Abstract
DNA methyltransferase inhibitors (DNMTi) decitabine and azacytidine are approved therapies for myelodysplastic syndrome and acute myeloid leukemia, and their combinations with other anticancer agents are being tested as therapeutic options for multiple solid cancers such as colon, ovarian, and lung cancer. However, the current therapeutic challenges of DNMTis include development of resistance, severe side effects and no or partial treatment responses, as observed in more than half of the patients. Therefore, there is a critical need to better understand the mechanisms of action of these drugs. In order to discover molecular targets of DNMTi therapy, we identified 638 novel CpGs with an increased methylation in response to decitabine treatment in HCT116 cell lines and validated the findings in multiple cancer types (e.g., bladder, ovarian, breast, and lymphoma) cell lines, bone marrow mononuclear cells from primary leukemia patients, as well as peripheral blood mononuclear cells and ascites from platinum resistance epithelial ovarian cancer patients. Azacytidine treatment also increased methylation of these CpGs in colon, ovarian, breast, and lymphoma cancer cell lines. Methylation at 166 identified CpGs strongly correlated (|r|≥ 0.80) with corresponding gene expression in HCT116 cell line. Differences in methylation at some of the identified CpGs and expression changes of the corresponding genes was observed in TCGA colon cancer tissue as compared to adjacent healthy tissue. Our analysis revealed that hypermethylated CpGs are involved in cancer cell proliferation and apoptosis by P53 and olfactory receptor pathways, hence influencing DNMTi responses. In conclusion, we showed hypermethylation of CpGs as a novel mechanism of action for DNMTi agents and identified 638 hypermethylated molecular targets (CpGs) common to decitabine and azacytidine therapy. These novel results suggest that hypermethylation of CpGs should be considered when predicting the DNMTi responses and side effects in cancer patients.
Highlights
DNA methyltransferase inhibitors (DNMTi) are widely used as chemical tools for hypomethylating the genome, with an aim to understand the role of DNA methylation in multiple processes (e.g., X-chromosome inactivation and DNA imprinting) and as an anti-cancer therapy (Minkovsky et al, 2015; Ramos M.P. et al, 2015; Bohl et al, 2018)
We hypothesized that DNMTi treatment causes hypermethylation in the genome, and we systematically investigated the extent of hypermethylated CpGs, their location, and biological role by analyzing cell line and primary tumor data
Most of the identified sites loss their hypermethylation by day 10 (Figure 1) suggesting that the observed hypermethylation is transient. Reanalysis of another methylation data for HCT116 cell line from the Han et al (2013) study validated our finding, as we found a corresponding increase in methylation level at 583 common CpGs after decitabine treatment (0.3 μM for 24 h) (Figure 1B)
Summary
DNA methyltransferase inhibitors (DNMTi) are widely used as chemical tools for hypomethylating the genome, with an aim to understand the role of DNA methylation in multiple processes (e.g., X-chromosome inactivation and DNA imprinting) and as an anti-cancer therapy (Minkovsky et al, 2015; Ramos M.P. et al, 2015; Bohl et al, 2018). Two DNMTi drugs, decitabine and Decitabine/Azacytidine Increases DNA Methylation azacytidine, have been approved for treating patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) (Döhner et al, 2017; Bohl et al, 2018), and are being tested as therapeutic options in multiple solid cancers (Fu et al, 2011; Singal et al, 2015; Lee et al, 2018). Attempts are being made to improve treatment efficacy (e.g., by testing combinations of DNMTis with other anticancer agents) and safety (e.g., by stratifying cancer patients into DNMTi responders and non-responders) (Nervi et al, 2015) of the drugs. The precise prediction of drugs that show synergy with DNMTi in combination as well as distinguishing the drug responders from the non-responders are challenging tasks and are currently compromised by our incomplete knowledge of the drug mechanisms of action in various cancer-types (Ramos F. et al, 2015; Wang et al, 2018)
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