Project Summary Abstract The Mre11-Rad50-Nbs1 complex is a pleiotropic sensor of DNA double strand breaks that promotes ATM signaling, cell cycle checkpoint activation, and DNA repair. We have recently shown that Mre11 is an essential component of the tumor suppressive DNA damage response (DDR) in a murine model of breast cancer. Mre11 pathway deficiency has also been reported in a significant fraction of human triple negative (estrogen/progesterone receptor negative, HER2 non-amplified) breast cancers (TNBC), which are characterized by rampant chromosomal instability and nearly universal inactivation of the p53 pathway. In this project, we will investigate a role for Mre11-mediated tumor suppression in murine models of TNBC initiated by c-Myc overexpression, Rb1 deletion, and/or p53 deficiency. In Aim 1, we will analyze the effect of Mre11 deficiency on cell cycle checkpoint activation in response to oncogene-induced DNA damage. Time lapse microscopy of DNA damage and cell cycle state transitions will be performed in preneoplastic primary mammary epithelial cells. In Aim 2, we will use single cell whole genome sequencing to quantify stochastic chromosomal aberrations that accumulate during oncogene-induced replication stress in primary mammary epithelial cells. The effect of Mre11 and/or p53 deficiency on the chromosomal instability phenotype at different stages of tumorigenesis will be analyzed. In Aim 3, we will engineer Mre11 mutations in a Rb1/p53-deficient murine TNBC model to measure effects on tumor latency. Gene expression and genomic scar signatures associated with Mre11 deficient breast cancers will be determined, and compared to signatures of Brca1 deficiency. A panel of pharmacological DDR pathway inhibitors will be tested to identify targetable synthetic lethal vulnerabilities of Mre11 deficiency. Collectively, this project will provide insight into p53-independent mechanisms of Mre11-mediated tumor suppression, and identify molecular signatures and therapeutic susceptibilities of Mre11 deficient breast cancer.