Cancer cells lacking functional BRCA1 have a profound defect in HDR that leads to genomic instability and renders them exquisitely sensitive to PARP inhibitors (PARPi) and interstrand crosslinkers (ICLs). There is great interest in using these agents to treat a larger spectrum of sporadic cancers with functional defects in DNA repair pathways. However, even BRCA1-mutant cancers can rapidly develop resistance to PARPi and ICLs leading to tumor progression. Understanding the molecular mechanism underlying the development of chemoresistance in cancers with defined defects in DNA repair will be critical for developing methods to overcome resistance and improve the outcome of patients with these cancers. We have recently identified an unexpected mechanism of chemo-resistance in BRCA1-mutant cells. Loss of the DNA- repair and checkpoint protein 53BP1 reverses the DNA repair defect present in BRCA1-mutant cells and induces resistance to both PARPi and cisplatin. Subsets of both hereditary BRCA1-associated breast cancers and sporadic BRCA1-like breast cancers show loss of 53BP1 expression. Moreover, loss of 53BP1 is associated with poor clinical outcome in these cancers. We hypothesize that abnormalities in 53BP1 function will alleviate the defect in homology-mediated DNA repair present in BRCA1-mutant and BRCA1-like cancers, and lead to chemo-resistance. To investigate this hypothesis we will: 1) Determine the functional elements of 53BP1 required to maintain the HDR defect present in BRCA1-mutant cells. 2) Determine the role of interacting partners of 53BP1 in regulating the DNA repair defect in BRCA1-mutant cells and 3) Determine whether or not acquired loss of 53BP1 in sporadic cancers is a compensating mutation for an underlying defect in HDR. The first two aims will be achieved through the analysis of genetically defined sets of murine breast cancer cell lines with clinically relevant null mutations in BRCA1. Several of these cell lines hav acquired chemo-resistance through somatic mutations in 53BP1. The role of DNA factors upstream and downstream of 53BP1 in regulating the DNA repair phenotype in Brca1-/- cell lines will be carefully defined and validated through use of specific DNA repair assays. Aim 3 will focus on the role of 53BP1 in regulating the DNA repair phenotype in the more common sporadic breast cancers in which BRCA1 is genetically intact but functionally impaired. Here human basal-like cell lines identified to have defects in 53BP1 function will be systematically analyzed to determine the molecular mechanism of 53BP1 dysfunction, and its effect on DNA repair and chemo-resistance. Any findings in cell lines will be validated in clinically annotated sets of human cancer specimens. By this approach we will gain a new understanding of how 53BP1 function impacts the DNA repair phenotype and sensitivity of both hereditary BRCA1-mutant and sporadic BRCA1-like cancers to PARPi and ICL chemotherapeutic agents. This will ultimately lead to new approaches to overcome resistance and specifically target these poor-prognosis cancers. PUBLIC HEALTH RELEVANCE: Human cancers with loss of BRCA1 function can be initially successfully treated with PARP inhibitors and certain chemotherapeutic agents such as cisplatin, but resistance can rapidly develop. In this project we aim to define how abnormalities in 53BP1 can alter the DNA repair mechanisms present in theses cancer and lead to chemoresistance. This will lead to new insight into the regulation of DNA repair mechanisms in human cancer with the ultimate aim of developing methods to overcome resistance and improve outcome.