Background: Glutaminase (GLS) levels are upregulated in prostate cancer [PMID: 28138303, 16897757]. The androgen receptor signaling pathway enhances the expression of SLC43A1, SLC1A4 and SLC1A5 (amino acid and glutamine transporters) in prostate cancer cells [PMID: 22007000, 24052624, 28507054]. We evaluated prostate cancer data from The Cancer Genome Atlas and found that GLS expression correlates with Gleason sum. We performed in vitro and in vivo experiments to evaluate the effect CB-839, a glutaminase inhibitor (GLS1 > GLS2), as monotherapy and in combination with other agents. Methods: We evaluated glutamine dependence of 3 prostate cancer cell lines [parental LNCaP (P0, hormone-sensitive), LNCaP-derived castrate resistant (CRPC) variant (P1, PMID: 34575033), and PC-3 (CRPC)] using media with and without glutamine, as well as clonogenic assays in regular media with BPTES (GLS1 inhibitor as a control) and CB-839. Combinatorial treatment and dose response studies were performed with CB-839 plus enzalutamide (ENZ), everolimus (EVE), or olaparib (OLA). We used NOD SCID female nude mice carrying P1 and PC3 prostate tumor xenografts to evaluate effects of CB-839 monotherapy, CB-839 + EVE, and EVE monotherapy. Results: P0, P1, and PC-3 all exhibited glutamine dependence. In glutamine-deplete media, all tested cell lines formed < 50% of colonies from baseline. Clonogenic assays with BPTES showed IC50 of 2-6 uM. CB-839 showed single agent activity in vitro with IC50 = 1 uM (P0), IC50 = 2 (P1), and IC50 < 0.1 uM (PC-3). The IC50 of CB-839 + ENZ in P1 cells was < 4uM CB-839 + 2uM ENZ. CB-839 + OLA showed no additive effect as OLA alone was very active (IC50 < 100 nM) in P1 cells. CB-839 + EVE showed single agent and synergistic effect (IC50 < 4 uM CB-839 + 0.05 uM EVE) in both P1 and PC-3 cells. In P1 tumor xenografts, treatment response was seen in CB-839 monotherapy, EVE monotherapy, and CB-839 + EVE treatment arms. In PC-3 xenografts, treatment response was seen in all groups, with greater effect in the EVE monotherapy and CB-839 + EVE treatment groups compared with CB-839 monotherapy. Conclusions: CB-839, EVE, and CB-839 + EVE all demonstrated activity in P1 and PC-3 castrate resistant prostate cancer tumor xenografts. Additive or synergistic activity was difficult to assess with combination therapy since each drug was effective as monotherapy. Additional studies are needed to evaluate the role of EVE monotherapy or CB-839 + EVE in prostate cancer. Research Sponsors: NCI CTEP UM1 Supplement CA186688-04, NCI University of Colorado Cancer Center Support Grant 5P30CA046934-30.