ATRX调控组蛋白甲基转移酶重分布介导脑胶质瘤耐药性的机制研究

Chemo resistance of temozolomide (TMZ), the first-line chemotherapeutic agent, is a major issue in the management of patients with glioma. Alterations of ATRX constitute one of the most prevalent genetic abnormalities in gliomas. Previously, we applied the mRNA array, methylation array, and ChIP-seq to find that higher level of ATRX was detected in TMZ resistant glioma cells compared with the parental cells. ATRX could recruit EZH2, a methyltransferase, to promote tri-methylation of H3K27 in FADD promoter region, which resulted in the suppressed expression of FADD. Based on the results, we aim to explore the molecular mechanism of TMZ resistance by upregulated ATRX regulating PARP1 stabilization in TMZ resistant glioma cells. We will reveal the biological mechanism that overexpression of ATRX in TMZ resistance glioma cells by applying chromatin immunoprecipitation assay, and explore whether knocking out ATRX could influence TMZ sensitivity and PARP1 stabilization in TMZ resistance glioma cells and ATRX stabilizes PARP1 by suppressing FADD expression. ChIP-seq and immunoprecipitation assay helps to explore ATRX facilitating tri-methylation of H3K27 in FADD promoter by recruiting EZH2. This study is expected to elucidate a new theoretical evidence for the mechanism of TMZ resistance medicated by ATRX in TMZ resistance glioma cells.

DNA损伤修复是影响脑胶质瘤患者化疗敏感性的重要因素。申请人前期利用表达谱芯片、甲基化芯片、高通量染色质沉淀测序等技术发现：耐药脑胶质瘤中异常高表达的ATRX通过招募甲基转移酶调节组蛋白甲基化，抑制FADD表达，进而促进PARP1稳定，调节替莫唑胺化疗敏感性。在此基础上，申请人拟利用染色质免疫共沉淀等技术探究ATRX表达异常升高的分子机制；通过生物学实验验证敲除ATRX对耐药胶质瘤细胞替莫唑胺敏感性以及PARP1蛋白稳定性的影响；运用高通量免疫共沉淀测序技术探究ATRX通过调节蛋白质甲基转移酶EZH2的重分布介导FADD启动子区组蛋白H3K27三甲基化抑制其表达并促进PARP1稳定的机制。本研究可揭示耐药脑胶质瘤中ATRX异常表达并促进PARP1稳定性介导脑胶质瘤替莫唑胺治疗抵抗的分子机制，为脑胶质瘤精准治疗提供新的理论依据。

脑胶质瘤是颅内最常见的成人原发性恶性肿瘤，与其他肿瘤类似，胶质瘤表现出明显的抗DNA损伤表型，这种表型是脑胶质瘤产生化疗抵抗的重要原因。尽管脑胶质瘤化疗提高了脑胶质瘤患者的总生存期，但恶性程度最高的胶质母细胞瘤（Glioblastoma，GBM）患者的化疗抵抗仍然是限制患者生存期的重要因素。申请人报道了ATRX基因在耐药型脑胶质瘤中抗替莫唑胺的作用，以及在特定分子分型的耐药性脑胶质瘤中使用PARP抑制剂的临床前景进行了初步探索。在此项研究中，研究人员在构建TMZ耐药的胶质瘤后，验证了耐药细胞中ATRX表达量高于亲代细胞。与此同时通过免疫组化发现ATRX缺失脑胶质瘤标本中DNA损伤修复指标表达量明显少于ATRX野生型标本。通过高通量RNA芯片，甲基化芯片以及多种数据库内基因表达分析发现耐药细胞中ATRX甲基化水平明显降低，同时转录因子STAT5b与去甲基化酶TET2与ATRX表达量呈显著的正相关性。多种免疫共沉淀实验验证TET2依赖STAT5b富集于ATRX启动子区。干扰STAT5b或TET2的表达使ATRX启动子区甲基化水平增高，抑制ATRX表达。使用CRISPR-Cas9系统敲除ATRX后，脑胶质瘤细胞显示出明显的DNA损伤和PARP1降解增强，同时PARP1抑制剂奥拉帕尼（Olaprib，Ola）可显著增强完整表达ATRX耐药细胞的TMZ敏感性。通过联合高通量染色质免疫共沉淀实验发现ATRX可能通过募集EZH2使FADD启动子区组蛋白H3K27三甲基化水平增强，抑制FADD表达，与此同时体内实验验证，敲除ATRX增强耐药型脑胶质瘤细胞的TMZ敏感性，联合使用Olaparib同样增强TMZ对ATRX功能完整的脑胶质瘤细胞的治疗效果。相关结果发表于生物医学1区杂志《Theranostics》( IF:11.6)