Targeting Cancer-Derived Adenosine: New Therapeutic …adenosine receptor ( ADORA2B ) gene expression represents a potential global mechanism of hypoxia response, interaction between

AUGUST 2014�CANCER DISCOVERY | 879

MINI REVIEW

Targeting Cancer-Derived Adenosine:New Therapeutic Approaches Arabella Young 1 , 2 , Deepak Mittal 1 , 2 , John Stagg 3 , and Mark J. Smyth 1 , 2

ABSTRACT CD73 generation of immunosuppressive adenosine within the hypoxic tumor micro-

environment causes dysregulation of immune cell infi ltrates, resulting in tumor

progression, metastases, and poor disease outcomes. Therapies targeted toward the adenosinergic

pathway, such as antibodies targeting CD73 and CD39, have proven effi cacy in mouse tumor models;

however, humanized versions are only in preliminary development. In contrast, A 2A adenosine receptor

antagonists have progressed to late-stage clinical trials in Parkinson disease, yet evidence of their

role in oncology is limited. This review will compare the merits and challenges of these therapeutic

approaches, identifying tumor indications and combinations that may be fruitful as they progress to

the clinic.

Signifi cance: High concentrations of immunosuppressive adenosine have been reported in cancers,

and adenosine is implicated in the growth of tumors. This brief review delineates the current treatment

strategies and tumor subtypes that will benefi t from targeting adenosinergic pathways, alone or in com-

bination with contemporary approaches to cancer treatment. Cancer Discov; 4(8); 879–88. ©2014 AACR.

Authors’ Affi liations: 1 QIMR Berghofer Medical Research Institute; 2 School of Medicine, University of Queensland, Herston, Queensland, Aus-tralia; and 3 Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Faculté de Pharmacie et Institut du Cancer de Montréal, Mon-tréal, Québec, Canada

Corresponding Author: Mark J. Smyth, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, 4006, QLD, Australia. Phone: 61-7-3845-3957; Fax: 61-7-3362-0111; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-14-0341

©2014 American Association for Cancer Research.

INTRODUCTION Immunosuppression within the tumor microenviron-

ment causes inability of host immune effectors to eliminate

transformed malignant cells. Although immune infi ltrate

is present in a proportion of most tumor types, variability

in immune constituents may dictate disease outcome. The

presence of regulatory T cells (Treg), tumor-associated macro-

phages (TAM), and myeloid-derived suppressor cells (MDSC)

drives a suppressive environment, which inhibits antitumor

immune defenses ( 1, 2 ). Furthermore, the ratio between

effector T cells and Tregs appears an important predictor

of tumor prognosis ( 3 ). Generation of anti-infl ammatory

cytokines and metabolites by the tumor together with these

suppressive cell types antagonize tumor immunity by inhibit-

ing the cytotoxic functions of natural killer (NK) and CD8 + T

lymphocytes, instead enhancing angiogenesis and promoting

tumor cell survival and progression.

Currently, the development of therapeutic modalities to

overcome effector immune cell dysfunction seems promising.

Targeted therapies and immunotherapies offer advantages

over conventional cytotoxic cancer treatments. Targeted ther-

apies limit the availability of critical metabolites necessary

for tumor survival, reducing off-target effects and minimiz-

ing unrestrained tissue damage. Immunotherapies aim to

improve the patient’s own antitumor immune responses by

releasing the suppressive restraints on effector immune cell

populations. The advantages of such an approach include

reduced toxicity and the development of memory responses

leading to long-term tumor eradication.

Promising preclinical investigations into the blockade of

immune checkpoint receptors, reduction of angiogenesis,

and inhibition of immunosuppressive metabolite signaling

in the tumor microenvironment have translated to oncology

practice. Inhibitory immune checkpoint molecules cytotoxic

T-like antigen-4 (CTLA4 ) and programmed death-1 (PD1),

expressed on dysfunctional tumor-infi ltrating lymphocytes,

have been highlighted as targets to reset the effector T-cell

immune response. The monotherapies ipilimumab (anti-

CTLA4) and nivolumab (anti-PD1) have shown success in

enhancing patient survival in a growing number of can-

cer types ( 4, 5 ). Nevertheless, tumors can deploy multiple

mechanisms to allow their survival by avoiding immunosur-

veillance. Increasingly evident is the need to target multiple

immunosuppressive mechanisms to overcome the high level

of redundancy that exists in eliciting immunosuppression,

if we are to increase patient life expectancy and quality. This

need was highlighted by the clinical effects of combining

ipilimumab and nivolumab, which showed even greater

tumor clearance for patients with nonresectable metastatic

melanoma ( 6 ).

Research. on November 30, 2020. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

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Young et al.MINI REVIEW

Targeting the immune system to instigate enhanced effec-

tor immune cell function through blockade of inhibitory

receptor signaling is not without risk. Immune checkpoint

receptors are critical regulators of immune system activity,

enabling self-tolerance and preventing the development of

autoimmune pathologies. Administration of ipilimumab and

nivolumab signifi cantly enhances tumor regression; however,

the risk of developing toxicity in the form of immune-related

adverse events (irAE) from unrestrained stimulation of the

immune response is also increased ( 6 ). Selection of com-

patible therapies that present synergistic antitumor effi cacy

in combination with minimal toxicity and autoimmunity

is a current clinical challenge. Identifi cation of alternate

immuno suppressive pathways, which may be targeted to

enhance clearance of malignancies with limited associated

irAEs, will be advantageous.

Recently, the adenosinergic pathway has risen to promi-

nence as a therapeutic target for immune activation. This

review details the effects of adenosine-generated immuno-

suppression in the tumor microenvironment, highlighting

the potential use of associated enzymes and receptors as

predictive biomarkers for treatment response and progres-

sion. Further, we dissect the pros and cons of targeting

the generation of adenosine versus adenosine receptors as a

therapeutic strategy. Finally, we identify potential synergistic

combinations that might reduce tumor growth and meta-

static burden.

ADENOSINE-ABUNDANT TUMOR MICROENVIRONMENT

Adenosine is generated in response to proinfl ammatory

stimuli such as cellular stress initiated by hypoxia or ischemia

( 7 ). Release of extracellular ATP undergoes conversion to

AMP by the enzyme CD39 and subsequent dephosphoryla-

tion of AMP to adenosine is catalyzed by CD73 ( Fig. 1 ). Land-

mark studies by Ohta and colleagues ( 8 ) have highlighted

the importance of adenosine for tumor escape. Adenosine

interacts with four G-protein–coupled receptor subtypes (A 1 ,

A 2A , A 2B , and A 3 ), allowing for diversity and cellular spe-

cifi city in signaling pathway activity. Each receptor possesses

variable affi nity to the adenosine ligand indicative of different

sensitivities and responsiveness to adenosine levels. In par-

ticular, the low-affi nity A 2B adenosine receptor initiates signal

transduction in comparatively limited physiologic condi-

tions. Together, the A 2A and A 2B adenosine receptors stimulate

G s /G olf -dependent adenylyl cyclase-driven cAMP accumula-

tion, enabling signal transduction to target genes. A 2B adeno-

sine receptors are also coupled to G q proteins, which can

activate JNK and p38 via phospholipase C–dependent or

–independent pathways ( 9 ). Positioning of adenosine recep-

tors, particularly A 2A and A 2B adenosine receptors that

promote an anti-infl ammatory response, on immune cells

reduces tissue injury in conditions of cellular stress, ena-

bling maintenance of tissue homeostasis. The persistence of

increased adenosine concentrations beyond the acute-injury

phase can, however, become detrimental to tissues by activat-

ing pathways that trigger immunosuppression or promote

an unremitting wound-healing process. Thus, the accumula-

tion of adenosine in the hypoxic tumor microenvironment

mediates immunosuppression, causing dysregulation of effec-

tor immune cell subsets, dampening the antitumor immune

response.

Rapid proliferation of tumor cells in a manner unsup-

ported by surrounding vasculature leads to the development

of hypoxic regions within solid tumors ( Fig. 1 ). Although

hypoxia increases cell death due to oxygen and nutrient depri-

vation, it also promotes selection of resistant, aggressive cancer

cells able to survive in unfavorable conditions. Hypoxia-resist-

ant malignant cells phenotypically possess stem-like features

with greater proliferative and migratory capacity. This corre-

lates with increased risk of metastasis, reduced responsiveness

to conventional therapies, and ultimately poor prognosis for

patients. Hypoxia-inducible factors (HIF) initiate differential

transcriptional regulation in response to hypoxic conditions,

infl uencing cellular metabolism and angiogenesis, in an effort

to ease oxygen requirements. Identifi cation of HIF1α-driven

CD73 (NT5E) and CD39 (ENTPD) transcriptional activation

in epithelia and endothelia provides an important link for

hypoxia-mediated adenosine production ( Fig. 1 ; ref. 10 ).

Modifi cation of the HIF1α binding site situated in the CD73

promoter abrogated transcriptional response indicative of a

direct regulatory relationship ( 10 ). Similarly, hypoxia-control-

led HIF1α-induced expression of the A 2B adenosine receptor

was identifi ed, representing a coordinated response that may

contribute to autocrine and paracrine adenosine signaling

( Fig. 1 ; ref. 11 ). Although modulation of CD73 , CD39 , and A 2B

adenosine receptor ( ADORA2B ) gene expression represents a

potential global mechanism of hypoxia response, interaction

between these markers and HIF1α within the tumor microen-

vironment is yet to be confi rmed.

Apart from the mobilization of adenosine via concentra-

tive and equilibrative nucleoside transporters, adenosine pro-

duction within the tumor microenvironment is essentially

mediated by CD39 and CD73 ectonucleotidase expression on

both non-hematopoietic and hematopoietic cellular subsets.

Predominant cell types expressing CD39 and CD73 include

endothelial cells and lymphocytes including immunosuppres-

sive Tregs, as well as the tumor itself ( Fig. 1 ). In humans, only

a small fraction of Tregs express CD73 in steady-state ( 12 ).

In response to IL2, however, the majority of human Tregs

express both CD39 and CD73 ( 13 ). Increased Treg abundance

in response to hypoxia via upregulated FOXP3 gene expres-

sion represents an additional mechanism of HIF1α regula-

tory control. Consequently, increased Treg infi ltrate within

the hypoxic tumor microenvironment is likely to enhance

adenosine-mediated immunosuppression ( 14 ). However,

tumors produce comparatively greater adenosine levels than

those generated by endogenous host CD73 ( 15, 16 ). Release

of tumor-derived exosomes expressing catalytically functional

CD39 and CD73 represents an additional mechanism of

tumor-mediated immunosuppressive adenosine generation

( 17 ). This is indicative of the critical importance and reliance

on adenosine production to enable immune evasion and

enhance tumor survival.

ADENOSINE-DRIVEN CELLULAR SIGNALING As demonstrated in Fig. 1 , elevated adenosine is instru-

mental in regulating an abundance of cellular responses in





Novel Adenosine-Based Cancer Therapeutics MINI REVIEW

Figure 1. Adenosine -mediated effects in the hypoxic tumor microenvironment. Release of extracellular ATP in the hypoxic tumor microenvironment is converted to adenosine by CD39 and CD73 ectonucleotidases. CD39 and CD73 are broadly expressed across a number of cell types. Modulation of their distribution can vary dependent on cellular activation and tissue localization. Adenosine enhances polarization of myeloid and T-cell subsets to proang-iogenic and immunosuppressive phenotypes, enhancing tumor growth and survival. High adenosine levels affect effector immune cells, NK cells, and CD8 + T cells, responsible for cytotoxic killing of aberrant malignancies due to inhibited expression of molecules that mediate cell death. DC, dendritic cells; IDO, indoleamine 2,3 dioxygenase; TCR, T-cell receptor.

DC TAM Treg

Increased

• VEGF, IL6, IL10, TGFβ, and IDO

• Modulation of MHC-I and -II antigen presentation

• M2 macrophage polarization, MDSC expansion

NK

CD8+ effector CD4+ effector

Increased

• Polarization to CD4+ Treg

• Proliferation

• Immunosuppression

Tumor cells

Pro–tumor survival

• Enhanced migration

and metastasis

• Chemoresistance

• Immune evasion

and proliferation

Decreased

• Granule exocytosis-

induced cytotoxicity

• Perforin and CD95L

molecule expression

• Cytokine production

IFNγ, TNFα, IL2,

IL12, MIP1α

• NK cell maturation

MDSC

Anti–tumor survival

• Inhibited

proliferation

• Enhanced caspase

expression

• Cell death

Increased

• Production of VEGF,

IL8, angiopoietin-2

• Neovascularization

and proliferation

Reduced • Th1 CD4+ polarization

• IFNγ, IL2, TNFαproduction

Endothelial cells

Hypoxic core

Adenosine A

Exosomes

Tregegg CD4+ effector

enos ee

Endoth

ee

MDSC

Reduced • Adhesion to tumor cells

• Cytotoxicity

• TCR signaling • Activity leading to

induced anergy

ATP AMP

Adenosine

CD39

CD73

Adenosine

receptors osi

Adenosine receptors

• A1 and A3 – inhibit cAMP release

• A2A and A2B – stimulate cAMP signaling

HIF1α • CD39

• CD73

• Adenosine • A2B AR

A1 A2A A2B

A3

ce

es

T D4D4

the tumor. Essentially, adenosine disables cytotoxic effector

functions of both NK and CD8 + T cells predominantly via

A 2A adenosine receptor signaling, enabling tumor immune

evasion and escape ( 7 ). Furthermore, signal transduction

through the A 2A adenosine receptor inhibits the Th1 CD4 +

T-cell response, limiting the cytokine environment neces-

sary to support these effector cell types. Adenosine polarizes

myeloid cells to develop into immunosuppressive pheno-

types such as M2 macrophages and tolerogenic dendritic

cells (DC) due to A 2A and A 2B adenosine receptor signaling,

respectively ( 18 ). In addition, adenosine enhances prolif-

eration of Tregs and granulocytic MDSCs, which further

affect T effector cell proliferation and function ( 14 , 19 , 20 ).

Release of cytokines and immune modulatory factors, such

as VEGF, IL6, IL10, and TGFβ, by these suppressive cell

types enhances tumor survival via heightened angiogenesis

and inhibited immunosurveillance, and thus the majority

of evidence suggests that adenosine is favorable to tumor

survival.

ADENOSINE MEDIATORS AS PUTATIVE BIOMARKERS

Overexpression of the ectonucleotidase enzyme CD73 has

been shown in different types of cancer, and several stud-

ies reported an association between high CD73 levels and

poor prognosis, increased risk of metastasis ( 21 ), and resist-

ance to chemotherapy ( 22, 23 ). In breast cancer, hypoxia-

independent mechanisms further drive CD73 upregulation.

Spychala and colleagues ( 24 ) fi rst described that the absence

of estrogen receptor (ER ) expression in human breast cancer

cell lines was inversely associated with high CD73 expression,

but the underlying mechanism is still not fully understood.

Using methylation-specifi c PCR and DNA pyrosequencing,

CD73 expression in breast cancer cell lines was inversely

associated with CD73 promoter CpG methylation ( 25 ). Inter-

estingly, CD73 promoter methylation was associated with

increased disease-free and overall survival and better out-

come after adjuvant chemotherapy. These results suggest that






CD73 promoter methylation could be used as a breast cancer

biomarker to predict metastasis and global clinical outcome.

We recently reported a large meta-analysis of CD73 gene

expression in breast cancer, gathering more than 6,000

patients, where CD73 gene expression is signifi cantly asso-

ciated with poor prognosis in patients with triple-negative

breast cancer (TNBC; ref. 23 ). CD73 expression in patients

with TNBC was also correlated with an increased resistance to

anthracycline chemotherapy due to adenosine-mediated sup-

pression of antitumor immunity. In addition to its immuno-

suppressive effects, we showed that CD73- derived adenosine

promotes tumor cell migration in vitro and metastasis in vivo

via the A 2B adenosine receptor ( 26 ). Most interestingly, the

A 2B adenosine receptor has recently been identifi ed as a target

of the metastasis-inducing transcription factor FOS -related

antigen 1 (FRA1) in TNBC ( 27 ). Ntantie and colleagues ( 28 )

further unraveled a signaling pathway linking A 2B adenosine

receptor signaling and tumor cell adhesion. Taken together,

these studies converge to suggest an important role for the

CD73–A2B axis in metastatic dissemination, independently

of immunosuppression. These fi ndings also highlight the

importance of developing these and other biomarkers of

adenosine generation and signaling as putative biomarkers of

TNBC, before any new therapeutic approaches.

TARGETED THERAPEUTICS AGAINST ADENOSINE GENERATION

Because adenosine is a metabolite, genetics can only be

used to study the adenosinergic pathway by targeting the

genes encoding for CD39, CD73 (that generate adenosine),

or the adenosine receptors (that consume adenosine). Initial

investigations determined that CD39- or CD73-defi ciency

improved antitumor immunity and survival by signifi cantly

reducing tumor growth and metastasis ( 29–32 ). Development

of bone marrow chimeras confi rmed that critical CD39 and

CD73 expression on both nonhematopoietic endothelial and

hemato poietic Treg cellular lineages mediated tumor growth

( 30 , 32 , 33 ). However, formation of experimental pulmonary

metastases in CD73-defi cient mice appeared dependent on

nonhematopoietic CD73 expression ( 30 ). Nonhematopoietic

endothelial expression of CD73 regulates angiogenesis, a

necessary requirement for the promotion of metastasis ( 29 ,

34 , 35 ). Reduction of immunosuppressive adenosine within

the tumor microenvironment enhances effector functions

of immune cell infi ltrate in a tumor-dependent manner.

Expansion of tumor antigen–specifi c CD8 + T cells infi ltrat-

ing the tumor and circulating within the periphery was

evident in CD73-defi cient mice ( 33 ). In contrast, deletion of

CD39 reduced Treg-mediated immunosuppression, enabling

increased NK cell infi ltration and cytotoxic activity in the

hepatic tumor microenvironment ( 32 ).

Mimicking these enhanced antitumor responses through

pharmacologic targeting of CD73 and CD39 identifi ed simi-

lar reductions in cancer progression. Small-molecular inhibi-

tors APCP and POM-1, which directly abrogate the catalytic

functions of CD73 and CD39, respectively, inhibited tumor

growth and metastasis ( 30 , 32 , 33 ). Effi cacy was negated when

treating their respective gene-targeted mouse strains, indica-

tive of inhibitor specifi city ( 32, 33 ). Intratumoral injection

of CD39 inhibitors also reestablished the proimmunogenic

activity of anthracyclines in mice ( 36 ). Blockade of CD39

enzymatic activity may lead to an increase in extracellular

ATP levels, enabling coactivation of infl ammasomes, promot-

ing tumor immunity. Antibody-directed immunotherapies

targeting CD73 demonstrated similar antitumor properties

( 26 ). Therapeutic effi cacy required the presence of a compe-

tent immune system, indicating that antibody blockade of

immunosuppressive adenosine generation enhanced immu-

nosurveillance. Cocultured immune cell subsets, NK, CD4 + ,

and CD8 + T cells, with ovarian carcinoma cell lines expressing

CD39 and CD73, displayed enhanced proliferative and lytic

capacity in vitro following the addition of anti-CD39 or anti-

CD73 ( 15 ). These effects were directly attributed to reduced

adenosine production within the culture system, enabling

improved immune responsiveness.

In addition to mediating the rate-limiting step for adeno-

sine production, CD73 also promotes cellular adhesion, par-

ticularly lymphocyte interactions with the endothelium, and

angiogenesis ( 34, 35 , 37 ). Furthermore, CD73 enhances the

migratory capacity of malignant cells, resulting in increased

risk of metastatic progression ( 26 , 38 , 39 ). Recently, anti-

human CD73 monoclonal antibody (mAb) therapy was

shown to reduce spontaneous metastasis of human breast

cancer xenografts, independent of primary tumor growth

( 39 ). Lack of a functional immune system within these mod-

els implies that metastasis was attenuated by an alternative

mechanism to the improved antitumor immunity previously

determined. An epitope-specifi c function was identifi ed, in

which recognition of CD73 on cancer cell lines promoted

clustering and internalization of CD73, preventing CD73-

mediated extravasation and colonization of potential meta-

static clones ( 39 ). Determining which patients will benefi t

from anti-CD73 treatment or contraindicated circumstances

will be necessary as this therapy moves forward to clinical

utility. In the setting of ER + breast cancer, the use of anti-

CD73 may facilitate the accumulation of extracellular AMP,

directly activating the A1 adenosine receptor, potentially

enhancing ER signaling and breast cancer growth ( 40, 41 ).

Although CD73-defi cient mice present no overt phenotypic

abnormalities, global CD39 defi ciency is associated with vari-

ous physiologic disturbances, including glucose intolerance,

insulin resistance, and increased levels of circulating fatty

acids ( 42 ). Importantly, CD39-defi cient mice exhibit signifi -

cantly increased bleeding times (>20 minutes compared with

1 minute) resulting from P2Y1 receptor desensitization, and a

vascular prethrombotic state in basal conditions ( 43 ). Perhaps

most worrisome is the propensity for CD39-defi cient mice

to spontaneously develop hepatocellular carcinoma, with a

70% incidence at 18 to 24 months of age ( 44 ). This seems to

result from enhanced mTOR signaling in hepatocytes as a

consequence of high extracellular ATP levels and P2 receptor

activation. RAS and PI3K may also be involved, because both

are regulated by P2 receptors. These studies emphasize the

need to further investigate the role of CD39 in cancer.

In summary, preclinical studies targeting CD39 or CD73

identifi ed the therapeutic potential and validity of these

approaches as strategies for improving antitumor immune

response ( Table 1 ). Both targets display enhanced expres-

sion in a broad spectrum of cancer types, as well as on






Table 1. Advantages and disadvantages for targeting adenosine generation (CD39 and CD73) and adenosine signaling (A 1 , A 2A , A 2B , and A 3 adenosine receptors) pathways

Therapeutic target—localization Potential advantages Potential disadvantages

CD39

• B cells, NK cells, activated T cells, par-

ticularly Tregs, endothelial cells, solid

tumors, and certain leukemias.

• Rate-limiting step converting ATP to AMP

• Increased expression in some cancer types

• Antibody therapies and small-molecule

inhibitors [polyoxometalate-1 (POM-1)]

improve antitumor immunity and in some

cases display anticancer potential in murine

models.

• Lack humanized antibodies

• POM-1 displays proven selectivity for

CD39, diffi cult to determine potential off-

target effects in humans.

• Possible side effects due to total loss of

CD39.

CD73

• Endothelium and epithelium, stromal

cells, Tregs, MDSC, B cells, CD8 + T cells,

tumor correlating with increased

metastasis.

• Multifunctionality of CD73

• Generation of adenosine from AMP

• Immunosuppressive environment

• Angiogenesis

• Involved in lymphocyte adhesion

• Migration of immune and tumor cells

• Inhibition promotes antitumor immunity.

• Direct relationship with several types of

cancers and proven anticancer properties.

• Use as a predictive biomarker to dictate

therapeutic effi cacy of adenosine-

mediated therapies.

• Current lack of humanized antibodies.

• Development of antibodies that target mul-

tiple functions of CD73 will be necessary

for maximal effi cacy.

• Highly abundant CD73 expression may

lead to alternative deleterious effects not

identifi ed in murine models.

• Given the above and the longer half-life of

intact mAb, smaller fragments or small-

molecule inhibitors might be required.

A 1

• Broad distribution, particularly abun-

dant in nerves, heart, and kidneys.

• A 1 adenosine receptor antagonists and ago-

nists have undergone clinical testing, i.e.,

Rolofylline, Tonapofyline, and GW493838.

• A 1 adenosine receptor shown to regulate

ER expression in certain breast cancer

subtypes.

• Disappointing clinical outcomes and

discontinuation of testing in the treatment

of nerve injury and prevention of heart and

renal failure.

• Areas of highest distribution on host tissue

are unrelated to practical cancer targets.

A 2A

• Broad distribution, particularly immune

cells NK, CD4 + , and CD8 + T cells, macro-

phages as well as endothelium.

• High-affi nity adenosine receptor expressed

on immune cells.

• Antagonism promotes antitumor immunity,

enhancing cytotoxic functions of CD8 + T

cells and NK cells.

• A 2A adenosine receptor antagonists and

display excellent safety profi les in clinical

testing for neurodegenerative diseases.

• Small-molecular inhibitors show appropri-

ate tissue penetrance and bioavailability

but short half-life ranging in hours.

• Therapeutic effi cacy specifi c to highly

hypoxic adenosine-mediated tumors.

A 2B

• Broad distribution, particularly en-

dothelium, MDSCs, DCs also apparent

in some cancer types.

• HIF1α-driven A 2B adenosine receptor ex-

pression in response to the hypoxic tumor

microenvironment.

• Antagonists display anticancer, antiangio-

genesis, and enhanced immune effi cacy.

• Low-affi nity adenosine receptor, may be

necessary to block others, particularly A 2A

adenosine receptor, in concert for effi cacy.

A 3

• Broad distribution, particularly the

nervous and cardiovascular system

as well as immune cell subsets, highly

expressed in liver and colon cancers.

• CF101 A 3 adenosine receptor agonist in

clinical trial for the treatment of autoim-

mune disorders such as psoriasis and

rheumatoid arthritis.

• CF102 A 3 adenosine receptor agonist in

clinical trial for treatment of liver cancer.

• Good safety profi le and increased survival

in initial clinical testing.

• Development of therapeutic resistance by

directly targeting cancer cells.

• Off-target effects have not been estab-

lished, potential agonists of immune cells

can lead to activation-induced cell death or

autoimmunity.






immunosuppressive targets within the tumor microenviron-

ment. However, given that these molecules collectively regu-

late infl ammation in cancer and other chronic diseases, the

safety of these therapeutic modalities in patients must be met

with caution. Nonetheless, there is currently a lack of human-

ized anti-human CD39 and CD73 antibodies available for

immediate progression toward clinical translation. Because

of the multifunctionality of CD73 (promoting tumor-asso-

ciated angiogenesis, and cellular adhesion and migration),

CD73 appears to be a most promising target. It is our con-

tention that therapies able to target both its catalytic and

noncatalytic roles would be desirable and are likely to display

the greatest effi cacy in the treatment of cancer.

BLOCKADE OF ADENOSINE SIGNALING ENHANCES ANTITUMOR RESPONSE

Expression of the A 2A adenosine receptor on immune cell

subsets has led to this receptor being primarily investigated

as a target to disrupt adenosine-mediated immunosuppres-

sion in the tumor microenvironment ( Table 1 ). Immunogenic

CD8 + T cell–regulated tumors were shown to display reduced

growth in A 2A receptor–defi cient mice ( 8 ). In addition, T cell–

specifi c knockdown of the A 2A and A 2B adenosine receptors in

combination reduced metastasis and enhanced survival ( 8 ).

Similarly, antagonism of both A 2A and A 2B adenosine receptors

with small-molecule inhibitors suppressed metastatic nodule

formation ( 38 ) against CD73-expressing tumors, indicative of

adenosine receptor reliance on adenosine generation within

the tumor microenvironment. A 2A adenosine receptor antag-

onism mediated its response via a lymphocyte-dependent

perforin-mediated mechanism, whereas metastatic progres-

sion was inhibited via the A 2B adenosine receptor independent

of NK and CD8 + T-cell functions ( 38 ). In contrast, another

study showed that orthotopic murine breast cancer growth

and subsequent development of spontaneous lung metastasis

was reduced by intratumoral A 2B adenosine receptor antago-

nism in an IFNγ-activated T cell–dependent manner ( 45 ).

Recently, A 2A adenosine receptor expression was localized

in the cell membrane of malignant lung adenocarcinoma tis-

sue ( 46 ). Blockade of the A 2A adenosine receptor was shown

to reduce tumor growth by enhancing apoptotic cell death

in vivo in an immunocompromised human xenograft model

( 46 ), indicative of possible alternate functions of A 2A adeno-

sine receptor antagonism. Similarly, A 2B adenosine receptor

inhibitors are able to directly infl uence tumor cell growth and

migration ( 30 ). Prostate cancer cell lines, in which A 2B was the

dominant adenosine receptor, demonstrated reduced prolif-

erative capacity following blockade of A 2B adenosine receptor

or endogenous reduction via siRNA knockdown indicative

of direct anticancer effects ( 47 ). Alternatively, promotion

of endothelium-derived angiogenesis by the A 2B adenosine

receptor in response to hypoxia is an alternative mechanism

for impeding tumor development ( 11 ). Gene-targeted A 2B

adenosine receptor mice display impacted VEGF secretion in

the tumor microenvironment, resulting in reduced neovascu-

larization and repressed tumor growth ( 48 ).

In contrast to the A 2A and A 2B adenosine receptors, which

activate signal transduction cascades via cAMP, the A 1 and

A 3 adenosine receptors downregulate cAMP, enhancing a

proinfl ammatory response. Consequently, targeting these

receptors via a different approach is necessary, with agonists

perhaps more suitable for abrogating tumor cell survival.

Currently, the A 3 adenosine receptor agonist CF102 is under-

going clinical testing for safety and effi cacy in treatment of

hepatocellular carcinoma, in which A 3 adenosine receptor

is highly expressed ( Table 1 ; ref. 49 ). Current indications

suggest this agonist is well tolerated by patients, inducing

modest survival benefi t through initiation of cancer cell

apoptosis ( 49, 50 ). However, targeting malignant cells directly

induces selection of A 3 adenosine receptor–null clones, which

potentially may escape therapeutic intervention. Although

A 3 adenosine receptors are present on immune cell subsets,

the effect of this therapy on the immune system has not been

distinguished. As adenosine receptors show overlapping cel-

lular specifi city and redundancy in pathway activity, it may be

necessary to target multiple receptor subtypes concomitantly

to potentiate an effective treatment response.

COMPARING SMALL-MOLECULE INHIBITORS WITH ANTIBODY-TARGETED THERAPIES

One of the driving forces behind the development of mAbs

in oncology is their historically higher approval rates com-

pared with small molecules. Part of the success of mAbs

comes from the fact that they generally have higher specifi city

and longer half-life, and can exert both direct on-target and

indirect immune-dependent antitumor effects, such as anti-

body-dependent cell-mediated cytotoxicity (ADCC ) and anti-

body-dependent cell-mediated phagocytosis (ADCP; Fig. 2 ).

On the other hand, small molecules are generally associated

with a broader biodistribution. Small molecules can easily dif-

fuse across physiologic barriers such as the blood–brain bar-

rier and plasma membranes. In contrast, mAbs largely rely on

tumor endothelium permeability to penetrate solid tumors,

and endocytic consumption affects mAbs biodistribution.

Upon antigen recognition, antigen–antibody complexes can

be rapidly internalized and degraded, and free antigen turned

over on the cell surface. As with any protein-based biologics,

mAbs also carry an increased risk of immunogenic responses,

even against humanized and fully human mAbs.

Because of the broad expression of CD73, including on

endothelial cells, administration of anti-CD73 mAbs

with ADCC activity may carry the risk of systemic toxici-

ties. Although this has not been reported in mice receiving

anti-mouse CD73, antiendothelial cell antibodies can induce

systemic sclerosis and other vasculitis diseases. In humans,

antiendothelial cell antibodies upregulate adhesion mole-

cules, increase leukocyte adhesion, and promote ADCC ( 51 ).

Through ADCC, anti-CD73 may also deplete lymphocyte sub-

sets. Accordingly, CD73 is expressed on 70% of circulating B

cells, 10% of CD4 + T cells, 80% of naïve CD8 + T cells, and 30% of

memory CD8 + T cells. Nevertheless, higher CD73 levels in the

tumor microenvironment compared with normal tissue might

offer a therapeutic window for ADCC-profi cient anti-CD73.

Compared with a small-molecule inhibitor, an anti-CD73

mAb offers the possibility to target both adenosine-depend-

ent and adenosine-independent CD73 functions ( Fig. 2 ).

Indeed, accumulating evidence suggests that CD73 is more






than just an adenosine-producing enzyme. Early studies have

shown that anti-CD73 mAbs (e.g., clone 1E9), in combination

with suboptimal mitogenic signals, can stimulate human

T-cell activation. Interestingly, immortalized human T cells

(Jurkat) expressing enzymatically inactive CD73 can still be

coactivated with anti-CD73. These fi ndings raise the possibil-

ity that a physiologic ligand for CD73 may be involved dur-

ing T-cell activation, although this is yet to be demonstrated.

Interaction with SRC protein kinases has been suggested as

a mechanism of signal transduction by CD73. One study

has shown that anti-CD73 mAbs can induce intracellular

kinase signaling in T cells, but they are unable to deliver a

tyrosine phosphorylation signal in endothelial cells ( 52 ). This

supports the view that CD73 molecules on different cells

can have different functions. Anti-CD73 targeting distinct

epitopes can also induce different effects. Although anti-

human CD73 mAb 1E9 promotes T-cell signaling and inhibits

CD73 enzymatic activity, mAb 4G4 induces CD73 shedding

from the T-cell surface and fails to promote mitogenic activ-

ity. 4G4 nevertheless enhances human lymphocyte adhesion

to endothelial cells ( 53 ). The anti-human AD2 mAb, on the

other hand, induces clustering and internalization of CD73,

but has minimal effect on enzymatic activity ( 39 ).

COMBINATORIAL POTENTIAL OF ADENOSINE METABOLISM INHIBITION

Combining therapies with distinct abilities to heighten

immune effector functions or limit angiogenic potential is

likely to enhance treatment effi cacy. In this section, we will

consider combinations where preclinical data support their

rapid translation to the clinic.

The mechanism of action for current conventional can-

cer therapeutics, chemotherapy and radiotherapy, is consid-

ered largely reliant on direct cytotoxicity of tumor cells via

genome instability and inability to repair vast amounts of

DNA damage. In addition, some of these therapies enhance

immunogenicity of tumor cells through increased MHC-I

Figure 2. Mechanism of action for antibody-mediated therapies versus small-molecule inhibitors. Antibody-based therapies operate via a range of mecha-nisms depicted in this fi gure and representing the different immune mechanisms that have been proposed to explain the therapeutic effect of anti-CD73 mAb. In particular, identifi ed epitope-specifi c functions of anti-CD73 mAbs include enzyme inhibition (A), shedding (B), and clustering and internalization (C), all of which restrict adenosine production by reducing enzyme availability. Additional antibody-mediated mechanisms such as ADCC (D) and ADCP (E) must be considered when formulating a humanized antibody for clinical utility. In contrast, small-molecule inhibitors elicit effi cacy primarily by blockade of catalytically active enzymatic sites as well as competitive inhibition of ligand binding on receptors (F). AR, adenosine receptor; FcR, Fc receptor.

A B C

D E F

Enzyme inhibition Shedding of target Clustering and internalization

ADCC

AMP

Adenosine

CD73

AMP

NK cell

Tumor cells

NK cell

FcR

Tumor

cell lysis

FcR

Antibody

ADCP

Perforin and granzyme

released

Tumor cells TTTTT

FcR

FcR

Phagocytosis

Small-molecule inhibitors

CD73 inhibitor

(APCP)

AMP

Adenosine

AMP

A2A AR

Adenosine

= immunosuppression

A2A AR

inhibitor

Reduced adenosine signaling

Enhanced effector and cytotoxic

functions

CD8+ T cells

Tregs

Macrophage






expression and initiate immunogenic cell death, stimulat-

ing an immune response, enabling patients to actively clear

residual malignant cells as well as distant metastases ( 54 ).

Cellular stress initiated by these therapeutic modalities

increases extracellular ATP release, leading to adenosine

production impeding the immune response ( 55 ). Induction

of CD73 and to a lesser extent CD39 by the anthracycline

doxorubicin was identifi ed in human breast cancer cell lines,

predominantly those lacking the ER ( 23 ). Patients with

TNBC expressing high levels of CD73 display increased risk

of relapse and poor prognosis following anthracycline treat-

ment. Similarly, transplantable TNBC murine models with

transduced CD73 confer increased anthracycline resistance

and reduced survival ( 23 ). This was abrogated following

combination treatment by doxorubicin and anti-CD73 or

A 2A adeno sine receptor inhibitor. Blockade of CD73 in com-

bination with doxorubicin robustly increases the frequency

of tumor-specifi c CD8 + T cells, correlating with the increased

IFNγ response essential for chemotherapeutic effi cacy ( 23 ,

54 ). Furthermore, chronic lymphocytic leukemia (CLL)

expressing CD73 was shown to have greater proliferative

capacity, particularly in localized perivascular regions ( 22 ).

Generation of adenosine by these cells attenuated both spon-

taneous and drug-induced apoptosis; however, inhibition of

A 2A adenosine receptor signaling rectifi ed CLL chemosensi-

tivity ( 22 ). Although antibodies and small-molecule inhibi-

tors against CD73 have yet to be tested in humans, clinically

tested therapies against downstream adenosine receptors

are available. Clinical investigations targeting A 2A adenosine

receptors have predominantly related to neurologic and

autoimmune disorders ( 56 ). Because of the excellent safety

profi le of these adenosine receptor antagonists, it is feasible

to consider a rapid translation toward clinical testing in

patients with cancer.

Manipulation of effector T-cell activity through blockade

of immune checkpoint receptors provides durable antitumor

responses in a proportion of patients ( 4, 5 ). Although both

ipilimumab and nivolumab extend patient survival, combi-

natorial treatments targeting multiple immunosuppressive

pathways appear necessary for optimal curative benefi t. The

development of toxicity in the form of irAEs due to systemic

stimulation of the immune system is of current concern

for clinicians. Because of excessive adenosine production in

the tumor microenvironment, it is expected that adenosine-

targeted therapies will specifi cally enhance antitumor immu-

nity with limited autoimmune pathologies. Nevertheless , due

to the central role of adenosine in dampening infl amma-

tion, great caution must be taken in early-phase trials while

targeting this pathway. Immune checkpoint blockade by

nivolumab and ipilimumab and inhibition of the adenosin-

ergic system display increased antitumor effi cacy via similar,

nonredundant enhancement of CD8 + T-cell responses ( 8 ,

23 , 57 ). Concurrent treatment schedules may synergistically

enhance antitumor effector function allowing for reduc-

tion in maximal dosage, potentially limiting development of

irAEs. Recently, anti-CD73 therapy in combination with both

anti-PD1 and anti-CTLA4 therapy was shown to synergisti-

cally reduce syngeneic tumor burden and extend survival via

IFNγ-dependent, Th1 CD4 + -driven expansion of tumor-spe-

cifi c CD8 + T cells ( 57 ). The adenosine analog NECA appeared

to heighten expression of PD1, but not CTLA4, on tumor-

specifi c T cells. However, this was attenuated by antagonism

of the A 2A adenosine receptor, potentially increasing anti-PD1

potency ( 57 ).

Blockade of the A 2A adenosine receptor signifi cantly

reduces metastatic burden and improves survival in experi-

mental and spontaneous metastases models ( 38 ). Use of

ipilimumab and nivolumab in advanced cancer settings, such

as the treatment of nonresectable metastatic melanoma, par-

allels these preclinical fi ndings. It is therefore conceivable

that immune checkpoint inhibitors and A 2A adenosine recep-

tors with corresponding, nonredundant functions may be

targeted to provide synergistic antimetastatic activity within

the clinic. However, screening for CD73 status of disease

must be considered to gain maximal effi cacy from adenosine-

targeted treatments ( 38 ). In particular, certain cancer types,

such as TNBC, in which CD73 expression correlates with

poor prognosis, should be targeted to examine potential effi -

cacy of therapeutic combinations involving the adenosinergic

pathway.

CONCLUSION Generation of extracellular adenosine is greatly dependent

on the ecto-enzyme CD73, which seems to be highly abun-

dant in the hypoxic tumor microenvironment.

CD73 acts as a prognostic biomarker for decreased sur-

vival, increased metastatic activity, and multidrug chemore-

sistance in various types of cancer. Protumorigenic effects of

CD73 are at least partly attributed to immunosuppression

via A 2A adenosine signaling in antitumor immune cells. The

recent clinical studies of A 2A adenosine receptor antagonists

in Parkinson disease are indicative of their potential safety

for rapid translation. In particular, preclinical evidence sug-

gests that A 2A antagonists may potentiate current chemo-

therapy and immunotherapeutic strategies. The development

of therapies that modulate adenosine receptor signaling in

the cancer setting must be taken forward cautiously, but are

likely to improve treatment effi cacy.

Disclosure of Potential Confl icts of Interest J. Stagg and M.J. Smyth declare a current sponsored research agree-

ment with Medimmune LLC. No potential confl icts of interest were

disclosed by the other authors.

Acknowledgments The authors would like to acknowledge Sherene Loi and Phillip

Darcy for helpful discussions and Shin Foong Ngiow for his assist-

ance with formulating fi gures.

Grant Support A. Young is supported by a Cancer Council Queensland Ph.D.

fellowship. M.J. Smyth is supported by a National Health and Medi-

cal Research Council of Australia (NHMRC) Australia Fellowship

(628623) and Program Grant (1013667). D. Mittal is supported by a

Program Grant from Susan G. Komen for the Cure (#IIR12221504).

J. Stagg is supported by the Canadian Institutes of Health Research

(CIHR) and the Famille Jean-Guy Sabourin Research Chair.

Received April 1, 2014; revised May 27, 2014; accepted May 29,

2014; published OnlineFirst July 17, 2014.






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2014;4:879-888. Published OnlineFirst July 17, 2014.Cancer Discovery Arabella Young, Deepak Mittal, John Stagg, et al. Targeting Cancer-Derived Adenosine:New Therapeutic Approaches

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Targeting Cancer-Derived Adenosine: New Therapeutic …adenosine receptor ( ADORA2B ) gene expression represents a potential global mechanism of hypoxia response, interaction between