GSE123928
DNA methylation analysis of melanoma cell lines SK28 and MEL501
Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment.  In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints.  As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed.  We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation.  Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, alters the transcriptome and methylome, and impairs xenograft tumor formation.  Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity.  These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target.  By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy. Overall design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SK28 and MEL501 melanoma cells with TDG knockdown by lentiviral shRNA and empty vector control, both in triplicate.  Briefly, genomic DNA was sequentially digested by SmaI and XmaI, which both recognize the sequence CCCGGG. SmaI is methylation sensitive, where XmaI is methylation insensitive. Distinct signatures, 5'-GGG at unmethylated sites or 5'-CCGGG at methylated sites were created by enzyme digestion and ultimately high througput sequencing was used to map these sites to the genome. Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts, and subsequently adjusted for differences in restriction enzyme efficiency using values obtained from spiked in standards.