Title: Plk1 as a prognostic biomarker for prostate cancer Abstract Because androgen receptor (AR) signaling is essential for development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC), androgen signaling inhibitors (ASI) are becoming the first line treatment for CRPC. However, the limited clinical success of ASIs makes it urgent to develop new approaches to treat ASI-resistant CRPC. Ionizing radiation is another major approach to treat CRPC with limited efficacy. Olaparib, a PARP1 inhibitor, is recently developed and used to target cancers with a defect in DNA repair, such as BRCA mutations. Unfortunately, the usage of olaparib in CRPC is profoundly limited by the fact that BRCA mutations only occur in low percentages of PCa. Thus, identifying additional critical regulators that control DNA damage response (DDR) is of significance as it will identify specific patient populations who will be responsive to olaparib. The objective is to define the role of polo-like kinase 1 (Plk1) in regulating DDR and to exploit its unique impact on the efficacy of olaparib for ASI-resistant CRPC patients. The central hypothesis is that Plk1 phosphorylation of Mre11, a component of MRN (Mre11/Rad50/Nbs1) complex, and MDC1 (mediator of DNA damage checkpoint 1), leads to premature termination of DNA damage checkpoint, reduced DNA repair, thus increased olabparib efficacy based on the concept of synthetic lethality. Our data show that Plk1 directly phosphorylates Mre11, whose activation is the first step in response to DNA damage and that Plk1 phosphorylation of Mre11 leads to recovery from DNA damage checkpoint and reduced DNA repair. We also show that MDC1, a protein that further amplifies DDR signals, is a Plk1 substrate. Our hypothesis will be tested by pursuing three Specific Aims - (1) to dissect how Plk1 phosphorylation of Mre11 regulates the MRN complex; (2) to test whether Plk1 phosphorylation of MDC1 contributes to premature termination of checkpoint; and (3) to determine whether Plk1 is a prognostic biomarker for PCa. These complementary aims will be accomplished using biochemical analyses of signaling intermediates and employing both cell culture systems and genetic strategies with inducible mouse models. The rationale for the research is that it will probe the importance of Plk1 to DDR and to examine whether Plk1 can be a predictable biomarker for the efficacy of olaparib in CRPC. This contribution is significant because, if positive, the results of the proposed study will support an immediate clinical trial to compare the efficacy of olaparib in CRPC patients carrying different levels of Plk1. The research is innovative as it approaches the disease from a novel Plk1 signaling pathway, challenging the traditional view that Plk1 functions solely to regulate mitotic events. These studies provide a new paradigm for therapies by identifying the key regulator of DDR that is critical for the efficacy of olaparib in CRPC.