Abstract 3622: DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma

Abstract In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome has not been thoroughly investigated. Since epigenetic alterations in DNA-methylation can impact the binding of factors involved in chromatin folding, such as CTCF as well as transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Here, using radiation, we show there are significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. However, it is difficult to study these alterations because breaks are introduced at different sites in each cell, resulting in irreproducible DNA methylation alterations between replicates and cell lines. To circumvent this issue, we have developed a “multi-cut” CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. We find that induction of mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation and we characterize the kinetics of overall cellular resolution of DNA damage using immunofluorescence markers and COMET assays. In order to assess repair efficiency (i.e., deletions, translocations, complete repair), we have designed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing of each cut site. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subject cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites. To understand how these alterations may contribute to treatment resistance after DNA damage, we show these alternating chromatin contacts result in new promoter and enhancer interactions, which may drive gene-expression alterations after DNA damage at nearby loci. These results provide a mechanistic view of the interplay between DNA damage, DNA methylation and genome re-organization in glioblastoma. Importantly, these studies shed light on how these alterations evolve and their effect on local gene expression and ensuing treatment resistance. Citation Format: Aram S. Modrek, Catherine Do, Zeyan Zhang, Yingwen Deng, Jerome Karp, Ravesanker Ezhilarasan, Giulia Cova, Matija Snuderl, Aristotelis Tsirigos, Jane Skok, Erik P. Sulman. DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3622.