The majority of current cancer immunotherapy strategies target and potentiate antitumor adaptive immune responses. Unfortunately, the efficacy of these treatments has been limited to a fraction of patients within a subset of tumor types, with an aggregate response rate of approximately 20% to date across all malignancies. The success of therapeutic inhibition of programmed death protein 1 (PD-1), protein death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) with immune checkpoint inhibitors (ICI) has been limited to “hot” tumors characterized by preexisting T cell infiltration, whereas “cold” tumors, which lack T cell infiltration, have not achieved durable benefit. There are several mechanisms by which “cold” tumors fail to generate spontaneous immune infiltration, which converge upon the generation of an immunosuppressive tumor microenvironment (TME). The role of the innate immune system in tumor immunosurveillance and generation of antitumor immune responses has been long recognized. In recent years, novel strategies to target innate immunity in cancer therapy have emerged, including therapeutic stimulation of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs); the DNA sensing cGAS/STING pathway; nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRP3; and the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs). In addition, therapeutic modulation of key innate immune cell types, such as macrophages and natural killer cells, has been investigated. Herein, we review therapeutic approaches to activate innate immunity within the TME to enhance antitumor immune responses, with the goal of disease eradication in “cold” tumors. In addition, we discuss rational immune-oncology combination strategies that activate both innate and adaptive immunity, with the potential to enhance the efficacy of current immunotherapeutic approaches.

Purpose: PTEN loss-of-function occurs in ~50% of metastatic, castrate-resistant prostate cancer (mCRPC) patients, and associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss-of-function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anti-cancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Experimental design: Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic and proteomic profiling, or ex vivo co-culture studies. Single-cell RNAseq on human mCRPC samples was performed using 10X Genomics platform. Results: Co-clinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent ~3-fold increase in anti-cancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anti-cancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Conclusions: Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in PTEN-deficient mCRPC patients.

<p>Supplementary Figure S1. The majority of Pb-Cre; PTENfl/fl Trp53fl/fl mice are de novo resistant to ADT. Supplementary Figure S2. ADT/PI3Ki combination therapy halts prostate tumor growth up to 14 days, followed by development of resistance in majority of Pb-Cre; PTENfl/fl Trp53fl/fl mice. Supplementary Figure S3. PI3Ki treatment with concurrent androgen depletion does not alter proliferation and survival of PTEN/p53-deficient murine PC cells in vitro. Supplementary Figure S4. ADT/PI3K inhibitor combination increases MHC-II and PD-1 expression on TAM within the TME of PTEN/p53-deficient murine PC. Supplementary Figure S5. PD-1 upregulation suppresses phagocytic capacity of activated TAM. Supplementary Figure S6. Ex vivo AD + PI3Ki + PD-1 antibody treatment activates MHCIIlo TAM when co-cultured with PTEN/p53-deficient murine prostate tumor cells. Supplementary Figure S7. The addition of PD-1 blockade to androgen depletion/PI3Ki therapy does not alter phagocytic capacity of PD-1 lo macrophages. Supplementary Figure S8. The combination of androgen depletion, PI3Ki and aPD-1 blockade does not alter phagocytic checkpoint expression on PTEN/p53-deficient prostate tumor cells. Supplementary Figure S9. Androgen depletion, singly and in combination with aPD-1, did not alter phagocytosis activity of inactivated MHC-IIlo/PD-1 lo and MHC-IIlo/PD-1 hi TAM subsets. Supplementary Figure S10. Androgen depletion, not PI3Ki or aPD1, directly enhances TAM activation within the TME of PTEN/p53-deficient PC. Supplementary Figure S11. PI3Ki does not alter phagocytosis/histone lactylation status of MHC-IIlo/PD-1 lo TAM and MHC-IIlo/PD-1 hi TAM. Supplementary Figure S12. PI3Ki inhibits lactate secretion from PTEN/p53-deficient prostate tumor cells within TME. Supplementary Figure S13. Direct ex vivo treatment of TAM with PI3Ki, singly and in combination with PD-1 antibody and/or androgen depletion does not alter their histone lactylation profile. Supplementary Figure S14. ADT + PI3Ki + aPD-1 induces tumor control in 60% of Pb-Cre; PTENfl/fl TP53fl/fl mice. Supplementary Figure S15. Depletion of activated TAM abrogates anti-cancer response elicited by ADT + PI3Ki + PD-1 antibody treatment in the PTEN/p53-deficient murine prostate GEMM tumors. Supplementary Figure S16. Long-term treatment of ADT + PI3Ki + aPD-1 activates Wnt/βcatenin pathway in murine PTEN/p53-deficient GEMM-derived SC1 cells. Supplementary Figure S17. Feedback Wnt/β-catenin-pathway activation within murine PTEN/p53-deficient GEMM-derived PC cells following long-term ADT + copanlisib + aPD1 treatment suppresses phagocytosis via increased histone lactylation within bone marrow derived macrophages (BMDM).</p>

<p>Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN/p53-deficient prostate tumors.</p>

<p>Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN/p53-deficient prostate tumors.</p>