Abstract Mortality from advanced prostate cancer remains a significant challenge facing clinicians due to the incurable nature of the disease. PARP inhibition (PARPi) is a novel strategy for the treatment of prostate cancer which exploits synthetic lethality by specifically targeting tumor cells which harbor DNA damage repair defects. PARPi, including Olaparib, represent the dawn of precision medicine for the treatment of prostate cancer and the strategy was approved in 2020. Despite this tremendous step forward, resistance to PARPi has already been clinically documented. We have developed novel PARPi resistant cell lines, LN- OlapR and 2B-OlapR, derived from LNCaP and C4-2B prostate cancer cell lines respectively. Our published and on-going studies demonstrate that PARPi’s induce DNA damage leading to p53 activation and subsequently to either cell death (cleaved-PARP) or p21-dependent senescence. RNA-sequencing of our OlapR models versus parental cells revealed a shared and significant increase in IGFBP3 expression. Preliminary data confirm overexpression of IGFBP3 in OlapR cells versus parental cells and shows that inhibition of IGFBP3 reduces OlapR cell viability, increases DNA damage, and promotes the efficacy of PARP inhibition. Our preliminary data also show that relative to parental cells, OlapR cells express significantly higher levels of phosphorylated EGFR and DNA-PKc. Targeting EGFR by either its specific EGFR siRNA or the small molecule inhibitor Gefitinib overcame olaparib resistance. The overall hypothesis is that overexpressed IGFBP3 mediates resistance by enhancing DNA repair through formation of a tripartite complex with EGFR and DNA-PKc, and blocking this signaling pathway by inhibiting EGFR activity with Gefitinib overcomes resistance to olaparib. The specific aims of this proposal are: 1. Define the role of IGFBP3 towards promoting Olaparib resistance in resistant cells. 2. Determine if IGFBP3 promotes DNA repair pathways in resistant cells. 3. Determine the anti-tumor response of targeting IGFBP3/EGFR in combination with Olaparib. This proposal will address a significant unmet research need. We will study novel models of PARPi resistance which will allow us to characterize mechanisms mediating PARPi sensitivity. We will demonstrate the role of IGFBP3 as a key mediator of resistance and we will explore novel therapeutic strategies which may be developed for clinical impact.