PROJECT SUMMARY Homologous recombination (HR), a pathway that repairs DNA double strand breaks (DSB), is frequently mutated in cancers. HR-deficient cancers are prone to genomic instability and are critically dependent on other DNA repair mechanisms for survival. Among them is the DNA damage sensor PARP, and PARP inhibitors have therefore proved an efficient therapy to eliminate HR-deficient cancers. However, some HR-deficient tumors do not respond to PARP inhibitors, and most tumors eventually relapse and become resistant to the drug. New therapeutic strategies are therefore urgently needed to treat HR-deficient cancers and overcome PARP inhibitor resistance. Among the potential strategies, growing attention has focused on alternative end-joining (Alt- EJ), a back-up DSB repair pathway that is dispensable in normal cells but critical to cellular survival when HR is compromised. While mounting evidence suggests that inhibition of Alt-EJ could be a powerful strategy to overcome the problem of resistance to PARP inhibitors, the Alt-EJ pathway remains poorly characterized, limiting the potential number of therapeutic targets that could be developed. To address this gap in knowledge, my lab has designed a novel approach to identify Alt-EJ factors using genome-wide CRISPR/Cas9 screens. Using this method as well as Alt-EJ assays based on repair reporters or Alt-EJ-mediated fusion of telomeres, we have identified the nuclease APE2 as a critical protein in Alt-EJ. This compelling discovery raises the exciting possibility that APE2 inhibition could be used to specifically target HR- deficient cancer cells, and thereby prevent or overcome resistance to PARP inhibitors. Several questions however need to be addressed first, as the exact function of APE2 in Alt-EJ and its role in HR-deficient cells remain elusive. First, we will characterize, at the molecular level, the function and mechanism of action of APE2 in Alt-EJ. We will determine: (1) the role of APE2 in Alt-EJ, (2) the domains and biochemical activities involved, and (3) the mechanism of its recruitment to DSBs. The interest here is both to significantly improve our fundamental understanding of the Alt-EJ repair pathway and to gain sufficient knowledge on APE2 to enable the future development of an inhibitor. Second, we will uncover the therapeutic potential of APE2 inhibition in HR-deficient cancer cells. (1) We will identify the repair function of APE2 that drives its synthetic lethality with HR, and (2) we will determine the potential of APE2 inhibition to synergize with PARP inhibitors and to prevent or overcome resistance to PARP inhibitors. Our novel discovery that APE2 plays a critical function in alternative end-joining, and the innovative tools that we have created to study APE2’s role in Alt-EJ places my lab in a unique position to successfully carry out this proposed research.