Bipolar androgen therapy (BAT) is a paradoxical approach for the treatment of castration-resistant prostate cancer (CRPC) whereby testosterone levels are rapidly cycled between supraphysiologic and castrate concentrations. Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Previous observations suggest that supraphysiological testosterone (SupraT) induce DNA double strand breaks (DSB). It has been speculated that if left unrepaired, DSBs may lead to cellular crisis and apoptosis. In this proposal, we provide novel evidence that unrepaired DSBs induced by SupraTs are routed to the autophagosomes where they activate cytoplasmic nucleic acid sensors that trigger the downstream interferon stimulated genes (ISGs) and innate immune pathways. Based on our preliminary data, we propose a novel idea that: i) Unrepaired DSBs induced by SupraTs are routed for specialized autophagic degradation termed nucleophagy; ii) SupraTs induced autophagosomal DNA can activate cytoplasmic DNA sensing pathways~ specifically the nucleic acid sensing pathway (cGAS-STING and RIG-I pathway); iii) Activation of nucleic acid sensing pathway by SupraTs would be more pronounced in prostate tumors that have DNA repair defect; and iv) BAT might activate innate and adaptive immune cells specially, in a subset of patients having DNA repair defect. In this proposal, we will determine the role of nucleic acid sensors in mediating immune signaling by SupraTs in PCa. Utilizing tumor biopsies from PCa patients receiving BAT, we will evaluate whether nucleic acid sensor mediated innate immune signaling serves as a molecular determinant of treatment response. To test our hypothesis, we will utilize several innovative tools and resources including CRISPR-Cas9 generated knockout cellular models, GeoMx Digital Spatial profiling of immune landscape in the tumor microenvironment, unique patient derived prostate cancer xenografts models, a humanized mouse models that has functional innate and adaptive immune cells, and serum and tumor biopsies from patients receiving BAT. We think these unique resources position us well to undertake the proposed work with immediate clinical impact. We have assembled a team of experts in basic and clinical prostate cancer biology and immunotherapy who will provide their unique expertise to successfully accomplish our goals. Successful completion of the proposed work will generate: a) mechanistic insights into modulation of immune response by SupraTs, b) valuable clinical insights into activation of immune cells by BAT, c) novel tumor- and serum-based markers that can be utilized for the development of biomarkers predictive of therapy response, and d) provide a rationale for strategically utilizing BAT to activate immune response and combine it with immunotherapeutics that empower the adaptive immune system such as immune checkpoint blockade and T-cell therapeutics to achieve synergy.

We have recently developed a novel and paradoxical approach to treat castration resistant prostate cancer patients by using supraphysiologic testosterone (SupraT) termed bipolar androgen therapy (BAT) as a therapy for this disease. The proposed work will provide mechanistic insights into how SupraT works at the molecular and cellular level. Successful completion of the project will inform us whether SupraT involves immune activation and immune editing of prostate tumors, an insight, which could transform the clinical management of patients with metastatic castration-resistant prostate cancer in the immediate term.