Abstract
The standard chemoradiation strategy used to treat GBM triggers cell death by inducing overwhelming numbers of DNA double-strand breaks (DSBs). Cancer cells escaping apoptosis activate error-prone DNA damage response mechanisms that have the potential to produce new mutations in critical driver genes, leading to the emergence of treatment-resistant subclones. Decitabine (DAC) is a DNA demethylating agent currently FDA approved for the treatment of higher-risk myelodysplastic syndrome. Early studies of DAC in GBM suggest a possible synergy with temozolomide (TMZ), but the mechanism remains unclear. Because methylated CpG sites are hypermutable and alter the accessibility of DNA to the damage repair machinery, we hypothesized that epigenetic priming with DAC may be used as a novel method to mitigate radiation- and TMZ-induced mutagenesis without sacrificing cytotoxicity. In this study, we examine how DAC alters patterns of treatment-induced DSBs in a region of TP53 particularly susceptible to acquiring deleterious missense mutations. GBM stem cells were isolated from human surgical resections and subjected to conventional GBM treatment with or without pre-treatment with DAC. We used a novel long-read bisulfite-converted DNA sequencing technique to quantify 5-methylcytosine (5mC) levels at CpGs within a 2 kb region coding for a portion of the TP53 DNA-binding domain. After DAC treatment, we observed an average decrease in 5mC levels by 25%. Interestingly, some CpGs at known mutational hotspots exhibited variable demethylation across samples. We developed an inverse PCR method to quantify DNA damage in the same region, and found that DAC pre-treatment resulted in a significant reduction in DSBs. Our preliminary experiments show that demethylation therapy with DAC prior to TMZ may have a protective effect against C:G>T:A transitions at CpG mutational hotpot sites in TP53. Our current follow-up studies are exploring CpG demethylation patterns in a larger region of TP53, and will correlate this with tumor evolutionary outcomes.