PROJECT SUMMARY The deadliest form of advanced-stage prostate cancer is treatment-resistant metastatic castration- resistant prostate cancer (mCRPC), which occurs in ~35% of patients after exposure to androgen receptor (AR)-directed therapies such as Abiraterone and Enzalutamide, resulting in a dismal median survival of only ~5.5 months. Unfortunately, detection of treatment-resistant disease typically occurs too late, when patients are already progressing on AR-directed drugs, after which they succumb rapidly to their disease. There is therefore a critical need to detect AR resistance more sensitively and earlier in the disease course. To address this, our team designed a novel cell-free DNA assay called Enhancer and Neighboring Loci of Androgen Receptor Sequencing (EnhanceAR-Seq), built upon the Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) platform, and demonstrated superb sensitivity for detecting AR gene body and enhancer locus alterations in plasma collected from mCRPC patients. Notably, we applied our EnhanceAR-Seq technology to mCRPC patient plasma and showed that the results correlate strongly with clinically verified resistance to AR-directed therapy, and significantly outperform circulating tumor cell (CTC) AR-V7 detection. Building upon this, the current proposal will evaluate whether our EnhanceAR-Seq technology can be used to predict both (Aim 1) pre-treatment and (Aim 2) on-treatment resistance to AR-directed therapy in mCRPC patients. Further, in Aim 3 we will investigate whether integrating EnhanceAR-Seq with genome-wide methylation sequencing can granularly refine our ability to predict treatment resistance and monitor both clonal and phenotypic evolution in mCRPC. We are uniquely positioned to accomplish this through our interdisciplinary team comprised of cell-free DNA genomicists, bioinformaticians, oncologists, and statisticians. Overall, this study has significant translational impact by establishing cell-free genomic and epigenomic biomarkers of tumor evolution and resistance to AR-directed therapy in men with lethal prostate cancer.