PROJECT SUMMARY Mutations arise as a result of exogenous and endogenous processes that leave characteristic imprints or signatures upon the genome. Systematic analysis of these mutational signatures led to the identification of >50 distinct types of single base substitutions (SBS) in human cancer genomes. Revealing the origins of individual signatures is critical for understanding cancer etiology, with potential implications for cancer prevention and therapy. Two of the most prevalent mutational signatures in cancer, termed SBS2 and SBS13, are present in >78% of cancer types and 56% of all cancer genomes, with a particular prominence in breast, bladder, and lung cancers. SBS2 and SBS13 are proposed to be caused by the endogenous APOBEC3 (A3) enzymes, which target ssDNA and RNA of viruses and retroelements as part of the innate immune defense. Correlations between A3 expression, driver gene mutations in A3-preferred contexts, and clinical outcomes suggest that A3 mutagenesis may play important roles in cancer etiology and evolution. Thus, there is strong rationale to understand the mechanisms of A3 activity. However, reliance on engineered model systems and correlative data have caused links between A3 enzymes, mutations in cancer, and cancer etiology to be poorly understood. We have identified human cancer cell lines with endogenous A3 mutagenesis and developed a workflow that enables us to quantify contributions of individual A3 members to mutations. Here, we propose to leverage this workflow to accomplish the following goals: 1) Identify A3 mutator enzymes in cancer types where A3 mutagenesis is prevalent and find biomarkers of their activity; 2) Investigate mechanisms modulating A3 mutagenesis; 3) Determine the functional relevance of A3 mutagenesis in therapy resistance and metastasis. Aim 1 will expand upon our characterization of human cancer cells with active A3 mutagenesis to identify A3 mutators in breast, bladder, and lung cancers. In parallel, we will directly assess the unknown specificity and sensitivity of assays to measure activities of individual A3 enzymes. These experiments may further confirm the speculative A3-etiology of a large number of cancer mutations and quantify contributions of individual A3 enzymes, thus nominating them as putative targets for therapeutic pursuit. Aim 2 builds on our preliminary data to investigate proposed modulators of A3 mutagenesis. These experiments have the potential to broaden the scope of therapeutic opportunities focused on cancer cell evolution. Aim 3 will assess the links between A3 enzymes, therapy resistance and metastasis in breast, bladder, and lung cancer cell lines. These experiments will test predictions from multi-dimensional associations that A3-mutagenesis is a disease-modifying process that can be therapeutically exploited at various stages of cancer evolution. Taken together, these studies will define the etiologies of highly prevalent mutational processes and identify strategie...