PROJECT SUMMARY Tumor heterogeneity is the main cause of resistance to current chemotherapy drugs as well as metastasis development, leading to patients' death. Within the same tumor from the same patient, tumor cells might be subtly or even dramatically different, making it harder to treat clinically. Understanding mechanisms driving cancer diversity is a critical step toward developing new strategies to attenuate tumor evolution and adaptation. Genomic instability is a prominent source of genetic diversity within tumors, generating a cell population subject to potential selection from a micro-environmental or therapeutic context. In recent years, next- generation sequencing technologies have begun to identify genomic signatures of DNA damage and errors in DNA repair processes, revealing new mechanisms causing an accumulation of mutations in cancer genomes. From the 30 mutational signatures identified across many cancer types to date, one is particularly dominant: the APOBEC signature. APOBEC3A (A3A) and APOBEC3B (A3B), two members of the APOBEC3 family, target TpC motifs on single-stranded DNA and are the major sources of the APOBEC mutational signature detected in patients' tumor samples. Our preliminary observation identified a discrepancy between A3A and A3B expression and mutation accumulation in cancer cells. On one hand, A3A is rarely found expressed, yet many of the tumors have a strong A3A-mutational signature. On the other hand, A3B is expressed in most cancer cells, but only a fraction has an A3B-mutational signature. Both A3A and A3B significantly increase mutations in tumors, but these observations have led us to propose that A3A and A3B expression is not a reliable way to assess the APOBEC status of cancer cells, as previously thought. We propose that A3A is tightly regulated at the transcription level and transiently expressed to generate mutations. Our study will explain why A3A is rarely found expressed in cancer but many cancers have a strong A3A mutational signature. In contrast, we propose that A3B is regulated at the protein level to protect the genome against A3B activity. Our goal is to uncover the molecular mechanisms that govern A3A and A3B regulation in cancer cells. Our overall hypothesis is that cells exploit two separate mechanisms to regulate A3A and A3B and to protect their genome against their activity. In addition, we propose that specific signals in cancer lead to the deregulation of these protective mechanisms, causing a surge of mutations. Our Specific Aims are to 1) define signaling pathways in cancer cells that regulate A3A expression and 2) identify protein complexes controlling A3B activity in cancer cells. Our long-term goal is to develop therapeutic strategies to suppress mutations in the genome caused by A3A and A3B, leading to tumor heterogeneity, metastasis, and drug resistance.