ABSTRACT Mutational signatures represent a footprint of tumor evolution and its endogenous and exogenous mutational processes. However, their functional impact on the proteome remains incompletely understood. We analysed the protein-coding impact of single base substitution signatures in 12,341 cancer genomes from 18 cancer types. Stop-gain mutations (SGMs) were strongly enriched in the signatures of tobacco smoking, APOBEC cytidine deaminases, and reactive oxygen species. These mutational processes affect specific trinucleotide contexts to substitute serine and glutamic acid residues with stop codons. SGMs are enriched in cancer hallmark pathways and tumor suppressors such as TP53, FAT1 , and APC . Tobacco-driven SGMs in lung cancer correlate with lifetime smoking history and highlight a preventable determinant of these harmful mutations. Our study exposes SGM expansion as a genetic mechanism by which endogenous and carcinogenic mutational processes contribute to protein loss-of-function, oncogenesis, and tumor heterogeneity, providing potential translational and mechanistic insights.

Mutational processes of tobacco smoking and APOBEC activity generate protein-truncating mutations in cancer genomes