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Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen–specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell–based immunotherapy for the treatment of tumor growth and metastasis.

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Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance.Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo. Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance.Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivo.Conclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987–2001. ©2018 AACR.

Supplementary Table 3

Mishra and Sahin patent info for Conflict of Interest

This file contains supplementary methods, 7 supplementary figures and 5 supplementary tables.

Supplementary Table S1

Supplementary Figure S1-11

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity–dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer.Significance:This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

Supplementary Table S2

Supplementary Table of Antibodies.

Supplemental Figure legends

Table summarizing the sources, properties and dilutions of antibodies used in this study.

Supplementary Figure 2. Myeloid deletion of Adora2a increases lung infiltration of NK cells and antigen specific T cells in mice with melanoma.

Supplementary Figure 1. Phenotypic analysis of myeloid dendritic cells isolated from spleen (SPL) and B16F10 tumors.

Supplementary Figure 5. Effect of A2AR signaling on T cell survival and effector differentiation in B16F10 tumors.

Supplementary Figure 1. Selective expression of Cre protein and deletion of A2AR mRNA from granulocytes derived from LysMCre-AdorA2Af/f mice.

Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a−/− mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector–memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell–specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector–memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector–memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. Cancer Res; 74(24); 7239–49. ©2014 AACR.

Supplementary Figure 4. Effects of T cell-selective A2AR deletion on expression of CD69 expression on tumor and spleen lymphocytes.

Supplementary Figure 2. Effects of T cell selective A2AR-deletion on B16F10 growth.

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f–LysMCre+/− mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C+ or Ly6G+ myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8+ T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors. Cancer Res; 74(24); 7250–9. ©2014 AACR.

Supplementary Figure Legends. Legends for supplementary Figures 1 and 2.

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity–dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer. Significance: This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

Hypoxia, metabolic activity, cell death and immune responses influence the adenosine concentrations in the extracellular space. Cellular responses to hypoxia and inflammation in myeloid cells promote activation of adenosine sensing circuit, which involves increased expression of ectoenzymes that converts phospho-nucleotides such as ATP to adenosine and increased expression of G protein-coupled adenosine receptors. Adenosine sensing circuitry also involves feedforward signaling, which leads to increased expression of hypoxia-inducible factor 1-alpha (HIF1 and feedback signaling, which leads to the suppression of inflammatory transcription factor, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) activation. In this review we will discuss how different subsets of myeloid cells sense adenosine accumulation and how adenosine sensing by myeloid cells influence progression of different immune-related conditions including cancer.

Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen–specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell–based immunotherapy for the treatment of tumor growth and metastasis.

In the present study, bio-oil produced from vacuum pyrolysis of woody biomass has been investigated as a source of chemical feedstock. Bio-based resins were produced using the bio- oil with phenol substitutions ranging from 10 to 30 wt%. The conventional GC/MS analysis was carried out for the evaluation of the chemical composition of bio-oil. TGA, DSC and FT-IR analyses were used in order to characterize the bio-oil-phenol-formaldehyde (BPF) resins. The bonding quality of wood samples bonded with the BPF resins was investigated under different pre-treatment conditions. The highest shear strength was observed for the control samples bonded with the laboratory PF resin. As the amount of bio-oil was increased up to 30 wt%, the shear strength of the samples decreased from 12.08 to 11.76 N/mm2. The bonding performance was not negatively affected by the combination of bio-oil under dry conditions. According to TS EN 12765 standard, the relevant performance requirements for bonded samples under dry conditions must be at least 10 N/mm2. Relating to the standard, all samples bonded with BPF resins obtained the requirements for durability class C1. Under wet conditions, the bonding performance was negatively affected by the addition of bio-oil. However, the BPF resins fulfilled the durability requirements for C1, C2, and C3 specified in EN 12765 (2002)., U radu je predstavljeno istraživanje mogućnosti upotrebe bioulja dobivenoga vakuumskom pirolizom drvne biomase kao izvora kemijske sirovine. Biosmole su dobivene zamjenom 10 – 30 % mase (ili težinskog udjela) fenola biouljima. Analiza kemijskog sastava bioulja provedena je GC/MS metodom. Za karakterizaciju biouljnih fenolformaldehidnih smola (BPF) primijenjene su TGA, DSC i FT-IR analiza. Kvaliteta spoja uzoraka drva slijepljenih BPF smolama ispitivana je pri različitim uvjetima predobrade. Najveća čvrstoća na smicanje postignuta je na kontrolnim uzorcima lijepljenim laboratorijskim PF smolama. S povećanjem udjela bioulja do 30 % mase (ili težinskog udjela), čvrstoća na smicanje smanjila se s 12,08 na 11,76 N/mm2. Prema normi TS EN 12765, čvrstoća na smicanje u suhim uvjetima treba biti najmanje 10 N/mm2. Kombinacija bioulja s fenolformaldehidnim smolama nije negativno utjecala na svojstva slijepljenog spoja u suhim uvjetima i svi uzorci lijepljeni BPF smolama zadovoljili su zahtjeve klase trajnosti C1. U vlažnim uvjetima dodatak bioulja negativno je utjecao na svojstva slijepljenog spoja. Međutim, BPF smole ispunile su zahtjeve trajnosti za klase C1, C2 i C3 propisane normom EN 12765 (2002).

Extracellular adenosine accumulates in tumors and causes suppression of immune cells. Suppressive adenosine signaling is achieved through adenosine A2A and A2B receptors, which are Gs coupled, and their activation elevates cAMP levels. Gs-coupled GPCR signaling causes cAMP accumulation, which plays an anti-inflammatory role in immune cells. Protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are two intracellular receptors of cAMP. In this study we showed that adenosine receptor signaling polarizes activated murine dendritic cells (DCs) into a tumor-promoting suppressive phenotype. Adenosine receptor signaling activates cAMP pathway and upregulates the negative regulators of NF-κB but does not influence phosphorylation of immediate inflammatory signaling molecules downstream of TLR signaling. Pharmacologic activation of both PKA and Epac pathways by specific cAMP analogues phenocopied the effects of adenosine signaling on murine DCs, such as suppression of proinflammatory cytokines, elevation of anti-inflammatory IL-10, increased expression of regulators of NF-κB pathway, and finally suppression of T cell activation. Inhibition of effector cytokine, IL-12p40 production, and increased immunosuppressive IL-10 production by adenosine signaling is significantly reversed only when both PKA and Epac pathways were inhibited together. Adenosine signaling increased IL-10 secretion while decreasing IL-12p40 secretion in human monocyte-derived DCs. Stimulation of both PKA and Epac pathways also caused combinatorial effects in regulation of IL-12p40 secretion in human monocyte-derived DCs. Interestingly, PKA signaling alone caused similar increase in IL-10 secretion to that of adenosine signaling in human monocyte-derived DCs. Our data suggest adenosine/cAMP signaling targets both PKA/Epac pathways to fully differentiate DCs into a suppressive phenotype.

The nucleoside adenosine accumulates extracellularly in solid tumors and inhibits CD8

The nucleoside adenosine accumulates extracellularly in solid tumors and inhibits CD8(+) T cells by activating adenosine receptors. The cytokine interleukin-7 (IL-7), which is produced by various tissues and tumors, promotes the survival and maintenance of T cells. Adenosine and IL-7 signaling are being clinically targeted separately or in combination with other therapies for solid tumor indications. Here, we found that IL-7 signaling promoted the accumulation of tumor-associated CD8(+) T cells, in part, by preventing adenosine-mediated immunosuppression. Inhibition of the transcription factor FoxO1 downstream of IL-7 receptor signaling was important for protecting CD8(+) T cells from suppression by adenosine. These findings have implications for the development of new approaches for cancer immunotherapies that target the adenosine pathway.

Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance. Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo. Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance. Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivo. Conclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987–2001. ©2018 AACR.

Hyaluronan (HA) is a glycosaminoglycan and one of the major extracellular matrix (ECM) components regulating cell proliferation, differentiation and motility. Given the importance of tumor-surrounding ECM, especially in drug uptake and immune cell infiltration, investigating the expression of HA-related genes in tumors and stroma and their effects on clinical outcome of different breast cancer subtypes under treatment is critical. Here we aimed to study the association of ECM and HA-related genes with disease progression, therapy response and anti-tumor immunity by analyzing gene expression datasets of breast cancer patients. We found that high stage, high grade and ER-negative/basal types of tumors have higher mRNA levels of HA synthesizing genes and lower mRNA levels of HA-degrading genes in general. Higher expression of HA synthesizing enzymes and lower expression of HA degrading enzymes (HA accumulating subset) in combination is associated with worse survival in breast cancer patients treated with systemic therapy. Among different HAS and HYAL genes, expression of HAS2, HYAL2 and PH20 (SPAM1) demonstrate the strongest association with clinical outcome in breast cancer patients treated with systemic therapies. Cumulative expression of a set of ECM-related genes cluster patients into groups with variable clinical outcome, and critically these results are recapitulated using an HA-related gene signature. Notably, although most of the patients in the HA accumulating subset are of basal subtype, there are also a relatively large fraction of luminal patients in the HA accumulating group with worse survival. Tumor and stromal specific analyses of ECM and HA-related gene signatures demonstrated that higher “tumor-specific” expression of a group of ECM-related genes predicts better therapy response that also correlates with higher immune cell infiltration. Interestingly, these patients also have higher tumor-specific levels of HA-related gene signature. On the other hand, higher “stromal-specific” expression of a different set of ECM-related genes predicts higher tumor grade that correlates with higher stromal levels of HA-related gene signature and a low immune cell infiltration. These results suggest that tumor and stromal cells might have differential expression of ECM and HA-related genes and that while tumor-specific HA levels are associated with therapy response and immune activation, stromal HA may predict higher tumor grade and lower immune cell infiltration. Overall, we demonstrated that ECM and specifically HA-related gene signatures may predict therapy response and clinical outcome in breast cancer and suggest that modulating HA may be beneficial together with systemic therapy. Citation Format: Ozge Saatci, Caglar Cekic, Ozgur Sahin. Analyses of Hyaluronan (HA)-regulating genes and HA-related ECM signature in different breast cancer subtypes [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 1560.

Extracellular adenosine accumulates in solid tumors due to direct release or due to dephosphorylation of ATP, ADP, and AMP by extracellular enzymes such as CD39 and CD73. Adenosine signaling promotes metastasis and suppress antitumor immune responses. CD73 expression is associated with poor survival and chemotherapy resistance in triple-negative breast tumors. Adenosine deaminase 2 is a serum protein that catalyzes the deamination of adenosine to inosine. Expression of Adenosine deaminase 2 increases in pathologic conditions such as inflammation and cancer. Here we show that expression of the gene coding for ADA2 (CECR1) in lung and breast tumors is associated with increased expression of gene signatures associated with immune checkpoint blockade responsiveness. Our analyses have also indicated that increased ADA2 expression is associated with favorable outcomes among Her2+ and triple-negative breast cancer patients. Further stratification based on the expression of adenosine-generating enzymes, CD39/CD73, revealed that increased ADA2 expression is particularly associated with increased survival among patients with elevated CD39 and CD73 expression. ADA2 expression is found to be higher in stroma areas of the tumor as compared with tumor cells. Stromal expression but not tumor expression of ADA2 is associated with increased T-cell costimulation and infiltration scores. To support clinical evidence in preclinical models and to show the potential of PEGylated Adenosine Deaminase 2 (PEGADA2) as a novel therapy, we have shown that an engineered high-catalytic activity PEGADA2 variant (PEGADA2HCA) can slow growth of EMT6 syngeneic breast tumors. PEGADA2HCA also completely reversed adenosine inhibition of human T-cell activation. PEGADA2HCA treatment in humanized mice caused a linear dose-exposure relationship after repeat dosing. These results provide a strong rationale for testing PEGylated Adenosine Deaminase 2 alone or in combination in a clinical setting. Citation Format: Ozge Saatci, Luz Marina Londoño, Renee Clift, Curt Thompson, Mike LaBarre, Ozgur Sahin, Caglar Cekic. Clinical correlates and rationale for PEGylated adenosine deaminase 2 as a novel cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A84.

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f–LysMCre+/− mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C+ or Ly6G+ myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8+ T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors. Cancer Res; 74(24); 7250–9. ©2014 AACR.

Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a−/− mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector–memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell–specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector–memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector–memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. Cancer Res; 74(24); 7239–49. ©2014 AACR.

Cellular stress or apoptosis triggers the release of ATP, ADP and other nucleotides into the extracellular space. Extracellular nucleotides function as autocrine and paracrine signalling molecules by activating cell-surface P2 purinergic receptors that elicit pro-inflammatory immune responses. Over time, extracellular nucleotides are metabolized to adenosine, leading to reduced P2 signalling and increased signalling through anti-inflammatory adenosine (P1 purinergic) receptors. Here, we review how local purinergic signalling changes over time during tissue responses to injury or disease, and we discuss the potential of targeting purinergic signalling pathways for the immunotherapeutic treatment of ischaemia, organ transplantation, autoimmunity or cancer.

The accumulation of high levels of adenosine in tumors activates A(2A) and A(2B) receptors on immune cells and inhibits their ability to suppress tumor growth. Deletion of adenosine A(2A) receptors (A(2A)ARs) has been reported to activate antitumor T cells, stimulate dendritic cell (DC) function, and inhibit angiogenesis. In this study, we evaluated the effects of intermittent intratumor injection of a nonselective adenosine receptor antagonist, aminophylline (AMO; theophylline ethylenediamine) and, for the first time to our knowledge, a selective A(2B)AR antagonist, ATL801. AMO and ATL801 slowed the growth of MB49 bladder and 4T1 breast tumors in syngeneic mice and reduced by 85% metastasizes of breast cancer cells from mammary fat to lung. Based on experiments with A(2A)AR(-/-) or adenosine A(2B) receptor(-/-) mice, the effect of AMO injection was unexpectedly attributed to A(2B)AR and not to A(2A)AR blockade. AMO and ATL801 significantly increased tumor levels of IFN-gamma and the IFN-inducible chemokine CXCL10, which is a ligand for CXCR3. This was associated with an increase in activated tumor-infiltrating CXCR3(+) T cells and a decrease in endothelial cell precursors within tumors. Tumor growth inhibition by AMO or ATL801 was eliminated in CXCR3(-/-) mice and RAG1(-/-) mice that lack mature T cells. In RAG1(-/-) mice, A(2B)AR deletion enhanced CD86 expression on CD11b(-) DCs. Bone marrow chimera experiments demonstrated that CXCR3 and A(2B)AR expression on bone marrow cells is required for the antitumor effects of AMO. The data suggest that blockade of A(2B)ARs enhances DC activation and CXCR3-dependent antitumor responses. The Journal of Immunology, 2012, 188: 198-205.

Adenosine deaminase 2 (ADA2) is a human serum protein that catalyzes the reaction of deamination of adenosine to inosine. Besides catalytic activity, ADA2 has a heparin-binding domain and a growth factor-like domain. Adenosine accumulates in the tumor microenvironment and suppresses anti-tumor immune responses, while promoting tumor cell metastasis. Previously, we showed that treatment of mice with PEGylated forms of ADA2 (PEG ADA2) can decrease the accumulation of tumor-associated adenosine. Here, we analyzed the TCGA RNA-seq database using OncoLnc tool and observed that high expression of the ADA2 gene (CECR1) in certain immunotherapy responsive tumors correlated with increased survival of patients. We then tested different engineered ADA2 variants, using the inactive enzyme variant as a control, for the reversal of adenosine-mediated responses in human T cells, mouse peritoneal macrophages, and mouse and human tumor cell lines. ADA2 variants with higher catalytic activity were able to reverse adenosine-mediated suppression of human T-cell proliferation, cAMP accumulation in tumors and human T cells, and polarization of macrophages into an immunosuppressive, pro-tumor phenotype, whereas the inactive ADA2 variant failed to cause any of these effects. In addition, the variant that lacks the ability to bind to heparin but has improved catalytic activity (PEG NME8058) showed similar responses to those with the catalytically improved variant with intact heparin-binding domain (PEG NME8062). These results suggest that the improved immune cell activity with ADA2 in the presence of adenosine requires the enzyme activity of this molecule. Next, we assessed PEG NME8062 in MC38 and CT26 colon carcinoma syngeneic tumors in C57BL/6 and Balb/c mice, and observed delayed growth in both tumors. Using the inactive enzyme variant, we have shown that anti-tumor effect of PEG NME8062 depends on enzyme activity. Delayed tumor growth was also associated with increased density of key anti-tumor immune cell populations in both tumor models. These data suggest that PEG ADA2 can reduce tumor growth in an enzyme-activity-dependent manner and promote anti-tumor immune responses in vitro and in vivo. These findings have important implications for targeting purinergic signaling for cancer therapy. Citation Format: Luz Marina Londoño-R, Jessica Cowell, Lin Wang, Qiping Zhao, Lei Huang, Chris Thanos, Michael J. LaBarre, Xiaoming Li, Caglar Cekic. PEGylated adenosine deaminase (ADA2) prevents adenosine-mediated increase in tumor growth and improves antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1755.

Inhibition of proximal TCR signals by adenosine A2A receptors (A2AR) is important for T cell development and maintaining T cell quiescence in vivo. Deletion of A2ARs causes accelerated growth of B16 and MB49 tumors, which is associated with reduced T cell accumulation in tumors and decreased CD127 expression and interleukin-7 responsiveness. Here we showed that ectopic expression of CD127 in A2AR deficient mice restores normal numbers of thymic populations and IL-7 responsiveness of T cells. Ectopic expression of CD127 in A2AR deficient mice also caused accumulation of similar numbers of T cells in the periphery as compared to A2AR proficient mice. Growth of B16 tumors in A2AR deficient mice was accelerated as compared to A2AR proficient mice. However, growth of B16 tumors was comparable between A2AR deficient or proficient mice ectopically expressing CD127. Adenosine A2A receptor deletion or ectopic CD127 expression did not change Vbeta selection suggesting changes in T cell repertoire may not be involved in increased tumor growth and reduced T cell numbers in A2AR deficient mice. Interestingly, tumor growth in A2AR and CD127 double deficient mice was slower as compared to CD127 deficient mice. Finally, adenosine preconditioning of engineered T cells significantly reduced lung colonization of B16OVA tumors and persistence of engineered T cells. Overall these results suggest that CD127 regulation by adenosine is important for T cell maintenance and functional responses in vivo and blockade of A2AR to improve anti-tumor immune responses should be further promoted by providing signals for T cell survival.

Polarization of T helper subsets into different functional phenotypes strongly affects the progression of immune-related diseases. One of the hallmarks of immune cell activation and inflammation is the elevation of extracellular adenosine and increased expression of adenosine A2A receptors in activated immune cells. Adenosine strongly inhibits activation of helper T cells by elevating cAMP concentrations, which activate PKA and EPAC proteins to regulate cellular responses. Here we showed that adenosine strongly inhibits T cell accumulation rather than differentiating into functional subsets in the presence of polarizing conditions. Different aspects of adenosine mediated T cell suppression were phenocopied by specific activation of PKA and/or EPAC pathways. Adenosine can suppress phospho-activation of Akt pathway to promote T cell quiescence and to inhibit immediate downstream events after TCR stimulation. One of the targets for Akt is Foxo1. Foxo1 is known to suppress T cell proliferation. Our results showed that adenosine receptor stimulation reduced inhibitory phosphorylation of Foxo1 downstream of Akt. Foxo1 inhibitor, AS1842856, completely reversed the inhibition of T cell accumulation by adenosine signaling. Our results suggest that adenosine by activating PKA and/or EPAC pathways and by sustaining Foxo1 activation suppresses T cell activation and accumulation. These findings have important implications to develop novel interventions to regulate helper T cell responses in different pathological conditions.

Metabolic and immune related activities converge as main triggers of adenosine accumulation in extracellular space. Adenosine by engaging adenosine A2A and A2B receptors strongly suppresses innate and adaptive immune responses. Although adenosine receptors are being targeted in preclinical and clinical studies how different danger signals are regulated by adenosine is poorly understood. Here we showed that adenosine receptor stimulation strongly inhibited inflammatory responses while sparing type-I interferon responses downstream of different danger signals in dendritic cells and macrophages. Mechanistically, danger signals associated with MyD88-dependent inflammatory pathways such as LPS and CpG but not the danger signals associated with IRF3/Type-I interferon pathways such as pA:U and cGAMP increase the expression adenosine A2A and A2B receptors. Adenosine was shown to increase the expression of NR4A nuclear hormone receptors to inhibit NF-κB activation. Although adenosine did not influence NF-κB phosphoactivation expression of anti-inflammatory NR4A1 was increased after adenosine receptor stimulation in the presence of TLR ligands known to activate MyD88 pathway but not in the presence of cGAMP and pA:U. Overall these results indicate that there is a differential modulation of danger signaling by adenosine rather than overall supression. Our results have important implications for developing combinatorial approaches to target adenosine and danger signaling pathways to cure immune-related diseases.

Activation of immune cells is associated with increased expression of adenosine receptors. Extracellular adenosine is present in primary and secondary lymphoid organs and limit the amplitude of immune responses suggesting that antagonists for adenosine receptors can be targeted to improve the efficacy of vaccines. Here we showed that vaccine adjuvant Monophosphoryl Lipid A (MPLA) strongly increased adenosine A2A and adenosine A2B receptor expression in primary macrophages and dendritic cells. Adenosine A2A but not adenosine A2B receptor blockade significantly increased both numbers and percentages of antigen specific endogenous T cells in both spleen and draining lymph nodes after primary immunizations with MPLA and ovalbumin. We observed a similar increase in both endogenous or adoptively transferred T cells in mice with myeloid-specific deletion of A2A receptors after primary immunization. Adenosine receptor blockade significantly increased the total IgG titers and IgG2c/IgG1 ratio after rechallenge with ovalbumin. These results suggest that adenosine A2A receptor blockade potentially improve vaccine efficacy by promoting both cellular and humoral immune responses and promoting Th1 type immunity. Our results also suggest that A2ARs on antigen presenting cells are important targets to improve vaccine efficacy by adenosine A2A receptor blockade. These findings have important implications for the design of novel and more efficacious vaccine formulations.

Extracellular adenosine concentrations and expression adenosine receptors are elevated after cell death, injury and inflammation. Antigen presenting cells are one of the primary targets for adenosine to suppress T and NK cell responses. However, molecular mechanisms for adenosine regulation of dendritic cells are poorly understood. Here we show that adenosine receptor stimulation strongly suppresses dendritic cell activation. Adenosine did not affect phosphoactivation of inflammatory signaling pathways such as MAPKs, NF-κB and IRF3. Adenosine receptor signaling increased intracellular cAMP accumulation. Specific cAMP analogs for EPAC and PKA pathways phenocopied the effects of adenosine on dendritic cells especially when used together. Adenosine-mediated suppression of inflammatory/effector cytokines is associated with increased expression of anti-inflammatory c-Fos and NR4A receptors. NR4A and cFos expression were also increased by EPAC and PKA-specific cAMP analogs. Addition of conditioned mediums from dendritic cells prestimulated with stable adenosine analog or PKA and EPAC specific cAMP analogs together inhibited the IFNγ production by activated T cells. Overall our data suggest that adenosine can potentially target both PKA and EPAC pathways and increase the expression of NR4A nuclear orphan receptors and cFos to regulate dendritic cell responses. Our findings have important implications for the development of novel therapies for immune related diseases such as cancer by targeting adenosine receptors.