Epigenetic alterations after DNA damage repair drive treatment resistance in glioblastoma

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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.