Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor, and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma [ChIP-Seq]

Background: Uterine leiomyosarcoma (uLMS) has a poor prognosis owing to its resistance to chemotherapy. Therefore, novel therapeutic targets for uLMS should be identified. Suppressor license of variegation 3-9 homolog 2 (SUV39H2) is a histone methyltransferase that promotes the repair of double-stranded DNA breaks by recruiting phosphorylated H2AX (?H2AX). In this study, we investigated the potential therapeutic targets of SUV39H2 in uLMS and the mechanism of synthetic lethality between PARP inhibitors and the SUV39H2 inhibitor OTS186935. Methods: First, we analyzed the mRNA and protein expression of SUV39H2 in the clinical tissues of uLMS, normal myometrium, and leiomyomas using real-time polymerase chain reaction and immunohistochemistry, respectively. Next, we conducted drug sensitivity assays for OTS186935 alone and in combination with olaparib, a poly (ADP-ribose) polymerase inhibitor, using the uLMS cell lines SK-LMS-1 and SK-UT-1. We performed Western blotting, immunofluorescence, and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate ?H2AX following OTS186935 treatment in addition to in vivo experiments using nude mice with subcutaneously implanted uLMS. Results: SUV39H2 expression in uLMS was significantly higher than that in the normal myometrium and leiomyomas. OTS186935 decreased the viability of both cell lines, and its combination with olaparib resulted in synthetic lethality in SK-UT-1 cells (combination index = 0.88). After treatment with OTS186935, ?H2AX accumulation decreased. ChIP-seq also showed downregulation of ?H2AX following OTS186935 treatment. Notably, the combination of OTS186935 and a PARP inhibitor was significantly more effective in vivo. Conclusion: OTS186935 inhibited double-stranded DNA break repair, as evidenced by ?H2AX downregulation by ChIP-seq and other assays. OTS186935 combined with olaparib induces synthetic lethality in patients with uLMS. Overall design: ChIP-seq was conducted for SK-UT-1 cell line under four conditions: 0.1% DMSO (negative control), 1 µM OTS186935, 0.5 µM doxorubicin, and a combination of 1 µM OTS186935 with 0.5 µM doxorubicin.

背景：子宫平滑肌肉瘤（Uterine leiomyosarcoma, uLMS）因化疗耐药而预后不佳，亟需发掘其新型治疗靶点。杂色抑制因子3-9同源物2（Suppressor license of variegation 3-9 homolog 2, SUV39H2）是一种组蛋白甲基转移酶，可通过招募磷酸化H2AX（γH2AX）促进DNA双链断裂修复。本研究旨在探讨SUV39H2在uLMS中的潜在治疗价值，以及聚腺苷二磷酸核糖聚合酶（poly(ADP-ribose) polymerase, PARP）抑制剂与SUV39H2抑制剂OTS186935之间的合成致死机制。
方法：首先，我们采用实时聚合酶链反应（real-time polymerase chain reaction）与免疫组化（immunohistochemistry）技术，分别检测uLMS、正常子宫肌层及子宫肌瘤临床组织中SUV39H2的mRNA与蛋白表达水平。随后，我们以uLMS细胞系SK-LMS-1和SK-UT-1为模型，开展OTS186935单药及与PARP抑制剂奥拉帕利（olaparib）联合给药的药物敏感性实验。此外，我们通过蛋白质印迹（Western blotting）、免疫荧光染色（immunofluorescence）及染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）检测OTS186935处理后的细胞内γH2AX表达变化，并利用皮下移植uLMS的裸鼠开展体内实验。
结果：uLMS组织中SUV39H2的表达水平显著高于正常子宫肌层与子宫肌瘤组织。OTS186935可降低两种细胞系的存活率，其与奥拉帕利联合给药可在SK-UT-1细胞中产生合成致死效应（联合指数=0.88）。经OTS186935处理后，细胞内γH2AX的蓄积水平显著下降；ChIP-seq结果同样证实OTS186935处理后γH2AX的富集程度下调。值得注意的是，OTS186935与PARP抑制剂联合给药在体内实验中展现出更显著的抗肿瘤效果。
结论：OTS186935可通过下调γH2AX表达（经ChIP-seq及其他实验验证）抑制DNA双链断裂修复过程，其与奥拉帕利联合给药可诱导uLMS产生合成致死效应。
实验整体设计：针对SK-UT-1细胞系设置4组处理条件：0.1%二甲基亚砜（dimethyl sulfoxide, DMSO，阴性对照）、1 μM OTS186935、0.5 μM多柔比星（doxorubicin），以及1 μM OTS186935与0.5 μM多柔比星联合给药组，对各组开展ChIP-seq检测。