The goal of this proposal is to better understand a novel signaling axis initiated in prostate cancer (PCa) patients by next-generation androgen receptor signaling inhibitors (ARSIs). The standard use of ARSIs in PCa patients help limit tumor progression, but inevitably result in the development of therapeutically resistant disease. We have identified a synthetic lethal relationship established when ARSI is combined with CD105 antagonist, carotuximab, based on the finding that ARSI promote CD105 up regulation by PCa epithelia and associated stromal fibroblasts, and bone morphogenic protein (BMP) signaling downstream of CD105 receptor signaling support multiple mechanisms of ARSI resistance. CD105 was found to be a mediator of androgen receptor splice variants, fibroblast growth factor receptor 3 (FGFR3) signaling, and treatment emergent small cell neuroendocrine (t-SCNC) that are all demonstrated to limit ARSI sensitivity. CD105 expression by PCa associated fibroblastic cells of the prostate and bone marrow also potentiated paracrine signaling by glutamine. We reported the role of glutamine as not only a metabolite supportive of tumor expansion, it was necessary and sufficient to impact t-SCNC and FGFR3 signaling. Importantly, either ARSI or carotuximab as single agents had no effect on altering tumor progression in the preclinical models tested. We recently reported on a phase 2 trial (NCT03418324) demonstrating feasibility of combining ARSI with carotuximab in re-sensitizing PCa patients to abiraterone and enzalutamide. Given these findings, we will test the hypothesis that for PCa demonstrating resistance to ARSI, the inhibition of CD105 in combination with apalutamide will provide extended sensitivity compared to apalutamide alone by the following specific aims: Aim 1 is a prospective analysis of advanced PCa patients with an ongoing randomized clinical trial receiving apalutamide in the presence and absence of CD105 inhibition (NCT05534646). During this study, we will obtain prospective blood for circulating tumor cells to understand how changes in biology relate to changes in clinical behavior. These efforts will leverage a novel nanotechnology platform originating from the innovative molecular analysis technologies program and whose current development is supported through the nanotechnology alliance in cancer. Aim 2 will reveal the mechanism of action for the apalutamide/carotuximab combination. We will use reverse translation approach to help identify the relevance of the novel glutamine-BMP/CD105 signaling axis in PCa stromal-epithelial androgen signaling and define the criteria for selecting patients who will benefit from the combination treatment. Pharmacodynamic responses in mice will be compared to that in men with castration resistant prostate cancer from pre- and post-treatment samples from the trial.