Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes.

SUMMARY Targeted cancer therapeutics select for drug-resistant rescue subclones (RSCs), which typically carry rescue mutations that restore oncogenic signaling. Whereas mutations underlying antibiotic resistance frequently burden drug-naive microbes with a fitness cost, it remains unknown whether and how rescue mutations underlying cancer relapse encounter negative selection prior to targeted therapy. Here, using mouse models of reversible, Wnt-driven mam-mary cancer, we uncovered stringent counter-selection against Wnt signaling overdose during the clonal evolution of RSCs. Analyzing recurrent tumors emerging during simulated targeted therapy (Wnt withdrawal) by multi-region DNA sequencing revealed polyclonal relapses comprised of multiple RSCs, which bear distinct but functionally equivalent rescue mutations that converge on sub-maximal Wnt pathway activation. When superimposed on native (i.e., undrugged) signaling, these rescue mutations faced negative selection, indicating that they burden RSCs with a fitness cost before Wnt withdrawal unmasks their selective advantage. Exploiting collateral sensitivity to oncogene overdose may help eliminate RSCs and prevent cancer relapse.