Induction, Prediction and Impacts of APOBEC3 Mutagenesis in Cancer - APOBEC3 (A3) cytosine deaminases are major mutators in cancer. A3-induced cytosine mutations in TC dinucleotides manifest genome-wide as single base substitution (SBS) mutational signatures 'SBS2' and 'SBS13', present in >80% of tumor types and >50% of all cancers. A3 mutagenesis causally contributes to therapy resistance in several cancer types, spurring the development of A3 inhibitors to extend therapy responses and improve outcomes. Analyses of A3-related mutations indicate that A3 mutagenesis frequently persists in metastasis and may fuel tumor evolution, potentially contributing to recurrence and resistance in many tumor types. However, the mutagenic activities of individual A3 enzymes in tumors and models, which often express multiple A3 enzymes, have been largely inferred from suboptimal surrogate readouts. As a result, the exact sources and driving mechanisms of A3 mutagenesis remain largely unknown, hindering the identification of its biomarkers and broader impacts on therapy resistance - critical for identifying patients who might benefit from A3 inhibition. We have developed an experimental strategy supported by a bioinformatics framework to track the de novo generation of SBS2/13 induced by individual A3 enzymes in cancer cell lines, moving beyond reliance on surrogate readouts. Our discoveries demonstrated that SBS2/13 signatures arise during episodic mutagenic events followed by silent periods within individual cell lineages. We subsequently found that APOBEC3A (A3A) is a primary cause of these events, with a minor contribution from APOBEC3B (A3B). These findings established the first causal links between endogenous A3 deaminases and SBS2/13 signatures in cancer and, supported by data from clinical samples, resolved the debate over the major A3 mutator in cancer. Our preliminary data, along with expanded tools to study A3 misregulation, provide key insights into the unknown endogenous drivers of episodic A3A misregulation and poorly defined prevalence and mechanisms of A3A and A3B induction by certain standard-of-care therapies. In Aim 1, we propose to define the unknown endogenous triggers and biomarkers of episodic A3A misregulation in select tumor types that frequently present with SBS2/13. We will exploit large-scale analyses of accumulated tumor molecular datasets, coupled with in vitro examinations using our new tools for assessing A3A misregulation. Aim 2 seeks to determine the unknown impacts of A3A and A3B induction by certain common drugs on mutagenesis and resistance, and whether this induction can be predicted in tumors. Building on our findings, we will combine studies in preclinical in vitro and in vivo models with large-scale analyses of A3- related mutations in available data from metastatic tumors with annotated treatments. This research will provide a comprehensive understanding of A3 misregulation, defining its mechanisms, biomarkers, and impacts, to support future investigations into the benefits of targeting A3A and A3B using inhibitors in development. Although most experiments will be performed in human ex vivo models, a limited number of CDX studies in mice are essential to evaluate findings in an integrated physiological environment that cannot be fully recapitulated in vitro. Tumor resistance depends on interactions between cancer and stromal cells, and drug effects on A3 activity are influenced by organism-level pharmacokinetics, metabolism, and physiology. These processes can only be accurately modeled in vivo.
