Heterogeneous contributions of APOBEC3 deaminases to substitution and indel mutagenesis in lung adenocarcinoma

APOBEC3 cytosine deaminases are major sources of cancer mutations. In lung adenocarcinoma (LUAD), APOBEC3 activity drives tumor evolution and therapy resistance, nominating these enzymes as therapeutic targets. Although APOBEC3A drives most APOBEC3-associated mutational signature burden across cancers, the relative contributions of individual paralogs in LUAD remain unclear. Here, we define the roles of endogenously misregulated APOBEC3 deaminases using CRISPR-Cas9 knockouts and whole-genome sequencing of 197 long-term propagated single-cell clones. APOBEC3 mutagenesis falls into three patterns, driven predominantly by APOBEC3A, by both APOBEC3A and APOBEC3B, or minimal despite detectable enzyme expression. Commonly used surrogate measures, including mRNA and protein levels and in vitro deaminase activity, do not predict these contributions. We also provide the first causal evidence that endogenous APOBEC3A generates the indel signature InD9a, linking it to uracil excision following cytosine deamination. These heterogeneous contributions highlight the need for biomarkers that resolve individual APOBEC3 activities to guide therapeutic targeting.