Epigenetic reprogramming of different cancer-related pathways at the single cell level after low-dose DNA demethylation therapy

The efficacy of DNA demethylation therapy is highly dependent on the dosing regimen. Administration of a drug at a dose much lower than the maximum tolerated dose (MTD) for a prolonged period shows a high therapeutic efficacy, despite its small demethylating effect. However, the mechanisms underlying the high therapeutic efficacy of low-dose treatment are still unclear. In this study, we aimed to reveal the mechanisms by analyzing decitabine (DAC)-treated individual cancer cells. To reveal the epigenetic reprogramming at the single cell level and its functional consequences, individual DAC-treated HCT116 cells (H3 clones) were cloned from DAC-treated bulk HCT116 cells. Genome-wide DNA methylation was analyzed using InfiniumHuman450 beadChip. In bulk cells, only partial reduction of DNA methylation levels was observed, and no genes were completely demethylated. In contrast, in cloned cells, a much larger reduction of DNA methylation levels was observed, depending upon genes. Promoter CpG islands of 233-470 genes of 1,188 hypermethylated genes in HCT116 were completely demethylated (Δβ ≥ 0.6) in some of the clones analyzed. Demethylated genes were highly variable among individual clones, and different cancer-related genes, such as those involved in the p53 pathway, WNT pathway, and cell cycle regulation, were completely demethylated, depending upon clones. For example, CDKN2A (p16) was completely demethylated only in a H3-32 clone, and this clone showed slower cell growth rate (14.6% of untreated cells), and aneuploid cells were detected possibly due to cellular senescence. These results showed that epigenetic reprogramming at the single cell level involves diverse cancer-related pathways, and this mechanism is likely to underlie the high efficacy of low-dose DNA demethylation therapy. Citation Format: Hideyuki Takeshima, Yukie Yoda, Naoko Watanabe, Mika Wakabayashi, Toshikazu Ushijima. Epigenetic reprogramming of different cancer-related pathways at the single cell level after low-dose DNA demethylation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 838.