Immune checkpoint inhibitors (ICI) are active in only 10-25% of metastatic, castrate-resistant prostate cancer (mCRPC) patients. Our co-clinical tumor microenvironment (TME) immune profiling studies in mCRPC patients and murine PC models demonstrated sparse immune infiltrates, with predominance of immunosuppressive myeloid cells, particularly tumor-associated macrophages (TAM). Therefore, therapeutic strategies to reprogram TAM to overcome immunosuppression within the metastatic prostate TME represents an area of critical unmet need. NLR family pyrin domain containing 3 (NLRP3) is an intracellular receptor of the NLR pattern recognition receptor family that results in activation of inflammatory cytokines and pyroptosis, a form of inflammatory cell death. We have demonstrated significantly higher NOD-like receptor 3 (NLRP3) expression within TAM of bone metastatic samples from PC patients on ADT -/+ standard-of-care therapies, relative to primary PC, other metastatic cancer types and benign bone marrow controls. Consistent with our findings in human PC bone metastatic samples, we observed high NLRP3 expression within activated TAM in the immunotherapy-refractory murine syngeneic subcutaneous c-Myc driven PC model.

To evaluate the therapeutic impact of NLRP3 activation in PC, we treated c-myc driven murine PC model with BMS-392959 (NLRP3 agonist, NLRP3a), which induced a tumor cell extrinsic, TAM-dependent anti-cancer innate immune response, resulting in tumor growth inhibition. Importantly, addition of androgen deprivation therapy (ADT) led to complete tumor clearance in 55% of mice and significantly prolonged survival, relative to ADT or BMS-392959 monotherapy. Ex vivo and in vitro mechanistic studies in murine tumor and bone metastatic PC samples revealed that BMS-392959 enhanced M2-to-M1 macrophage polarization and phagocytosis of tumor cells. To determine the mechanism by which ADT enhances anti-tumor efficacy of NLRP3 agonist, we performed bulk RNAseq analysis on data collected from Stand Up to Cancer West Coast Dream Team (n=99 patients, NCT02432001), which demonstrated a negative correlation between AR and inflammasome activity within the TME of metastatic PC patients. Consistent with human metastatic PC RNAseq analysis, murine in vivo and ex vivo studies demonstrated that androgen depletion enhanced NLRP3 expression, and TAM-mediated phagocytosis of tumor cells in combination with BMS-392959, relative to monotherapy controls. Collectively, our results credential the NLRP3 inflammasome as an AR-regulated macrophage phagocytic checkpoint that can be inducibly expressed and activated in TAM following ADT and NLRP3a treatment, respectively, resulting in TAM-mediated phagocytosis and tumor control.

Our proposal hypothesis is that activation of NLRP3-mediated innate immunity will overcome TAM-mediated immunosuppression and sensitize advanced PC to ICI. To test this hypothesis, we propose the following aims. First, we will investigate NLRP3 expression and inflammasome activity across immune cell lineages/stromal/tumor cells and perform spatial/nearest neighbourhood analysis within metastatic PC microenvironment, before and after ADT. Second, we will elucidate the mechanism(s) by which ADT enhances NLRP3 expression and inflammasome activation within TAM. Third, we will evaluate mechanism and therapeutic efficacy of NLRP3 agonist, singly and in combination with ADT and ICI, in syngeneic subcutaneous vs. orthotopic prostate vs. metastatic murine PC models. Collectively, the proposed studies will provide the mechanistic rationale for NLRP3 inflammasome activation-based immuno-oncology combinations for advanced PC treatment.

This is a multi-disciplinary translational research project that will employ a diverse range of methods and technologies, including single-cell RNA sequencing, flow cytometry, multiplex immunofluoresence on prostate cancer samples from patients with metastatic disease and relevant mouse models of prostate cancer. In addition, the student will carry out mechanistic and therapeutic studies in mouse models, along with relevant ex vivo and in vitro assays.

All required software for data analysis will be provided by the lab/mentor.

The student will have the opportunity to present their work in our weekly lab meetings, Hem/Onc seminar series, and poster/oral presentations at national/international conferences