Single-nucleotide-resolution genomic maps of O6-methylguanine from the glioblastoma drug temozolomide

Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to cell cycle arrest and apoptosis. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Characterizing genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT. O6-MeG signatures were highly similar to mutational signatures from patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes in TMZ-exposed cells and naked DNA proposing an indirect influence of transcription to O6-MeG formation. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and nucleotide sequence. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers. Overall design: We used a novel DNA modification sequencing stragegy, O6-MeG-seq, to assess O6-methylguanine in LN-229 cells exposed to temozolomide. We used an MGMT-transfected LN-229 cell line and the transfection control cell line further referred to as wild-type (WT). They were exposed to temozolomide or solvent control once (1x) or repetitively (3x). Additionally, naked DNA from LN-229 WT and MGMT-transfected cells was exposed to temozolomide.There are 3 biological replicates per condition