MGMT阴性胶质瘤替莫唑胺获得性耐药中介导多种DNA损伤修复蛋白共同入核转运的机制研究

Temozolomide (TMZ) is an alkylating agent used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, acquired drug resistance is frequently induced. One of the major mechanisms of cancer drug resistance is MGMT protein. However, more than half of glioma patients with negative MGMT expression, also produce acquired resistance, which may be caused by multiple DNA damage repair pathways. If we can not only block each repair pathway, but also avoid the use of multiple drugs, it would be efficient and safe. We previously found that, long-term use of TMZ to MGMT negative cells leads to nuclear enrichment of multiple proteins of different repair pathways, such as nucleotide excision repair, base excision repair and mismatch repair, with almost the same time course. In addition, function inhibition of DHC2 as a motor for intraflagellar retrograde transport can significantly increase the sensitivity to TMZ. These results suggest that different repair pathways involved in TMZ resistance may be regulated by the same mechanism. This project will select XPC, PARP1, MSH6, all of which are highly enriched in nuclei by TMZ treatment, and play respectively a key role in the three repair pathways mentioned above, to identify the key protein which regulate their transportation to nuclei in vivo and in vitro through using high-throughput methods. Afterwards, the function of this key protein will be inhibited to verify its role mediating transportation of DNA repair proteins (XPC, PARP1, MSH6) into nuclei and acquired drug resistance, with cell and animal models, and verified in clinical specimens. This project will reveal the mechanism of acquired resistance of MGMT negative glioma to TMZ and provide some new therapeutic target candidates which would benefit most of the glioma patients.

替莫唑胺（TMZ）损伤DNA造成肿瘤细胞死亡，是胶质瘤首选药物，MGMT蛋白介导其耐药。然而过半胶质瘤患者MGMT表达阴性，却产生获得性耐药，可能是由于多种DNA损伤修复途径协同作用。若同时阻断各种修复途径，则可避免多药联用，安全高效。我们发现TMZ持续杀伤后残存的MGMT阴性细胞，核苷酸切除修复、碱基切除修复、错配修复等途径的多种蛋白同步性在胞核富集；抑制发挥逆转运功能的蛋白可加剧DNA损伤、细胞死亡。提示不同修复途径蛋白入核可能共用相同调控机制。本项目拟选择上述核富集蛋白中三种修复途径的重要蛋白PARP1、XPC、MSH6，用高通量技术筛选调控它们入核的关键转运蛋白。进而干预该关键蛋白功能，证明其能转运多种修复蛋白入核、修复损伤DNA，导致细胞获得性耐药，在动物模型和临床标本验证并设计靶向性多肽药物。本项目将为MGMT阴性胶质瘤综合治疗提供新手段，克服获得性耐药，使多数胶质瘤患者获益。

替莫唑胺获得性耐药是胶质母细胞瘤临床治疗面临的主要挑战。目前认为胶质母细胞瘤对替莫唑胺的治疗抗性主要归因于O [6] -甲基鸟嘌呤-DNA甲基转移酶（MGMT）修复替莫唑胺诱导的DNA损伤。然而，一些MGMT缺陷的胶质母细胞瘤仍然对替莫唑胺有抗药性，其潜在的分子机制仍不清楚。我们发现DYNC2H1（DHC2）在MGMT缺陷的复发性胶质母细胞瘤标本中表达更高，其表达与MGMT启动子甲基化胶质母细胞瘤患者的无进展生存期密切相关。 此外，在体外和体内沉默DHC2增强了替莫唑胺诱导的DNA损伤，并显著提高了替莫唑胺治疗MGMT缺陷性胶质母细胞瘤的效率。 我们应用亚细胞蛋白质组学和体外分析的组合，发现DHC2参与DNA修复蛋白的核定位，如XPC和CBX5，并且XPC或CBX5的敲低导致替莫唑胺诱导的DNA损伤增加。总之，我们的研究结果证明了DHC2介导DNA修复蛋白的核转运是MGMT缺陷性胶质母细胞瘤中获得性替莫唑胺治疗抵抗的关键机制。本项目的研究为改善MGMT缺陷性胶质母细胞瘤的治疗提供了新的思路和治疗靶点。本项目发表SCI收录论文8篇，其中中科院小类分区1区论文2篇，获得国家发明专利授权1项。

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