR) DNA repair-deficient tumors (e.g., BRCA1/2-mutated). Mutation of BRCA1/2 often leads to a defective HR repair pathway and significantly increases the risk of developing HGSOC [2]. The three approved PARPi (olaparib, rucaparib, and niraparib) were initially used for recurrent HGSOC with BRCA1/2 mutations as a third- or fourth-line therapy, but the SOLO1 clinical trial showed that first-line maintenance olaparib reduced risk of disease progression or death by 70% in newly diagnosed cases [3]. Known mechanisms of PARPi resistance, such as BRCA1/2 reversion mutations, restore HR but are found in only a small proportion of resistant cancers [6,7,8,9], suggesting that PARPi resistance has other causes that have yet to be explored [10]
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