Carcinogen-induced DNA adducts amplify APOBEC mutagenesis through nucleotide excision repair

Extrinsic and intrinsic factors induce mutations in somatic cells that play a key role in cancer development. Tobacco smoke contains carcinogens that contribute to cancers of the lung and head & neck. The APOBEC family of DNA cytosine deaminases is a prominent endogenous source of mutation, generating characteristic signatures (SBS2 and SBS13) that often co-occur with the tobacco-associated signature SBS4 in tumors from smokers. Here, we challenge the prevailing view that mutational processes act independently and additively. We show that 4-nitroquinoline 1-oxide (NQO), a widely used model for tobacco carcinogenesis, sensitizes cells to APOBEC3B (A3B)-mediated mutagenesis, resulting in a significant increase in oral carcinoma development in vivo. NQO exposure markedly amplifies APOBEC-associated SBS2 mutagenesis, without a corresponding increase in NQO-associated SBS4 mutations. Moreover, a large proportion of A3B-catalyzed SBS2 mutations occur as strand-coordinated pairs within 32 nucleotides of each other, supporting a model in which nucleotide excision repair (NER) of NQO-induced DNA adducts generates short single-stranded DNA intermediates that are accessible to APOBEC-catalyzed deamination. These APOBEC mutation pairs - termed didyma (Greek for twins) - exhibit distinct genomic features consistent with a NER-initiated mechanism, differing from other types of clustered mutations (omikli and kataegis). A3B exhibits processive deamination on NER-like tracts in vitro. Didyma formation in human cell lines and yeast depends on NQO exposure, catalytically active A3B, and functional NER (XPA in human cells and Rad14 in yeast). Computational analyses of lung and head & neck tumor genomes show that both APOBEC mutagenesis and didyma are elevated in cancers from smokers compared to non-smokers. Elevated levels are also observed in normal lung tissues from smokers prior to tumor development. Additional tumor types without obvious links to smoking also exhibit didyma. Collectively, these results indicate that exogenous DNA adduct-forming carcinogens can amplify mutagenesis by endogenous APOBEC enzymes and that mutational processes can interact synergistically to shape tumor genomes during cancer initiation and progression.