PROJECT SUMMARY Gross chromosomal rearrangements (GCRs), a type of genome instability, are often seen in inherited and sporadic cancers and are an important driver of malignant progression. For example, hereditary breast and ovarian cancers resulting from BRCA1 and BRCA2 germline mutations suffer from homologous recombination repair (HRR) defects that increase GCRs in model systems. Our previous studies have established a comprehensive network of genome instability suppressor (GIS) genes in yeast and demonstrated in silico that human homologs of these yeast GIS genes are genetically and/or epigenetically altered across many cancer types. From yeast genetic studies, we have also identified and rank-ordered synthetic lethal (SL) partners of non-essential GIS genes. The feasibility of targeting SL genetic interactions for rational cancer therapy has been supported by the application of PARP inhibitors for maintenance therapy of breast and ovarian cancers with BRCA1 or BRCA2 defects. To exploit additional SL interactions for cancer therapy, this proposal aims to leverage the wealth of knowledge on GIS genes and their SL partners developed in yeast to guide the development of therapeutics that target human GIS gene defects that cause GCRs in cancer. Specifically, the proposed studies will focus on the human Flap Endonuclease 1 (FEN1), the homolog of yeast RAD27, which has the highest number of SL-interactions with GIS genes of a variety of functions. FEN1 processes Okazaki fragments during lagging strand DNA synthesis and acts in long-patch base excision repair but is itself non- essential. To develop this nuclease as a target for treating cancers with defects in BRCA1, BRCA2 and other FEN1-SL partner GIS genes, we propose to carry out the following lines of research: AIM 1) To expand ongoing CRISPR-dropout screens and validation studies in cell lines and mice to (a) identify human genes in which defects cause sensitivity to our proprietary FEN1-inhibitors of highly potency and specificity and (b) define FEN1 SL-partner genes as well as cancer omics signatures that can be targeted with FEN1 inhibitors to induce SL; AIM 2) To combine informatics and cell-based validation approaches for a deep-dive into cancer omics and gene essentiality data to identify the spectrum and signatures of cancers amenable to therapeutic targeting with FEN1 inhibitors; AIM 3) To apply an array of genetics-based approaches to investigate mechanisms for acquired resistance to FEN1 inhibitors and to compare resistance to FEN1 versus PARP inhibitors; and, AIM 4) To determine the effects of FEN1 inhibition on DNA replication, fork stability, and chromosome integrity in BRCA1 and BRCA2 mutant cells to test the hypothesis that FEN1 inhibitors induce irreparable damage to replication forks in HRR-deficient cancer cells to cause lethality. These mechanistic studies will be extended to other validated FEN1-SL partner genes. These projects will greatly accelerate the development of FEN...