PROJECT SUMMARY/ABSTRACT The overarching scientific premise of our parent grant is to thoroughly investigate the impact of BRCA2 and DNA damage repair (DDR) gene alterations on the progression of lethal, untreatable prostate cancer. Our aim is to identify and develop innovative combination treatments, such as targeted immunotherapy and radiotherapy for BRCA2 deficient prostate cancer patients. However, the impact of alterations in non-BRCA2 HRR genes on mCRPC progression is not well understood. In the proposed supplemental project, we aim to elucidate the molecular mechanisms underlying the impact of non-BRCA2 DDR alteration on mCRPC progression. We will use CRISPR to generate isogenic pairs of prostate cells and organoids by individually eliminating 14 non-BRCA2 DDR genes. We will perform knockout efficiency and assess the impact of individual non-BRCA2 DDR genes on HRR alteration using Sanger sequencing and DNA damage assays, respectively. Using these models, we will evaluate whether the loss or elimination of any of these non-BRCA2 DDR genes can effectively promote castration-resistant prostate cancer progression. Transcriptomic analysis from the isogenic pairs of cells will help us discover targetable signaling pathways and develop novel therapeutics for lethal prostate cancer. Recent studies have also highlighted that inhibiting the androgen receptor (AR), such as with enzalutamide, increases the risk of DNA damage by disrupting the protective effects of androgens on genomic stability. Our team presented the first report of the loss of KDM5D, a chromosome Y histone demethylase and androgen receptor (AR) interacting protein, which augments prostate tumor growth and castration resistance. We also showed that the loss of KDM5D leads to replication stress due to impaired DNA damage repair. We aim to investigate the molecular mechanisms underlying the AR-KDM5D pathway-mediated DNA damage repair, which contributes to the progression of prostate cancer to a lethal state. Our research has identified that the loss of KDM5D leads to the activation of ATR, a pivotal DNA damage sensor gene. Based on this finding, our study aims to explore the effects of combining AR inhibitors with ATR inhibitors on the progression of prostate cancer, particularly in cases where KDM5D is lost. This proposal presents a unique opportunity for mentorship and training in functional genomics, molecular biology, and prostate cancer research. It offers the candidate valuable career development prospects. Additionally, the project allows the candidate to utilize extensive experimental model systems and access unique resources from the parent grant. These resources are essential for addressing the significant research gap regarding the molecular mechanisms underlying DDR deficiency-induced lethal therapy resistance in the progression of prostate cancer. Ultimately, this research aims to identify novel therapeutic targets for combatting this deadly disease.