Radiotherapy is Associated With Global Methylation Alterations in Patient Derived Glioblastoma Cell Lines.

Purpose/Objective(s) Despite maximal surgical resection, radiotherapy, chemotherapy and re-treatment at re-occurrence, median overall survival time of glioblastoma (WHO grade IV, IDH wild-type) is estimated to be ∼16 months. In glioma, DNA methylation states are the most predictive marker of overall survival and response to therapy. Our understanding of how epigenetic states, such as DNA methylation, are “mis-repaired” after DNA damage repair is scant, hampering our ability to understand how treatment associated DNA methylation alterations may drive tumor resistance and growth. Materials/Methods Three different patient derived glioma stem cell (GSC) lines, in duplicates, were treated with 20 Gy in 10 fractions and allowed to recover prior to DNA methylation analysis with 850K methylation arrays. To analyze the methylation array data, we used RnBeads (version 2.4.0) and R (version 3.6.1) packages. We further focused our analysis to various genomic regions, including CpG islands, promoters, gene bodies and CTCF motifs to understand how methylation alterations may differ between these and other genomic contexts. Results We found differential methylation (pre-treatment vs. radiation treatment) changes among the genomic regions examined. Interestingly, we found differential methylation changes at CTCF motifs, which play important DNA-methylation dependent roles in gene expression and chromatin architecture regulation. Hierarchical clustering, PCA and MDS analysis amongst CpG islands, promoters, gene bodies and CTCF domains did not reveal strong inter-sample differences that segregated the samples on the basis of treatment status, suggesting radiation associated methylation alterations are context dependent. Conclusion Radiation treatment is associated with wide-spread alterations of DNA methylation states in this patient derived glioblastoma model. Such alterations may drive gene expression changes, or genomic architecture alterations, that lead to treatment resistance in the recurrent setting. Author Disclosure A.S. Modrek: None. R. Ezhilarasan: None. M. Snuderl: None. E.P. Sulman: None.