The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells
Background: DNA methylation is a key regulator of gene expression in mammals. The covalent DNMT1 inhibitors, 5-azacytidine and decitabine, are commonly used in research to reduce DNA methylation levels, but their severe cytotoxicity limits their demethylation effects and complicates experimental interpretation. Recently, GlaxoSmithKline developed a non-covalent DNMT1 inhibitor, GSK-3484862. We aimed to assess whether GSK-3484862 induces demethylation more effectively than 5-azacytidine and decitabine. Murine embryonic stem cells (mESCs) are an ideal model for this experiment due to their high DNA methylation levels and tolerance for substantial methylation loss.
Results: We evaluated the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) and Dnmt1/3a/3b triple knockout (TKO) mESCs with various concentrations of the compound from two commercial sources. We found that concentrations of 10 µM or lower were well tolerated after 14 days of culture. Known DNA methylation targets, including germline genes and GLN-family transposons, were upregulated within 2 days of GSK-3484862 treatment. In contrast, 5-azacytidine and decitabine resulted in weaker gene upregulation and significant cell death. Whole-genome bisulfite sequencing revealed that GSK-3484862 treatment induced substantial DNA methylation loss, with global CpG methylation levels dropping from nearly 70% in WT mESCs to under 18% after 6 days of treatment. The methylation patterns in treated cells closely resembled those observed in Dnmt1-deficient mESCs.
Conclusions: GSK-3484862 induces significant demethylation in mESCs with minimal non-specific toxicity, offering a promising alternative to traditional covalent DNMT1 inhibitors.