Role of the repair of 5-hydroxymethylcytosine residues in occurrence of drug-tolerant persister cells in cancer

Collaborative Research Program for 2025-2027

Post-replicative methylation of cytosine at the C5 position (5mC) together with its erasure are
essential epigenetic processes in the course of organism development, cell differentiation, genomic imprinting and suppression of mobile elements. The methylation status of DNA is a result of balance between methylation and demethylation, which can undergo dramatic changes during organism development, cell differentiation and cancer. DNA demethylation can occur either in passive or by active process via direct enzymatic removal of 5-ethylcytosine (5mC) residues from DNA. In latter, the TET family proteins (TET1, 2 and 3) catalyze the sequential conversion of 5mC in the DNA first to 5hmC and then further to 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC). Human mismatch-specific Thymine-DNA glycosylase (TDG) catalyzes the removal of 5fC and 5caC residues, but not 5hmC, in DNA and their replacement with regular cytosine via base excision repair (BER) pathway. The global levels of 5mC and 5hmC are significantly reduced in many cancer types and decreased level of 5hmC correlated with tumor aggressiveness. Although, it was thought that 5hmC is resistant to excision repair, recently we demonstrated that TDG exhibits the slow excision kinetics towards 5hmC residues in duplex DNA. We suggest that DNA demethylation and regulation of gene expression may also proceed via DNA glycosylase-catalyzed removal of 5hmC. Cancer recurrence is observed when a subset of tumor cells, referred to as persister cells, survive chemotherapy. Most of persister cells do not divide in the presence of anticancer drug, however a subpopulation of these cells can re-enter the cell cycle during treatment and start to proliferate. Recent data suggest that non-genetic mechanisms such as epigenetic changes in the chromatin might have an important role in the development of a persistent state. Here, we propose that re-establishment of the pattern of DNA hydroxymethylation in cancer persister cells is linked to metabolic reprogramming and drug tolerance. Our data suggest that 5hmC, similar to 5fC and 5caC residues, can be removed in the BER pathway albeit at low rate and this in turn may lead to metabolic heterogeneity among human tumors via epigenetic alterations of the expression of enzymes involved in cellular metabolism. The aim of the present study is to understand the role TDG-catalyzed excision of 5hmC in cell differentiation, particular in adaptive changes of the patterns of gene expression in cancer cells exposed to DNA damage.