A context dependent hierarchy of APOBEC3A and APOBEC3B mutators 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 the major portion of APOBEC3-associated mutational signature burdens across cancers, the relative contributions of individual APOBEC3 paralogs in LUAD remain unclear, limiting effective targeting. Here, we define the roles of endogenously misregulated APOBEC3 deaminases in LUAD using CRISPR-Cas9 knockouts and whole-genome sequencing of 197 long-term-propagated single-cell clones. We show that APOBEC3A and APOBEC3B both generate mutations but display marked heterogeneity, ranging from single-enzyme to dual activity or minimal mutagenesis, which is not predicted by commonly used mRNA and protein levels, or deaminase assays. Using these genetically defined systems, we provide the first causal evidence that endogenous APOBEC3A and APOBEC3B generate the indel signature InD9a in tumors, linking it to uracil excision following cytosine deamination. Together, these findings reveal heterogeneous APOBEC3 contributions to LUAD mutagenesis and highlight the need for biomarkers that resolve individual APOBEC3 activities for their effective targeting.