Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

Inactivation of PTEN occurs frequently in advanced human prostate cancer and can promote an immunosuppressive tumor microenvironment (TME). Extracellular adenosine can be produced by tumors which can in turn modulate anti-inflammatory and immunosuppressive effects via interactions with the A2a receptor (A2aR). Here, we used a transgenic mouse model of Pten-deficient prostate cancer to characterize adenosine-mediated immunosuppression in the TME and evaluate the effects of A2aR blockade. Transcriptomic analysis of normal wildtype prostate and Pten-deficient prostate tumors revealed enriched signatures for genes downregulated after a A2aR knockdown or upregulated after A2aR stimulation. Moreover, androgen withdrawal led to increased expression of the A2aR-stimulated responsive genes Cxcl2/3/5, Il1b, S100a8, Ptgs2 and Thbs1. Immunohistochemical staining of CD73, the ecto-enzyme responsible for producing extracellular adenosine, showed heterogeneous staining ranging from negative to focally strong in epithelial cancer cells and was strongly expressed in immune cells. A2aR was also strongly expressed on immune infiltrates. We used the oral A2aR antagonist AZD4635 to determine the immunosuppressive activity of extracellular adenosine and evaluate its antitumor activity in the context of castration-naïve (CNPC), castration-sensitive (CSPC) and castration-resistant (CRPC) prostate cancer. Four weeks of treatment with AZD4635 led to a 12.1% (P=0.140) reduction of tumor burden compared to control mice. Notably a subset of these mice (4/8) had tumor reductions greater than 20%. For the CSPC/CRPC models, mice were orchidectomized and treated with apalutamide with or without AZD4635 for four or eight weeks to represent CSPC or CRPC phenotypes, respectively. In these settings, treatment with AZD4635 improved the reduction of tumor burden by 16.4% (P=0.165) and 16.8% (P=0.123) in CSPC and CRPC, respectively. Despite similar reduction rates between the cohorts, a greater proportion of AZD4635 treated mice were below the median distribution in the CRPC setting compared to the CSPC setting, 50% (4/8) vs 70% (7/10), respectively. Immunophenotyping showed an increase in the abundance of dendritic cells in tumors and tumor draining lymph nodes of AZD4635-treated mice in the CPNC setting. Whereas a shift in M2 to M1 macrophage, decrease of PD1hi CD8 T cells and an increase in genes related to cytotoxic lymphocyte activity (Gzma, Gzmb, Prf1, Klrg1, and B3gat1) were observed in AZDD4635 treated mice in the CRPC setting. Decreased expressions of immunosuppressive genes related to T regulatory cells (Ctla4, Il2ra, Itga2, Tigit, Foxp3) were noted in both CSPC and CRPC settings. Our findings show that targeting extracellular adenosine with AZD4635 is effective in a subset of mouse Pten-deficient tumors and provides evidence that suggests context-specific immune modulating activity by extracellular adenosine in prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Alwin Schuller, Kazuto Nishio, Hirotsugu Uemura. Targeting A2aR in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1071.

Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents; however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.

The gut microbiome is now known to influence host immune function, malignancies, and response to therapy. We previously showed that sequencing PD-L1/JAK1/2 blockade prior to androgen deprivation therapy (ADT) could promote immune activity to potentiate antitumor responses in mouse Pten-deficient prostate cancer. Here, we investigate the associations between the treatment pairings of PD-L1 antibody (aPD-L1, clone D265A, mouse/IgG1 kappa) blockade and the JAK1/2 inhibitor AZD1480 with ADT in mouse Pten-deficient prostate cancer and fecal gut microbiomes. We used 16S rRNA amplicon sequencing to survey fecal samples of tumor bearing conditional Pten-knockout (KO) mice treated for four weeks with aPD-L1 and AZD14870 alone and as combination therapy in intact mice, and as concurrent (Conc) therapy with ADT (via surgical castration), or sequenced as adjuvant (Adj) or neoadjuvant (NeoAdj) therapy. In this model, PD-L1 blockade was ineffective as monotherapy in all treatment settings and as combination therapy in intact and Conc combination settings and hyperprogression was observed in some mice treated with aPD-L1 monotherapy in intact and Adj settings. In the NeoAdj setting, aPDL1/AZD1480 therapy demonstrated superiority over monotherapy. Significant differences in microbial composition were observed between groups (P<0.001), drugs (P<0.001), castration status (P<0.001) and treatment responses (P<0.001). AZD1480 as monotherapy or in combination with aPD-L1 was associated with the greatest changes in microbial composition followed by surgical castration. In the neoadjuvant setting Allobaculum, Lactobacillus, Sutturella, Turcibacter and Prevotella were associated with responders whereas Hellicobacter, Oscillospora, and Ruminococcus gnavus were associated with hyperprogression. Functionally, enrichments in fatty acid biosynthesis/metabolism, tetracycline biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and glycolysis/gluconeogenesis were revealed in responders whereas lipopolysaccharide biosynthesis, glycosaminoglycan degradation, fructose and mannose metabolism, histidine metabolism, glyoxylate and dicarboxylate metabolism, and the citrate cycle (TCA) were enriched in low responders. This study provides insight into the complex interactions between gut microbiota and cancer-burdened hosts, and reveals associations between gut microbial composition and treatment responses to androgen deprivation, immune modulation via JAK1/2 inhibition and PD-L1 blockade in a mouse model of Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1780.

Commensal gut bacteria are essential for maintaining intestinal homeostasis, however, aging, environmental factors and pathological conditions can cause changes in microbial composition resulting in dysbiosis. Specific microbial communities can alter host immune functions and are implicated in promoting carcinogenesis, tumor progression and therapeutic resistance. In this study we use a genetically engineered mouse model of prostate cancer to characterize changes in gut microbiota. 16S marker sequencing data was used to perform a comprehensive profile of the fecal microbiomes of conditional Pten-knockout mice harboring prostate tumors versus wildtype mice with normal prostate. Unsupervised clustering of microbiota was performed by hierarchical clustering. Community composition (beta diversity) was determine by principal components analysis, principal coordinates analysis (PCoA) and Adonis (permutational manova (PERMANOVA)) and the Shannon index was used to determine alpha diversity. The linear discriminant analysis effect size method was used to identify features associated with cancer and multiple linear regression was performed to determine relevant features of fecal bacteria collected from the proximal and distal colon. Predictive biomarkers associated with prostate cancer were identified with the Wilcoxon rank test. Weighted correlation network analysis (WGCNA) was performed to identify clusters of taxa associated with the presence of prostate cancer. Taxon set enrichment analysis (TSEA) was carried out to identify taxon sets associated with host genetic variations, and host intrinsic and extrinsic factors. Overall, the presence of prostate cancer did not affect diversity, however, significant compositional differences were observed between the cohorts (Bray-Curtis Adonis, P=0.003). At the family level S24_7, Odoribacteraceae, and Peptococcaceae were associated with cancer bearing mice while Bifidobacteriaceae, Peptostreptococcaceae, Coriobacteriaceae, Ruminococcaceae and Erysipelotrichaceae were associated with healthy mice (P<0.05). Prevotella, Lactobacillus reuteri and various unclassified Clostridiales, S24_7 and Rikenellaceae were identified as predictive biomarkers associated with cancer. Clusters of highly correlated taxa were identified in both cohorts and their functional influences are summarized. Overall, our study provides an in-depth overview of fecal microbiome in the context of mouse prostate cancer and provides the foundation to further investigate the interactions with disease progression and cancer therapy. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sako, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Prostate cancer alters gut microbiota in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3340.