Download Mini review Targeting Cancer-Derived Adenosine: New Therapeutic

Published OnlineFirst July 17, 2014; DOI: 10.1158/2159-8290.CD-14-0341
Mini review
Targeting Cancer-Derived Adenosine:
New Therapeutic Approaches
Arabella Young1,2, Deepak Mittal1,2, John Stagg3, and Mark J. Smyth1,2
CD73 generation of immunosuppressive adenosine within the hypoxic tumor microenvironment causes dysregulation of immune cell infiltrates, resulting in tumor
progression, metastases, and poor disease outcomes. Therapies targeted toward the adenosinergic
pathway, such as antibodies targeting CD73 and CD39, have proven efficacy in mouse tumor models;
however, humanized versions are only in preliminary development. In contrast, A2A 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.
Abstract
Significance: 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 benefit from targeting adenosinergic pathways, alone or in combination with contemporary approaches to cancer treatment. Cancer Discov; 4(8); 1–10. ©2014 AACR.
INTRODUCTION
Immunosuppression within the tumor microenvironment causes inability of host immune effectors to eliminate
transformed malignant cells. Although immune infiltrate
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 macrophages (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-inflammatory
cytokines and metabolites by the tumor together with these
suppressive cell types antagonize tumor immunity by inhibiting 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.
Authors’ Affiliations: 1QIMR Berghofer Medical Research Institute;
2
School of Medicine, University of Queensland, Herston, Queensland, Australia; and 3Centre de Recherche du Centre Hospitalier de l’Université de
Montréal, Faculté de Pharmacie et Institut du Cancer de Montréal, Montréal, Québec, Canada
Corresponding Author: Mark J. Smyth, QIMR Berghofer Medical Research
Institute, 300 Herston Road, Herston, 4006, QLD, Australia. Phone: 61-7-38453957; Fax: 61-7-3362-0111; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-14-0341
©2014 American Association for Cancer Research.
Targeted therapies and immunotherapies offer advantages
over conventional cytotoxic cancer treatments. Targeted therapies limit the availability of critical metabolites necessary
for tumor survival, reducing off-target effects and minimizing 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-infiltrating lymphocytes,
have been highlighted as targets to reset the effector T-cell
immune response. The monotherapies ipilimumab (antiCTLA4) and nivolumab (anti-PD1) have shown success in
enhancing patient survival in a growing number of cancer types (4, 5). Nevertheless, tumors can deploy multiple
mechanisms to allow their survival by avoiding immunosurveillance. 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).
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Targeting the immune system to instigate enhanced effector 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 significantly 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 compatible therapies that present synergistic antitumor efficacy
in combination with minimal toxicity and autoimmunity
is a current clinical challenge. Identification of alternate
immuno­suppressive pathways, which may be targeted to
enhance clearance of malignancies with limited associated
irAEs, will be advantageous.
Recently, the adenosinergic pathway has risen to prominence as a therapeutic target for immune activation. This
review details the effects of adenosine-generated immunosuppression in the tumor microenvironment, highlighting
the potential use of associated enzymes and receptors as
predictive biomarkers for treatment response and progression. 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 metastatic burden.
ADENOSINE-ABUNDANT TUMOR
MICROENVIRONMENT
Adenosine is generated in response to proinflammatory
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 dephosphorylation of AMP to adenosine is catalyzed by CD73 (Fig. 1). Landmark studies by Ohta and colleagues (8) have highlighted
the importance of adenosine for tumor escape. Adenosine
interacts with four G-protein–coupled receptor subtypes (A1,
A2A, A2B, and A3), allowing for diversity and cellular specificity in signaling pathway activity. Each receptor possesses
variable affinity to the adenosine ligand indicative of different
sensitivities and responsiveness to adenosine levels. In particular, the low-affinity A2B adenosine receptor initiates signal
transduction in comparatively limited physiologic conditions. Together, the A2A and A2B adenosine receptors stimulate
Gs/Golf -dependent adenylyl cyclase-driven cAMP accumulation, enabling signal transduction to target genes. A2B adenosine receptors are also coupled to Gq proteins, which can
activate JNK and p38 via phospholipase C–dependent or
–independent pathways (9). Positioning of adenosine receptors, particularly A2A and A2B adenosine receptors that
promote an anti-inflammatory response, on immune cells
reduces tissue injury in conditions of cellular stress, enabling maintenance of tissue homeostasis. The persistence of
increased adenosine concentrations beyond the acute-injury
phase can, however, become detrimental to tissues by activating pathways that trigger immunosuppression or promote
an unremitting wound-healing process. Thus, the accumulation of adenosine in the hypoxic tumor microenvironment
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mediates immunosuppression, causing dysregulation of effector immune cell subsets, dampening the antitumor immune
response.
Rapid proliferation of tumor cells in a manner unsupported 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 deprivation, it also promotes selection of resistant, aggressive cancer
cells able to survive in unfavorable conditions. Hypoxia-resistant malignant cells phenotypically possess stem-like features
with greater proliferative and migratory capacity. This correlates 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,
influencing cellular metabolism and angiogenesis, in an effort
to ease oxygen requirements. Identification 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).
Modification of the HIF1α binding site situated in the CD73
promoter abrogated transcriptional response indicative of a
direct regulatory relationship (10). Similarly, hypoxia-controlled HIF1α-induced expression of the A2B adenosine receptor
was identified, representing a coordinated response that may
contribute to autocrine and paracrine adenosine signaling
(Fig. 1; ref. 11). Although modulation of CD73, CD39, and A2B
adenosine receptor (ADORA2B) gene expression represents a
potential global mechanism of hypoxia response, interaction
between these markers and HIF1α within the tumor microenvironment is yet to be confirmed.
Apart from the mobilization of adenosine via concentrative and equilibrative nucleoside transporters, adenosine production 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 immunosuppressive 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 expression represents an additional mechanism of HIF1α regulatory control. Consequently, increased Treg infiltrate 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 instrumental in regulating an abundance of cellular responses in
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Novel Adenosine-Based Cancer Therapeutics
Increased
• VEGF, IL6, IL10, TGFβ, and IDO
• Modulation of MHC-I and -II antigen presentation
• M2 macrophage polarization, MDSC expansion
DC
TAM
T
MINI REVIEW
Increased
• Polarization to CD4+ Treg
• Proliferation
• Immunosuppression
MDSC
Reduced
• Th1 CD4+ polarization
• IFNγ, IL2, TNFα
production
CD4
D4+ effector
Treg
eg
Adenosine receptors
• A1 and A3 – inhibit cAMP release
• A2A and A2B – stimulate cAMP signaling
A1
NK
A nos e
Adenosine
A3
A2A
CD8+ effector
Decreased
• Granule exocytosisinduced cytotoxicity
• Perforin and CD95L
molecule expression
• Cytokine production
IFNγ, TNFα, IL2,
IL12, MIP1α
• NK cell maturation
Increased
• Production of VEGF,
IL8, angiopoietin-2
• Neovascularization
and proliferation
A2B
Adenosine
os
Adenosine
receptors AMP
ATP
Reduced
• Adhesion to tumor cells
• Cytotoxicity
• TCR signaling
• Activity leading to
induced anergy
Endothelial cells
Endoth
Pro–tumor survival
• Enhanced migration
and metastasis
• Chemoresistance
• Immune evasion
and proliferation
CD73
CD39
Hypoxic core
Tumor cells
ce
•
•
•
•
HIF1α
CD39
CD73
Adenosine
A2B AR
Exosomes
es
Anti–tumor survival
• Inhibited
proliferation
• Enhanced caspase
expression
• Cell death
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 proangiogenic 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.
the tumor. Essentially, adenosine disables cytotoxic effector
functions of both NK and CD8+ T cells predominantly via
A2A­ adenosine receptor signaling, enabling tumor immune
evasion and escape (7). Furthermore, signal transduction
through the A2A adenosine receptor inhibits the Th1 CD4+
T-cell response, limiting the cytokine environment necessary to support these effector cell types. Adenosine polarizes
myeloid cells to develop into immunosuppressive phenotypes such as M2 macrophages and tolerogenic dendritic
cells (DC) due to A2A and A2B adenosine receptor signaling,
respectively (18). In addition, adenosine enhances proliferation 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 studies reported an association between high CD73 levels and
poor prognosis, increased risk of metastasis (21), and resistance to chemotherapy (22, 23). In breast cancer, hypoxiaindependent mechanisms further drive CD73 upregulation.
Spychala and colleagues (24) first 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-specific PCR and DNA pyrosequencing,
CD73 expression in breast cancer cell lines was inversely
associated with CD73 promoter CpG methylation (25). Interestingly, CD73 promoter methylation was associated with
increased disease-free and overall survival and better outcome after adjuvant chemotherapy. These results suggest that
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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 significantly associated 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 suppression of antitumor immunity. In addition to its immunosuppressive effects, we showed that CD73-­derived adenosine
promotes tumor cell migration in vitro and metastasis in vivo
via the A2B adenosine receptor (26). Most interestingly, the
A2B adenosine receptor has recently been identified 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 A2B 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 findings 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-deficiency
improved antitumor immunity and survival by significantly
reducing tumor growth and metastasis (29–32). Development
of bone marrow chimeras confirmed that critical CD39 and
CD73 expression on both nonhematopoietic endothelial and
hemato­poietic Treg cellular lineages mediated tumor growth
(30, 32, 33). However, formation of experimental pulmonary
metastases in CD73-deficient 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 infiltrate in a tumor-dependent manner.
Expansion of tumor antigen–specific CD8+ T cells infiltrating the tumor and circulating within the periphery was
evident in CD73-deficient mice (33). In contrast, deletion of
CD39 reduced Treg-mediated immunosuppression, enabling
increased NK cell infiltration and cytotoxic activity in the
hepatic tumor microenvironment (32).
Mimicking these enhanced antitumor responses through
pharmacologic targeting of CD73 and CD39 identified similar reductions in cancer progression. Small-molecular inhibitors APCP and POM-1, which directly abrogate the catalytic
functions of CD73 and CD39, respectively, inhibited tumor
growth and metastasis (30, 32, 33). Efficacy was negated when
treating their respective gene-targeted mouse strains, indicative of inhibitor specificity (32, 33). Intratumoral injection
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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 inflammasomes, promoting tumor immunity. Antibody-directed immunotherapies
targeting CD73 demonstrated similar antitumor properties
(26). Therapeutic efficacy required the presence of a competent immune system, indicating that antibody blockade of
immunosuppressive adenosine generation enhanced immunosurveillance. 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 antiCD73 (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 adenosine production, CD73 also promotes cellular adhesion, particularly 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, antihuman 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 models implies that metastasis was attenuated by an alternative
mechanism to the improved antitumor immunity previously
determined. An epitope-specific function was identified, in
which recognition of CD73 on cancer cell lines promoted
clustering and internalization of CD73, preventing CD73mediated extravasation and colonization of potential metastatic clones (39). Determining which patients will benefit
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 antiCD73 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-deficient mice present no overt phenotypic
abnormalities, global CD39 deficiency is associated with various physiologic disturbances, including glucose intolerance,
insulin resistance, and increased levels of circulating fatty
acids (42). Importantly, CD39-deficient mice exhibit significantly 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-deficient 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
identified the therapeutic potential and validity of these
approaches as strategies for improving antitumor immune
response (Table 1). Both targets display enhanced expression in a broad spectrum of cancer types, as well as on
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Table 1. Advantages and disadvantages for targeting adenosine generation (CD39 and CD73) and adenosine signaling
(A1, A2A, A2B, and A3 adenosine receptors) pathways
Therapeutic target—localization
CD39
• B cells, NK cells, activated T cells, particularly Tregs, endothelial cells, solid
tumors, and certain leukemias. CD73
• Endothelium and epithelium, stromal
cells, Tregs, MDSC, B cells, CD8+ T cells,
tumor correlating with increased
metastasis.
Potential advantages
Potential disadvantages
• Rate-limiting step converting ATP to AMP
• Increased expression in some cancer types
• Antibody therapies and small-molecule
• Lack humanized antibodies
• POM-1 displays proven selectivity for
• 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
• Current lack of humanized antibodies.
• Development of antibodies that target mul-
CD39, difficult to determine potential offinhibitors [polyoxometalate-1 (POM-1)]
target effects in humans.
• Possible side effects due to total loss of
improve antitumor immunity and in some
CD39.
cases display anticancer potential in murine
models. cancers and proven anticancer properties.
• Use as a predictive biomarker to dictate
A1
• Broad distribution, particularly abundant in nerves, heart, and kidneys. A2A
tiple functions of CD73 will be necessary
for maximal efficacy.
• Highly abundant CD73 expression may
lead to alternative deleterious effects not
identified in murine models.
• Given the above and the longer half-life of
intact mAb, smaller fragments or smallmolecule inhibitors might be required.
therapeutic efficacy of adenosinemediated therapies. • A1 adenosine receptor antagonists and ago- • Disappointing clinical outcomes and
nists have undergone clinical testing, i.e.,
Rolofylline, Tonapofyline, and GW493838.
• A1 adenosine receptor shown to regulate
ER expression in certain breast cancer
subtypes.
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. • Broad distribution, particularly immune • High-affinity adenosine receptor expressed • Small-molecular inhibitors show appropricells NK, CD4+, and CD8+ T cells, macrophages as well as endothelium.
A2B
• Broad distribution, particularly en-
dothelium, MDSCs, DCs also apparent
in some cancer types. A3
• Broad distribution, particularly the
nervous and cardiovascular system
as well as immune cell subsets, highly
expressed in liver and colon cancers. on immune cells.
• Antagonism promotes antitumor immunity,
enhancing cytotoxic functions of CD8+ T
cells and NK cells.
• A2A adenosine receptor antagonists and
display excellent safety profiles in clinical
testing for neurodegenerative diseases. ate tissue penetrance and bioavailability
but short half-life ranging in hours.
• Therapeutic efficacy specific to highly
hypoxic adenosine-mediated tumors.
• HIF1α-driven A2B adenosine receptor ex-
• Low-affinity adenosine receptor, may be
• CF101 A3 adenosine receptor agonist in
• Development of therapeutic resistance by
pression in response to the hypoxic tumor
microenvironment.
• Antagonists display anticancer, antiangiogenesis, and enhanced immune efficacy.
clinical trial for the treatment of autoimmune disorders such as psoriasis and
rheumatoid arthritis.
• CF102 A3 adenosine receptor agonist in
clinical trial for treatment of liver cancer.
• Good safety profile and increased survival
in initial clinical testing. necessary to block others, particularly A2A
adenosine receptor, in concert for efficacy.
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.
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immunosuppressive targets within the tumor microenvironment. However, given that these molecules collectively regulate inflammation 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 humanized anti-human CD39 and CD73 antibodies available for
immediate progression toward clinical translation. Because
of the multifunctionality of CD73 (promoting tumor-associated angiogenesis, and cellular adhesion and migration),
CD73 appears to be a most promising target. It is our contention that therapies able to target both its catalytic and
noncatalytic roles would be desirable and are likely to display
the greatest efficacy in the treatment of cancer.
BLOCKADE OF ADENOSINE SIGNALING
ENHANCES ANTITUMOR RESPONSE
Expression of the A2A adenosine receptor on immune cell
subsets has led to this receptor being primarily investigated
as a target to disrupt adenosine-mediated immunosuppression in the tumor microenvironment (Table 1). Immunogenic
CD8+ T cell–regulated tumors were shown to display reduced
growth in A­­2A­ receptor–deficient mice (8). In addition, T cell–
specific knockdown of the A2A and A2B adenosine receptors in
combination reduced metastasis and enhanced survival (8).
Similarly, antagonism of both A2A and A2B 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. A2A adenosine receptor antagonism mediated its response via a lymphocyte-dependent
perforin-mediated mechanism, whereas metastatic progression was inhibited via the A2B 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 A2B adenosine receptor antagonism in an IFNγ-activated T cell–dependent manner (45).
Recently, A2A adenosine receptor expression was localized
in the cell membrane of malignant lung adenocarcinoma tissue (46). Blockade of the A2A 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 A2A adenosine receptor antagonism. Similarly, A2B adenosine receptor
inhibitors are able to directly influence tumor cell growth and
migration (30). Prostate cancer cell lines, in which A2B was the
dominant adenosine receptor, demonstrated reduced proliferative capacity following blockade of A2B adenosine receptor
or endogenous reduction via siRNA knockdown indicative
of direct anticancer effects (47). Alternatively, promotion
of endothelium-derived angiogenesis by the A2B adenosine
receptor in response to hypoxia is an alternative mechanism
for impeding tumor development (11). Gene-targeted A2B
adenosine receptor mice display impacted VEGF secretion in
the tumor microenvironment, resulting in reduced neovascularization and repressed tumor growth (48).
In contrast to the A2A and A2B adenosine receptors, which
activate signal transduction cascades via cAMP, the A1 and
A3 adenosine receptors downregulate cAMP, enhancing a
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proinflammatory response. Consequently, targeting these
receptors via a different approach is necessary, with agonists
perhaps more suitable for abrogating tumor cell survival.
Currently, the A3 adenosine receptor agonist CF102 is undergoing clinical testing for safety and efficacy in treatment of
hepatocellular carcinoma, in which A3 adenosine receptor
is highly expressed (Table 1; ref. 49). Current indications
suggest this agonist is well tolerated by patients, inducing
modest survival benefit through initiation of cancer cell
apoptosis (49, 50). However, targeting malignant cells directly
induces selection of A3 adenosine receptor–null clones, which
potentially may escape therapeutic intervention. Although
A3 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 cellular specificity 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 compared with small molecules. Part of the success of mAbs
comes from the fact that they generally have higher specificity
and longer half-life, and can exert both direct on-target and
indirect immune-dependent antitumor effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP; Fig. 2).
On the other hand, small molecules are generally associated
with a broader biodistribution. Small molecules can easily diffuse across physiologic barriers such as the blood–brain barrier 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 toxicities. 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 molecules, increase leukocyte adhesion, and promote ADCC (51).
Through ADCC, anti-CD73 may also deplete lymphocyte subsets. 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-proficient anti-CD73.
Compared with a small-molecule inhibitor, an anti-CD73
mAb offers the possibility to target both adenosine-dependent and adenosine-independent CD73 functions (Fig. 2).
Indeed, accumulating evidence suggests that CD73 is more
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Novel Adenosine-Based Cancer Therapeutics
A
B
Enzyme inhibition
Adenosine
MINI REVIEW
Shedding of target
C
Clustering and internalization
AMP
AMP
CD73
Antibody
D
E
ADCC
ADCP
F
Small-molecule inhibitors
Tregs
Adenosine
NK cell
CD73 inhibitor
(APCP)
FcR
AMP
FcR
Tumor cells
Macrophage
Tumor cells
T
AMP
CD8+ T cells
NK cell
FcR
Perforin and granzyme
released
A2A AR
FcR
Tumor
cell lysis
A2A AR
inhibitor
Adenosine
= immunosuppression
Phagocytosis
Reduced adenosine signaling
Enhanced effector and cytotoxic
functions
Figure 2. Mechanism of action for antibody-mediated therapies versus small-molecule inhibitors. Antibody-based therapies operate via a range of mechanisms depicted in this figure and representing the different immune mechanisms that have been proposed to explain the therapeutic effect of anti-CD73
mAb. In particular, identified epitope-specific 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 efficacy 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.
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 findings raise the possibility that a physiologic ligand for CD73 may be involved during 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 antihuman 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 efficacy. In this section, we will
consider combinations where preclinical data support their
rapid translation to the clinic.
The mechanism of action for current conventional cancer therapeutics, chemotherapy and radiotherapy, is considered 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
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Young et al.
MINI REVIEW
expression and initiate immunogenic cell death, stimulating 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 identified 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 treatment. 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
A2A adeno­sine receptor inhibitor. Blockade of CD73 in combination with doxorubicin robustly increases the frequency
of tumor-specific CD8+ T cells, correlating with the increased
IFNγ response essential for chemotherapeutic efficacy (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 spontaneous and drug-induced apoptosis; however, inhibition of
A2A adenosine receptor signaling rectified CLL chemosensitivity (22). Although antibodies and small-molecule inhibitors against CD73 have yet to be tested in humans, clinically
tested therapies against downstream adenosine receptors
are available. Clinical investigations targeting A2A adenosine
receptors have predominantly related to neurologic and
autoimmune disorders (56). Because of the excellent safety
profile 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, combinatorial treatments targeting multiple immunosuppressive
pathways appear necessary for optimal curative benefit. 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 adenosinetargeted therapies will specifically enhance antitumor immunity with limited autoimmune pathologies. Nevertheless, due
to the central role of adenosine in dampening inflammation, great caution must be taken in early-phase trials while
targeting this pathway. Immune checkpoint blockade by
nivolumab and ipilimumab and inhibition of the adenosinergic system display increased antitumor efficacy via similar,
nonredundant enhancement of CD8+ T-cell responses (8,
23, 57). Concurrent treatment schedules may synergistically
enhance antitumor effector function allowing for reduction 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 synergistically reduce syngeneic tumor burden and extend survival via
IFNγ-dependent, Th1 CD4+-driven expansion of tumor-specific CD8+ T cells (57). The adenosine analog NECA appeared
OF8 | CANCER DISCOVERY August 2014
to heighten expression of PD1, but not CTLA4, on tumorspecific T cells. However, this was attenuated by antagonism
of the A2A adenosine receptor, potentially increasing anti-PD1
potency (57).
Blockade of the A2A adenosine receptor significantly
reduces metastatic burden and improves survival in experimental and spontaneous metastases models (38). Use of
ipilimumab and nivolumab in advanced cancer settings, such
as the treatment of nonresectable metastatic melanoma, parallels these preclinical findings. It is therefore conceivable
that immune checkpoint inhibitors and A2A adenosine receptors 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 efficacy from adenosinetargeted treatments (38). In particular, certain cancer types,
such as TNBC, in which CD73 expression correlates with
poor prognosis, should be targeted to examine potential efficacy 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 abundant in the hypoxic tumor microenvironment.
CD73 acts as a prognostic biomarker for decreased survival, increased metastatic activity, and multidrug chemoresistance in various types of cancer. Protumorigenic effects of
CD73 are at least partly attributed to immunosuppression
via A2A adenosine signaling in antitumor immune cells. The
recent clinical studies of A2A adenosine receptor antagonists
in Parkinson disease are indicative of their potential safety
for rapid translation. In particular, preclinical evidence suggests that A2A antagonists may potentiate current chemotherapy 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 efficacy.
Disclosure of Potential Conflicts of Interest
J. Stagg and M.J. Smyth declare a current sponsored research agreement with Medimmune LLC. No potential conflicts 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 assistance with formulating figures.
Grant Support
A. Young is supported by a Cancer Council Queensland Ph.D.
fellowship. M.J. Smyth is supported by a National Health and Medical 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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Novel Adenosine-Based Cancer Therapeutics
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Targeting Cancer-Derived Adenosine: New Therapeutic
Approaches
Arabella Young, Deepak Mittal, John Stagg, et al.
Cancer Discovery Published OnlineFirst July 17, 2014.
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