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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Cancer Immunol Res. 2014 Jul;2(7):598–605. doi: 10.1158/2326-6066.CIR-14-0075

Hostile, Hypoxia-A2-Adenosinergic Tumor Biology as the Next Barrier to the Tumor Immunologists

Michail V Sitkovsky 1, Stephen Hatfield 1, Robert Abbott 1, Bryan Belikoff 1, Dmitry Lukashev 1, Akio Ohta 1
PMCID: PMC4331061  NIHMSID: NIHMS662225  PMID: 24990240

Abstract

The hypoxia-driven and A2A or A2B adenosine receptors (A2AR/A2BR)-mediated (“Hypoxia-A2-Adenosinergic”) and T cell autonomous immunosuppression was first recognized as critical and non-redundant in protection of normal tissues from inflammatory damage and autoimmunity. However, this immunosuppressive mechanism is high-jacked by bacteria and tumors to misguidedly protect pathogens and cancerous tissues. The inhibitors of Hypoxia-A2-Adenosinergic pathway represent the conceptually novel type of immunological co-adjuvants to be combined with cancer vaccines, adoptive cell transfer and/or blockade of immunological negative regulators in order to further prolong survival and minimize side effects. In support of this approach are preclinical studies and findings that some human cancers are resistant to chemotherapies and immunotherapies due to the tumor-generated extracellular adenosine and intracellular cAMP-elevating A2AR and A2BR on anti-tumor T and NK cells. Among co-adjuvants are i) antagonists of A2AR/A2BR; ii) extracellular adenosine-degrading drugs; iii) inhibitors of adenosine generation by CD39/CD73 ecto-enzymes and iv) inhibitors of the hypoxia-HIF-1 alpha signaling. It is emphasized that even after the multi-combinatorial blockade of immunological negative regulators the anti-tumor T and NK cells would be still vulnerable to inhibition by hypoxia and A2AR and A2BR. The advantage of combining these co-adjuvants with the blockade of the CTLA4-A and/or PD-1 is in expectations of additive or even synergistic effects of targeting both immunological and physiological tumor-protecting mechanisms. Yet to be tested is the potential capacity of co-adjuvants to minimize the side effects of blockade of CTLA-4 and/or PD1 by decreasing the dose of blocking antibodies or by eliminating the need in dual blockade.

Introduction

The recent advances in using cancer vaccines, adoptive cell transfer or blockade of the negative immunological regulators CTLA-4 and/or PD1 are reflected in the approvals by FDA and represent the hope for many (1–7). However, there is still room for improvement in terms of further prolongation of survival and lessening the adverse side effects (5, 6, 8–10).

These goals may be accomplished only after careful and rigorous considerations and testing of other important and not yet targeted immunosuppressive mechanisms that may limit the clinical outcomes of the current immunotherapies of cancer even after the depletion of all known immunological negative regulators, such as CTLA-4/PD-1 blockade or T regs.

The Hypoxia-A2-Adenosinergic immunosuppression, transcription and redirection of the effector functions of anti-pathogen and anti-tumor immune cells

The concept of targeting the physiological, i.e. cell metabolism and local tissue oxygen tension-dependent and A2A and A2B adenosine receptor-mediated immunosuppression in inflamed and cancerous tissues is the basis of discussed here therapeutic strategy (Fig. 1) (11–18). This type of immunosuppression in TME seems to be a misguided application of the likely to be evolutionary old, critical and non-redundant negative feedback immunosuppressive mechanism that is otherwise life-saving by protecting normal tissues from the excessive collateral damage during the anti-pathogen immune response (13,14,18). The identification of this indispensable immune-regulatory pathway may have provided one of the explanations of the co-existence of tumors and anti-tumor immune cells in the same cancer patient (19) as due to the A2AR adenosine receptor–mediated inhibition of tumor-reactive T cells in tumor microenvironment (TME) (12, 15).

Fig. 1. The Hypoxia-A2-Adenosinergic immunosuppression, transcription, and redirection of effector functions of anti-pathogen and anti-tumor T cells.

Fig. 1

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Described are the upstream and down-stream stages of this pathway in hypoxic and extracellular adenosine-rich microenvironments of inflamed and cancerous tissues (16). It is believed that the collateral damage to vasculature in inflamed microenvironments by overactive immune cells during the anti-pathogen immune response results in interruption of local blood supply, decrease in local oxygen tension and abnormal local tissue hypoxia (13,18). Tumors are hypoxic because of different reasons that are inflamed tissues i.e. due to the abnormal and chaotic tissue geometry and insufficient vascularization, among others (46). The hypoxia-driven stabilization of Hypoxia Inducible Factor (HIF-1alpha) transcription factor (64) leads to the CD39/CD73 ecto-enzymes-mediated generation of extracellular adenosine (11, 17,20,37,40,44). Adenosine then signals through the Gs protein coupled A2A and A2B adenosine receptors (11,30,31) and triggers the accumulation of intracellular cAMP. The binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase (PKA) results in a cascade of phosphorylation events that inhibits TCR-triggered signaling pathway and therefore inhibits the pro-inflammatory effects of T cells (23–29). In addition, the Cyclic AMP Response Element (CRE)-binding protein CREB is participating in transcription of gene products that have CRE after being phosphorylated by PKA (79), while HIF-1alpha is participating in transcription of genes that have the Hypoxia Response Element (HRE) (64). Another immunosuppressive molecule, adenosine A2B receptor was also shown to be regulated by transcriptional activity of HIF-1a (45). The Hypoxia-A2-Adenosinergic transcription may at least partially explain the redirection of immune response and the “infectious” tolerance by Tregs (16). The increased expression of CD73 on the Tregs surface (80) may generate the extracellular adenosine that would further enhance their suppressor activities in an autocrine manner as well as add to the immunosuppressive effects of tumor-produced adenosine on CD8+ T cells in paracrine manner.

It must be emphasized that hypoxia-A2-adenosinergic signaling is not only an immunosuppressive pathway that inhibits the e.g. TCR-triggered pro-inflammatory IFN-gamma production (Fig. 1). This pathway is also redirecting immune response by facilitating the switching for example Th1 toward Th2 pattern of cytokine secretion and toward suppressor phenotype as discussed in detail in (16). Accordingly, the increased levels by A2aR or A2BR signaling levels of intracellular cAMP may inhibit the IFN-gamma production in CD8+ or CD4+ T cells, while promoting transcription of genes that are expressing the cAMP response elements (CRE) or the hypoxia response elements (HRE). This, in turn, may lead to a synthesis of immunosuppressive molecules and development of Tregs (16) (Fig. 1). The facilitated by hypoxia-A2-adenosinergic signaling generation of anti-inflammatory mediators or development or functions of suppressor T regs may have provided an explanation of the “infectious tolerance” of T regs (16) in hypoxic and extracellular adenosine-rich inflamed tissues and in TME (16, 20, 21).

The power and versatility of the A2-Adenosinergic immunosuppression was taken advantage of and thereby strongly validated by Staphylococcus aureus and other bacteria that have evolved to synthesize extracellular adenosine in order to escape host immune responses by inhibiting anti-bacterial effector functions of neutrophils through their A2AR/A2BR (22).

The key molecules of the Hypoxia-A2-Adenosinergic signaling pathway

The description of the upstream and downstream stages of Hypoxia-A2-Adenosinergic pathway in Figs 1 and 2 and below follows the history of the search for the molecular mechanism of physiological immunosuppression which started with the bet on the importance of the intracellular cAMP. Intracellular cyclic AMP is the high fidelity intracellular immunosuppressor that inhibits virtually all tested TCR triggered effector functions of T cells-although with different efficacy-through activation of the cAMP dependent protein kinase (PKA) (23–29).

Fig. 2. Fig. 2A. The anti-Hypoxia-A2-Adenosinergic immunotherapeutic co-adjuvants that inhibit the upstream and down-stream stages of the Hypoxia-A2-Adenosinergic pathway.

Fig. 2

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The shown anti-hypoxia—A2-adenosinergic drugs exist since fortuitously they have been developed for other therapeutic indications. 1) The inhibitors of hypoxia-HIF1 alpha stage –including the inhibitors of HIF-1alpha-can be used in order to weaken the hypoxia and promote the destabilization and degradation of HIF-1alpha in anti-tumor T and NK cells. 2) Blockers or inhibitors of CD39 ecto-ATPase/ADPase and CD73 5′-nucleotidase may be used to prevent the accumulation of extracellular adenosine in TME and thereby decrease the intensity of immunosuppressive signaling through A2AR or A2BR. 3) The commercially available stabilized adenosine deaminase preparations may be tested to degrade the extracellular adenosine. Alternatively, the enzymes such as adenosine kinase may be tested for the ability to decrease the levels of extracellular adenosine by re-phosphorylating it into AMP. 4) Antagonists of A2A adenosine receptor compete with the tissue-produced adenosine for binding to the same binding site of adenosine receptor, but –in contrast to endogenously generated adenosine, antagonists do not activate the A2AR receptor to increase the intracellular cAMP levels.

The elevation of intracellular levels of cyclic AMP in T cells in hypoxic and extracellular adenosine-rich tissues is triggered after the binding of the extracellular adenosine to Gs protein coupled, cAMP-elevating A2AR (high affinity) and A2BR (low affinity) receptors (30,31). Our focus on studying the A2AR in T cell-mediated immunity was due to the observations that murine T cells preferentially express the A2AR (32–34), which is likely to be activated in the lower ranges (~50nM) of extracellular adenosine in inflamed and cancerous tissues in vivo.

T cells do not have “spare” A2AR receptors (i.e. no “receptor reserve”) (35), suggesting that the number of A2AR molecules per T cell is the limiting factor in determining the maximal cAMP response of T-lymphocytes to adenosine. This important property may help in minimizing the immunosuppressive effects of extracellular adenosine. It was also shown that T cells have a “memory” of signaling through A2AR (36) so that T cells are experiencing the A2AR-triggered suppression long after the exposure to the short-lived in vivo extracellular adenosine has ended.

Extracellular adenosine is generated by at least two known mechanisms, i.e. from intracellular ATP or from extracellular ATP due to activities of extracellular adenosine-generating tandem of ecto-enzymes CD39 (ecto-ATPase/ADPase) and CD73 (ecto-5′-Nucleotisase), which act in a tandem, as recently reviewed in (17). The CD39 and CD73 ecto-enzymes were shown to be important in limiting the inflammatory damage (20, 37–40) by generating the extracellular adenosine and thereby enabling the down-stream signaling by the adenosine→A2AR (11).

The hypoxia→ hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated events have been also implicated into upstream events that trigger the anti-inflammatory CD39/CD73→ [Adenosine]High→A2AR/A2BR signaling. The first evidence of the inhibitory role of Hypoxia-HIF-1alpha in T cells was obtained in HIF-1alpha−/−Rag2−/− chimeric mice, which are characterized by HIF-1alpha deficiency only in cells of adaptive immune system (41). These studies revealed not only that HIF-1alpha regulates the lymphocyte development and functions, but that it also protects from autoimmunity and inflammatory tissue damage. The immunosuppressive role of HIF-1alpha in T cells was then confirmed using mice with targeted deletion of HIF-1alpha gene only in T cells. The genetic “knock-down” of HIF-1a in T cells of these mice prevented the inhibition of T cells in hypoxic inflamed tissues, increased anti-bacterial response and improved mice survival (42). These observations also established that effects of HIF-1alpha are T cell autonomous.

The immunosuppressive role of HIF-1alpha in T cells was also indirectly supported by studies of the effects of the pharmacological weakening of tissue hypoxia and of the Hypoxia→HIF-1alpha signaling by systemic oxygenation (43). The observations of increased immune response and exacerbation of collateral inflammatory damage in these experiments extended the earlier genetic evidence HIF-1 was shown to induce the expression of CD73 (Ecto-5′-nucleotidase), a membrane-bound glycoprotein that generates the immunosuppressive adenosine due to HIF-1a binding to HRE sites in CD73 gene promoter and the inhibition of HIF-1α expression by antisense oligonucleotides led to inhibition of hypoxia-inducible CD73 expression (44). Another immunosuppressive molecule, the adenosine A2B receptor was also shown to be regulated by transcriptional activity of HIF-1a (45)

The Hypoxia-A2-Adenosinergic protection of tumors from anti-tumor immune cells

Demonstrations that inflammatory damage-associated interruption in local blood supply and ensuing local tissue hypoxia and adenosine accumulation were able to recruit the activities of anti-inflammatory A2AR on T cells in normal tissues (11), led to a straightforward assumption that hypoxic and extracellular adenosine-rich cancerous tissues may have high-jacked this mechanism in order to inhibit the incoming A2AR-expressing anti-tumor T cells (12, 15).

Indeed, many solid tumor microenvironments are hypoxic (46) and tissue hypoxia is conducive to the generation of extracellular adenosine (47). Tumors were shown to contain extracellular adenosine among many other molecules (48) although the intracellular cAMP-elevating A2 adenosine receptors were explicitly excluded as possible candidates to mediate the immunosuppression by adenosine in tumors (48–50).

The key test to confirm or to disprove the role of extracellular adenosine and of A2AR receptor as a mediator of immunosuppression in tumors was in comparisons of anti-tumor immune response in A2AR gene deficient mice and their WT littermates. Based on insights of the role of adenosine→A2AR in immunosuppression in inflamed tissues it was expected that A2AR gene deficient mice will have much stronger and longer-lasting anti-tumor immunity. This expectation was validated by findings that the genetic deletion of A2AR resulted in much stronger anti-tumor immunity, rejection of established tumors and survival of A2AR-deficient mice as compared with control group of tumor-bearing A2AR-expressing mice (12, 15).

The ability to re-capitulate the anti-tumor effects of genetic deficiency in A2AR by pharmacological maneuvers pointed to the feasibility and promise of the novel therapeutic approach of unleashing the anti-tumor T cells from the hypoxia-A2-adenosinergic inhibition using small molecules.

Anti-hypoxia-A2-adenosinergic co-adjuvants that may enable the realization of full anti-tumor capacities of current immunotherapies of cancer

The pharmacological temporary “knock-down” of A2AR using synthetic and natural A2AR antagonists, or using the A2AR expression-decreasing pretreatment of T cells before their adoptive transfer with siRNA also led to a much stronger anti-tumor effects of endogenously developed or adoptively transferred tumor-reactive T cells (12) including the lesser neovascularization of tumors, stronger rejection of lung metastases and stronger inhibition of tumor growth. Similar effects were observed with effects of the negatively selected A2ARlow anti-tumor T cells –that were resistant to inhibition by adenosine (51).

The ability of antagonists of A2A adenosine receptors to interrupt the latest stage of the Hypoxia→CD39/CD73→[Adenosine]High→A2A-adenosinergic signaling and unleash the anti-tumor effects (Fig. 2) provided both pharmacological in vivo confirmation of the role of A2AR in anti-tumor immunity shown by genetic deletion of A2AR and provided the proof-of-principle (12) for the therapeutic use of the conceptually novel immunological co-adjuvants that block the physiological negative regulators of anti-tumor immunity (Figs.2) (11, 12).

Fig. 2 shows different types of treatments that target the individual up-stream and down-stream stages of the Hypoxia → CD39/CD73 → [Adenosine]High → A2AR/A2BR→ cAMP signaling in order to mitigate the immunosuppression in TME.

Inhibitors of cAMP-dependent protein kinase A (PKA) were considered first as a potential approach to prevent the inhibition of anti tumor T cells (23–29). These efforts were abandoned since such inhibitors may have the unacceptable side effects due to an important role of cAMP binding site in many fundamental biological processes.

A2A adenosine receptor antagonists

In contrast to targeting the PKA, the much more fruitful was an approach to block the intracellular cAMP-elevating high affinity A2A receptor (30,31) since T cells did preferentially express this type of adenosine receptor (30–34). The biological effects of antagonists of adenosine receptors are due to their competition with the tissue-generated endogenous extracellular adenosine for binding to the same site on A2AR, but-in contrast to the natural ligand of A2AR, i.e. adenosine, the antagonist does not trigger the intracellular cAMP accumulation.

This, in turn, prevents the inhibition of T cells by adenosine. In addition, by blocking the A2AR the antagonists may accomplish the shortening of the “memory” of exposure of T cells to immunosuppressive signaling through A2AR (36). Effects of A2AR antagonists are facilitated by the lack of spare A2AR receptors (i.e. no ‘receptor reserve’ on T cells (35), thereby allowing antagonists to further minimize the immunosuppressive effects of extracellular adenosine.

Even the short-lived “first generation” of synthetic A2AR antagonists had the anti-tumor immunity by unleashing the anti-tumor T cells (12) and subsequent extensive and well-controlled studies revealed potent anti-tumor effects of longer lived A2AR antagonists (52,53). These observations led authors to advocate clinical trials of existing immunotherapies of cancer in combination with the currently available synthetic A2AR antagonists (53).

The anti-tumor use of synthetic A2AR antagonists in combination with immunotherapies of cancer was the unexpected outcome of fundamental studies of anti-pathogen immune responses and autoimmunity (11–18). That alone would be sufficient to justify the large-scale research and development of this class of synthetic drugs, but, fortuitously, these drugs have been developed by neurobiologists due to an interest in the role of the A2AR in central nervous system and their original promise in slowing the progression of Parkinson’s Disease.

There are several selective synthetic antagonists of A2AR, which were shown to be safe in Phase II and III clinical trials of Parkinson’s Disease (30,54,55) and one such A2AR antagonist, KW6002 (istradefyline), is approved for treatment of Parkinson’s Disease patients in Japan. These drugs can be easily re-purposed and tested in combinations with existing immunotherapies of cancer. In contrast to the use of A2AR antagonists the considerations of clinical utility of antagonist of A2B adenosine receptor are premature due to insufficient preclinical data, its low affinity to adenosine and possibility of cardiovascular site effects.

Extracellular adenosine-generating or degrading enzymes

Approaches to target CD39 and CD73 ecto-enzymes, which function in a tandem to generate the extracellular adenosine have been developed in an innovative research by Simon Robson and collaborators in studies of CD39 ( 20,56,57) and in studies of John Stagg and Mark Smith and their collaborators in studies of CD73 (58–61) and by Zhang and co-authors (52,62). Drugs that degrade the already formed and accumulated in TME extracellular adenosine-such as adenosine deaminase (63) (ADAGEN, Enzon) – and drugs that inhibit the extracellular adenosine accumulation in TME by CD39/CD73 ecto-enzymes may provide yet another tool in inhibiting the CD39/CD73→A2A/A2B axis. Future studies may reveal relative advantages and disadvantages of using the anti-CD39 or anti-CD73 blocking monoclonal antibody as compared with small molecules inhibitors in order to decrease the intra-tumoral levels of extracellular adenosine.

Inhibitors of TME Hypoxia-HIF-1alpha

Drugs that may inhibit the Hypoxia-HIF-1alpha signaling are in high demand due to the well-established understanding of the pro-tumor effects of hypoxia (46) and HIF-1alpha (64). Promisingly, the inhibitors of HIF-1alpha including the digoxin and acriflavine (65) (66) were shown to decrease the lung metastasis in an orthotopic breast cancer model. Other HIF-1alpha inhibitors such as sirtuin-7 and ganetespib, - a new therapeutic candidate to target TNBC - were also found to have anti-tumor activities (67,68).

Confirmations of importance of hypoxia-A2-adnosinergic immunosuppression in studies of human cancers

The original observations of critical role of hypoxia-A2-adnosinergic immunosuppression in tumor protection (12, 15) have been confirmed by several groups of tumor immunologists in different models of tumor rejection in careful and extensive studies of effects of A2AR genetic deletion in mice, A2AR antagonists and/or of genetic deletion or pharmacological inhibition of upstream stages of adenosine generation by CD39/CD73 ecto-enzymes (53,60,62,69,70–72).

However, the most impressive are clinical implications of the recent extensive analysis of gene-expression data from more than 6,000 triple negative breast cancers. These studies provided evidence for correlation between i) the inhibition of anti-tumor T and NK cells by A2AR and A2BR, ii) high levels of expression of extracellular adenosine-generating ecto-enzyme CD73 on human triple negative and chemotherapy-resistant breast cancers and iii) the poor prognosis of patients with such tumors (61).

Anti-hypoxia-A2-adenosinergic co-adjuvants may also block other immunosuppressive pathways

The hypoxia-A2-adenosinergic pathway may be not only the evolutionary oldest, but also the most influential in recruiting other immunosuppressive pathways. Indeed it is hard to come up with older biochemical entities/parameters than the lack of oxygen (anoxia, hypoxia) or adenosine as was discussed in (16). It was proposed and case was made that A2AR and A2BR adenosine receptors, HIF-1, the cAMP response element (CRE)- and hypoxia response element (HRE)-mediated transcription in Treg and effector cells have key roles in governing the functions of Treg and effector cells (16) (Fig. 1).

Thus, blocking the hypoxia-A2-adnosinergic signaling is expected to at least partially block many other immunosuppressive mechanisms since it is already established that hypoxia-A2-adnosinergic pathway is also recruiting other immunosuppressive molecules such as cyclooxygenase-2 and eicosanoid mediators (73–75). This pathway is also implicated in the development and functions of Tregs (16, 20, 76). Interestingly, both the CD73-mediated generation of extracellular adenosine and A2AR were required for suppressive effects of Tregs through a PD-1-dependent mechanism in kidney ischemia-reperfusion injury model (77). It was also shown that activation of A2AR recruited the negative immunological regulators PD-1 and CTLA-4 on T cells (78).

Conclusions and expectations

Hypoxia-A2-adenosinergic immunosuppression negates the anti-tumor effects of tumor reactive T and NK cells and published data and insights from yet to be published studies identified it as an important remaining barrier to accomplish the effective tumor rejection. The immunosuppressive adenosine→A2AR/A2BR signaling can be weakened by anti-hypoxia-A2-adenosinergic co-adjuvants thereby enabling the full anti-tumor potential of current immunotherapies of cancer.

As follows from analysis of the Figs.1,2 the inhibition of hypoxia-A2-adenosinergic immunosuppression is expected to improve tumor rejection by tumor-reactive T cells that have been induced by any other immunotherapeutic protocol, including the mono- or dual therapies with CTLA-4 or PD1 blockade. This expectation was supported by recent important observations of stronger anti-tumor effects of CTLA-4 or PD1 blockade if combined with lowering the levels of extracellular adenosine and thereby inhibiting Adenosine→ A2AR and A2BR signaling (53, 60). It was shown that inhibition of accumulation of extracellular adenosine and A2AR/A2BR signaling by blocking the anti-CD73 mAb does indeed enhance the anti-tumor activity of dual CTLA-4 and PD-1 blockade mAbs in models of transplanted and chemically induced mouse tumors (60). It would be interesting to test whether the efficacy of A2AR antagonists could be further increased by lowering the concentration of extracellular adenosine in TME by drugs that either inhibit the accumulation of extracellular adenosine or degrade the already formed extracellular adenosine.

The cancer vaccine-induced tumor-reactive T cells, adoptively transferred tumor-reactive T cells and blockers of CTLA-4/PD1 and the anti-hypoxia-A2-adenosinergic co-adjuvants are highly complementary treatments that help each other. Indeed, the inhibition of even all known immunological negative regulators and depletion of Tregs will still leave T cells vulnerable to multi-faceted and powerful immunosuppression by tumor hypoxia and A2AR. However, the inhibitors of hypoxia-A2-adenosinergic pathway may have a favorable “side effect” by decreasing the intensity of many other immunosuppressive mechanisms such as Tregs, CTLA-4, TGF-beta and cyclooxygenase-2, eicosanoid mediators-mediated immunosuppression. This, in turn, may allow to lower the level of injected blockers of CTLA-4 or PD-1 and thereby to decrease the side effects. Taken together the available data strongly justify the targeting the hypoxia → adenosine →A2AR/A2BR pathway to prevent the inhibition of anti-tumor T cells and NK in the tumor microenvironment.

Footnotes

Disclosure of potential conflict of interest:

M.V.S. has ownership interests (including patents) in Redoxtherapies, which are charged by the Office of Technology Transfer of the National Institute of Health, USA to translate the developed at NIH technology into medical treatments.

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