ATRX/p53调控ATM信号通路影响脑胶质瘤DNA损伤修复的机制研究

Glioma is the most common primary malignant tumor of the brain. Traditional therapy has limit in prolonging the survival of patients with malignant glioma. Recent studies have showed that the accumulation of genomic variation is so important for the glioma development process, including ATRX and TP53 gene mutations. However, their mechanism regulating glioma malignancy and the TMZ sensitivity remains to be mining. Our previous bioinformatics analysis showed that ATRX expression was closely correlated with cell cycle, DNA repair, DNA damage response and DNA replication, including some key genes in ATM signaling pathway. We have built the ATRX knockout glioma cell lines using the CRISPR-Cas9 system. Western Blot showed that ATRX loss significantly inhibited the phosphorylation of ATM induced by TMZ. Comet assay also showed that ATRX loss was associated with increasing DNA damage caused by TMZ. However, it needs further research that the mechanism of ATRX and p53 regulating the ATM signaling pathway and the effect of ATRX deficient on the biological behaviors of TP53 mutant glioma cells..In this study, we mainly focus on the role of ATRX in TP53 mutant gliomas, and will carry out the following work: (1) to investigate the effect of ATRX on TP53 mutant glioma cell replication stress, DNA damage checkpoint activation, repair protein recruitment, cell biological behavior and TMZ sensitivity; (2) to illustrate the mechanism of ATRX regulating histone modifications to promote the phosphorylation of ATM; (3) to confirm the transcriptional regulation of p53 on the ATM gene; (4) to explore the effect of ATRX on DNA replication stress, DNA damage checkpoint activition, repair protein recruitment, biological behavior and TMZ sensitivity of glioma cells in vivo. Based on the previous work, the project focus on the mechanism that ATRX and p53 regulate ATM signaling pathway, affect brain glioma DNA damage repair ability and the first-line chemotherapy drugs TMZ sensitivity. Our work will provide some theoretical and experimental basis for the research of ATRX and TP53 variation and the individual treatments for the specific subtype of glioma.

脑胶质瘤中ATRX和TP53突变是重要的遗传变异事件。课题组前期研究发现ATRX和TP53与DNA损伤修复相关的ATM信号通路密切相关，利用CRISPR-Cas9敲除脑胶质瘤细胞ATRX可明显抑制TMZ诱导的ATM磷酸化，增加细胞基因组损伤，同时在ATM基因启动子区亦发现p53蛋白的转录结合位点。由于特定类型脑胶质瘤中ATRX和TP53几乎同时发生突变，本研究将进一步探索ATRX敲除对TP53突变的脑胶质瘤细胞复制压力、DNA损伤检查点调控及修复、细胞生物学行为和TMZ敏感性的影响；解析ATRX和p53调控ATM信号通路的具体机制；并通过建立TP53突变ATRX缺失的脑胶质瘤动物模型对其进行验证。旨在阐明ATRX和p53调控ATM信号通路，影响DNA损伤修复，揭示TP53突变ATRX缺失型脑胶质瘤细胞的生物学行为及TMZ敏感性，为脑胶质瘤精准医疗提供新的理论依据和实验证据。

本项目已按计划完成所有研究内容，取得多个创新型研究成果，具体研究成果主要体现在以下几方面：1.发现ATRX参与到胶质瘤的DNA复制和修复过程，利用CRISPR-Cas9基因编辑技术介导的ATRX基因敲除，可抑制细胞增殖、侵袭和血管生成拟态。此外，ATRX敲除后，胶质瘤细胞对替莫唑胺化疗敏感性上升，是由于缺乏ATRX对组蛋白H3K9三甲基化状态的维持，降低了DNA双链损伤时ATM蛋白的乙酰化水平，导致替莫唑胺介导的ATM磷酸化的水平降低，进而抑制了ATM通路下游蛋白的激活和DNA损伤修复的进程，因此胶质瘤细胞对替莫唑胺的敏感性上升；2.为进一步研究ATRX在胶质瘤耐药机制方面中所起的作用，我们通过基因表达、DNA甲基化分析及各种体内外实验证明STAT5b/TET2复合物可以通过DNA去甲基化上调ATRX表达。ATRX/EZH2复合物促进H3K27me3在FADD启动子区富集从而下调FADD表达水平提高PARP1稳定，进而促成胶质瘤的DNA损伤修复，公共数据库数据分析显示ATRX表达较低的患者总体生存预后较好，能从TMZ治疗中获益更多；4.我们通过诱导耐替莫唑胺胶质瘤细胞，并对其进行了lncRNA芯片检测发现了一种未曾报道的lncRNA，即lnc-TALC，并通过一系列实验发现其通过竞争性结合miR-20b-3p来激活Stat3/p30复合物从而调节c-Met通路和组蛋白H3甲基化进而促进MGMT的表达和GBM对替莫唑胺的抗性，构建了一种双向纳米抑制剂—BIP-MPC-NP，可通过减弱EGFR和MET信号通路的激活进而影响胶质瘤对TMZ的敏感性。此外，通过数据库分析、高通量测序的多组学联合分析，发现了LGALS1、IGFBP2、MDK在调控胶质瘤微环境方面具有重要作用。通过对WNT/β-Catenin 、SHH/GLI1、HMGB1/RAGE/IL-8等信号通路的研究中发现NEAT1、QKI、NETs可作为胶质瘤的重要潜在治疗方向。在本项目的支持下，课题组发表SCI文章10篇，其中最高影响因子12.1；申请专利1项；项目研究过程中参加国内外学术交流4次；培养博士研究生1名，硕士研究生2名。