Epigenetic alterations after DNA damage repair drive treatment resistance in glioblastoma

项目摘要/摘要 在这个K08职业发展奖中，首席调查员(PI)的目标是确定DNA甲基化是如何 DNA损伤后的改变，并研究这些变化对3D染色质组织、基因的影响 调节和治疗耐药。PI研究了胶质母细胞瘤的这一过程，这是一种难以治疗的恶性肿瘤 脑瘤。培训和指导活动将不仅有助于实现科学目标，而且还将促进校长 通过解决知识差距和通过课程工作扩大培训，促进调查员的职业发展， 会议、网络和由一名主要导师、联合导师和四名教职员工组成的专家指导团队 顾问。建议的研究将通过K08阶段的培训加以解决，并作为科学的 申请人作为独立调查员的职业生涯的基础。应聘者将获得必要的技能 通过精选的具有非重叠专业知识和课程作业的导师完成目标。中环 假说是，DNA损伤修复后，局部表观遗传状态没有正确恢复，导致 表观遗传学改变、基因表达变化和治疗耐药。这一假设是用以下方法检验的 以人患者来源的胶质母细胞瘤细胞培养为模型系统。这个项目的基本原理是 观察到随机DNA甲基化改变可以用辐射损伤模型检测到，以及 核酸内切酶损伤会改变局部DNA甲基化状态。这一过程背后的机制和 然而，它在癌症中的发生程度尚不清楚。这一假设很难用随机方法进行检验。 损伤，如辐射，或传统的核酸内切酶损伤模型，无法削减甲基化 DNA，并且有固定和有限数量的位点。为了绕过这个问题，调查员开发了一种 重复性诱导全基因组双链断裂研究DNA甲基化的CRISRP-Cas9工具 DNA损伤部位周围的改变和基因组组织。核心假说将由两个人来检验 特定的目的是(I)测试DNA甲基化和基因组组织变化是如何在受损的DNA上进化的 以及(Ii)测试在辐射应激期间，基因组重组因子是否可以作为治疗的靶点。这 培训方案具有创新性，因为它(I)开发了绘制DNA甲基化和3D染色质图谱的工具 DNA损伤后的组织改变和(Ii)这一过程与治疗耐药性有关。这个 这项拟议的研究的意义在于，它填补了表观遗传学、DNA损伤修复和 了解治疗对癌细胞的影响。成功完成这些研究将提供 对DNA损伤、DNA甲基化和基因组重组之间相互作用的可翻译见解 胶质母细胞瘤。

PROJECT SUMMARY/ABSTRACT In this K08 career development award, the principal investigator (PI) aims to determine how DNA-methylation is altered after DNA damage and investigate the impact of these changes on 3D chromatin organization, gene regulation and treatment resistance. The PI studies this process in glioblastoma, a difficult to treat malignant brain tumor. Training and mentoring activities will facilitate meeting not only scientific goals, but also the principal investigator's career development by addressing gaps in knowledge and expanding training through coursework, meetings, networking, and an expert mentoring team consisting of a primary mentor, co-mentor, and four faculty advisors. The research proposed will be addressed through this K08 phase of training and serve as the scientific basis for the applicant's career as an independent investigator. The candidate will acquire skills necessary to complete the aims through selected mentors with non-overlapping expertise and coursework. The central hypothesis is that, after DNA damage repair, the local epigenetic state is not restored correctly, leading to epigenetic alterations, gene expression changes and treatment resistance. This hypothesis is tested using human patient-derived glioblastoma cell cultures as a model system. The rationale for this project is the observation that stochastic DNA methylation alterations can be detected with radiation damage models, and endonuclease damage can alter local DNA methylation states. The mechanism underlying this process and the extent to which it occurs in cancer, however, is not known. This hypothesis is challenging to test using stochastic damage, such as radiation, or traditional endonuclease damage models, which are unable to cut methylated DNA, and have a fixed and limited number of sites. To circumvent this issue, the investigator developed a CRISRP-Cas9 tool to reproducibly induce genome-wide double strand breaks to study DNA methylation alterations and genome organization around sites of DNA damage. The central hypothesis will be tested by two specific aims to (i) test how DNA methylation and genome organizational alterations evolve at damaged DNA loci, and (ii) test if genome re-organization factors can be targeted therapeutically during radiation stress. This training proposal is innovative because it (i) develops tools to map DNA methylation and 3D chromatin organization alterations following DNA damage and (ii) implicates this process in treatment resistance. The significance of this proposed research is that it fills knowledge gaps in epigenetics, DNA-damage repair, and the understanding of the effects of treatment on cancer cells. Successful completion of these studies will provide translatable insight into the interplay between DNA damage, DNA methylation and genome re-organization in glioblastoma.