摘要
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.
摘要译文
背景荧光组蛋白-赖氨酸-N-甲基转移酶1和2（EHMT1 / 2，又名GLP / G9A）催化组蛋白H3赖氨酸9（H3K9me2）的二甲基化，并在基因表达的表观遗传沉默中起作用。 EHMT1 / 2在DNA修复中也具有直接作用，并且与几种癌症的化学抗性有关。对化疗和PARP抑制剂（PARPi）的耐药性是高级别浆液性卵巢癌（HGSOC）致死的主要原因，但表观遗传的贡献尚不清楚。结果为确定HGSOC中PARPi耐药性的表观遗传机制，我们采用了无偏探性技术，包括RNA-Seq和组蛋白修饰的质谱分析。与敏感细胞相比，耐PARPi的HGSOC细胞显示出H3K9me2的整体增加，并伴有EHMT1 / 2的过表达。在耐PARPi的体内患者衍生的异种移植（PDX）模型中也观察到EHMT1 / 2过表达。 EHMT1 / 2的遗传或药理破坏使HGSOC细胞对PARPi敏感。细胞死亡分析表明，EHMT1 / 2破坏不会增加PARPi诱导的细胞凋亡。功能性DNA修复分析表明，EHMT1 / 2的破坏会消除同源重组（HR）和非同源末端连接（NHEJ），而磷酸化组蛋白H2AX的免疫荧光染色则显示DNA损伤的大幅增加。碘化丙锭染色和细胞周期的流式细胞术分析表明，PARPi处理增加了S和G2期的PARPi耐药细胞的比例，而用EHMT1 / 2抑制剂处理的细胞保留在G1中。与PARPi和EHMT1 / 2抑制剂的共同处理产生中间表型。细胞周期调节剂的免疫印迹表明，EHMT1 / 2和PARP的联合抑制作用会降低特异性细胞周期蛋白的表达和有丝分裂标记物的磷酸化。这些数据表明DNA损伤和改变的细胞周期调节作为致敏机制。用EHMT1 / 2抑制剂处理的PARPi耐药细胞的RNA-Seq显示出显着的基因表达变化，其丰富的促存活途径在PARPi耐药的情况下仍未探索，包括PI3K，AKT和mTOR。结论本研究表明破坏EHMT1 / 2使HGSOC细胞对PARPi敏感，并提出了通过DNA损伤和细胞周期失调的潜在机制。 RNA-Seq识别可能改变PARPi抗性的几种未探索的途径。 EHMT1 / 2和调控基因的进一步研究将有助于开发成功治疗HGSOC的新型治疗策略。
Zachary L. Watson[1]Tomomi M. Yamamoto[1]Alexandra McMellen[2]Hyunmin Kim[3]Connor J. Hughes[4]Lindsay J. Wheeler[5]Miriam D. Post[1][6]Kian Behbakht[1][5]Benjamin G. Bitler[1]. Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma[J]. Clinical Epigenetics, 2019,11(1)