Immune checkpoint blockade (ICB) in metastatic castrate-resistant prostate cancer (mCRPC) patients has limited efficacy. PTEN loss-of-function (LOF) occurs in >50% of mCRPC patients and is associated with de novo and acquired resistance to ICB. We have previously demonstrated that the sparse immune infiltrate within PTEN LOF PC is characterized by a lack of T-cell infiltration, a necessary substrate for ICB, and a predominance of immunosuppressive myeloid cells. The overall vision is that activation of innate immune pathways within myeloid suppressive cells in the tumor microenvironment (TME) will enhance immune-responsiveness in PTEN-LOF mCRPC. We have recently shown that cyclic GMP-AMP Synthase (c-GAS)/Stimulator of INterferon Genes (STING) pathway, which is activated in response to cytosolic double-stranded DNA double strand breaks (DSBs), is frequently silenced in cancer cells. We demonstrated that activation of the c-GAS/STING pathway within the myeloid compartment of the TME is critical for generating a robust immune infiltrate. However, effective generation of DNA DSBs is insufficient to generate an immune cell infiltrate, which is consistent with lack of response to PARP inhibition (PARPi) plus ICB observed in our murine models and clinical studies. Probing more deeply into this disconnect, we found that failed STING pathway activation within macrophages was responsible for resistance. Furthermore, PI3K activity was preventing STING pathway activation in macrophages, and PI3K inhibition (PI3Ki) plus DNA damage with PARPi led to ICB responsiveness in PTEN-proficient, but not in isogenic PTEN-knockout PC, suggesting that additional immunosuppressive mechanisms are driven by PTEN LOF. Our central hypothesis is that PTEN-LOF PCs are de novo resistant to DNA DSB sensing c-GAS/STING pathway activation, which can be overcome by direct STING agonist/PI3Ki combination, leading to ICB sensitization.

Aim 1. Elucidate the cancer cell extrinsic mechanism(s) by which PTEN-deficient PCs are de novo resistant to DNA-sensing STING pathway activation. Co-culture assays of PTEN-proficient PC cells have revealed that PARPi/PI3Ki combination therapy activates the c-GAS/STING pathway within macrophages, which is mediated via DNA DSB-containing microvesicles (MVs) released from PARPi-treated tumor cells, coupled with c-GAS de-repression within PI3Ki-treated macrophages. Critically, MVs released from PARPi-treated PTEN-knockout PC cells lack DNA DSBs, suggesting a block to DNA DSB association with MVs. In this aim, we will dissect the mechanism by which PI3K prevents STING pathway activation within myeloid suppressive cells and investigate why DNA DSBs fail to associate with MVs released from PTEN-knockout cancer cells. Collectively, these studies will elucidate the mechanistic basis for the deleterious effects of macrophage PI3K signaling and tumor PTEN LOF on STING activation within myeloid suppressive cells in the TME.

Aim 2. Assess the therapeutic impact of direct STING agonists, singly and in combination with PI3Ki, in sensitizing PTEN-deficient murine CRPC to ICB. Our immune profiling studies reveal a significant increase in MDSCs in the TME of PTEN-knockout vs. PTEN-proficient murine PC, which can be partly overcome with direct STING agonist or PI3Ki. Furthermore, STING agonist activates macrophages, CD4+ effector and CD8+ T cells in the TME of PTEN-deficient PC, relative to their PTEN-proficient counterparts. Given our findings that PI3Ki can deplete T regulatory cells within the TME, we also anticipate that the STING agonist-induced CD4+FOXP3+ T regulatory cell infiltration will be overcome by concomitant PI3Ki. The central hypothesis of this aim is that deliberate STING activation within myeloid suppressive cells using a systemic STING agonist (in combination with PI3Ki), but not indirect DNA damage-induced STING activation with PARPi/PI3Ki, will activate myeloid suppressive cells and sensitize PTEN-deficient CRPC to ICB. We will elucidate the relative anti-tumor efficacy of systemic STING agonist/PI3Ki vs. PARPi/PI3Ki combinations in PTEN-deficient murine models of CRPC, with or without ICB, which will provide proof-of-concept for the proposed clinical trial in Aim 3B.

Aim 3. Evaluate the TME in prior and planned investigator-initiated clinical trials of PARPi/ICB and STING agonist/PI3Ki/ICB, respectively, in mCRPC patients. We conducted a combination trial of rucaparib (PARPi) and nivolumab (PD-1 antibody) in mCRPC patients. Consistent with our preclinical data, we observed inadequate anti-tumor responses. Metastatic biopsy samples from this trial will be used to perform extensive functional and immunophenotypic analysis of the TME using bulk/single-cell RNA sequencing, multiplex cyclic immunofluorescence and flow cytometry. This unbiased immune profiling will assess the relatively understudied myeloid compartment and allow us to interrogate patient-derived samples to corroborate the lack of T-cell infiltration and myeloid compartment activation observed in preclinical systems. Based on our preclinical data, and an International ImmunoOncology Network collaboration with Bristol Myers Squibb, we also plan to conduct a phase 1/2 clinical trial of BMS-986301 (systemic STING agonist), copanlisib (PI3Ki), and nivolumab in mCRPC patients, and will correlate response with PTEN status. These co-clinical studies will provide the mechanistic foundation for the next wave of immunotherapeutic strategies to eradicate PTEN-deficient mCRPC.

The strength of this project lies in our ability to conduct co-clinical investigation of response/resistance mechanisms for immuno-oncology (IO) combinations in murine CRPC models outlined in Aims 1 and 2, with parallel interrogation of these mechanisms in mCRPC patient biopsy samples obtained in Aim 3. Furthermore, we will elucidate the anti-tumor mechanism and efficacy of novel STING agonist-based IO combination strategies in murine PTEN-deficient CRPC models and human mCRPC clinical trials in Aims 2 and 3, respectively, thus providing the mechanistic foundation for the next wave of clinical trials. Taken together, these studies will lead to the clinical development of novel IO combination therapies to eradicate PTEN-deficient mCRPC.

All required software will be provided by lab/mentor.

The student will have the opportunity to present in research laboratory meetings, Hematology/Oncology seminar series and external national and international conferences.