Project Summary Genomic instability is a hallmark of cancer. The genomic instability in cancer cells arises from multiple sources, such as defects in cell-cycle control, DNA replication, and DNA repair. Recent cancer genomics studies have revealed that the mutation signatures in different cancers are distinct, reflecting the different types of oncogenic stress that cancer cells have experienced during the process of tumorigenesis. In particular, the mutation signatures associated with APOBEC3A/B cytosine deaminases are prevalent in several cancer types, suggesting that these APOBEC proteins are important drivers of mutation in a subset of cancers. Interestingly, recent studies have suggested that APOBEC3A/B proteins may act during DNA replication on the lagging strand of replication forks, raising a question as to whether APOBECs induce replication stress in cancer cells. In our preliminary studies, we have found evidence that APOBEC3A/B, through their cytosine deaminase activity, induce a unique type of replication stress in cancer cells and activate the ATR-mediated replication stress response. Furthermore, we found that cancer cells harboring high APOBEC activity are increasingly sensitive to ATR inhibition. These findings have led us to hypothesize that APOBECs impose a unique type of “mutator- driven” replication stress in cancer cells, which renders the cancer cells harboring high APOBEC activity susceptible to ATR inhibition. In our proposed studies, we will systematically investigate how APOBECs induce replication stress, how ATR suppresses the APOBEC-induced genomic instability, and how we can specifically target the unique APOBEC stress in cancer cells. The completion of these studies may provide a new view of the replication stress in cancer cells, and bring about a novel therapeutic strategy that may significantly improve the treatment of several cancer types.