ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain

Abstract

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5'-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. SIGNIFICANCE STATEMENT: The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.

Fig. 1

Cellular ATP turnover. The extracellular turnover of purines is composed of several steps: 1) the release of endogenous ATP after lytic cell injury or via nonlytic processes of vesicular exocytosis (VE), P2X7 receptors, ATP-binding cassette (ABC), connexin (Cx), and pannexin (Panx) hemichannels; 2) the initiation of signaling events via nucleotide (P2XR and P2YR) and adenosine receptors; 3) the further metabolism of nucleotides and nucleosides; and 4) the intracellular uptake of nucleotide-derived adenosine via ENTs and CNTs. Purine-inactivating pathways include NTPDase1/CD39, ENPP1, ecto-5′-nucleotidase/CD73, and ADA (lower right). Acute changes in the ratio between nucleoside tri- and diphosphates, together with feed-forward inhibition of CD73 through ATP and ADP, can influence the directional shift from adenosine-producing pathways toward the resynthesis of ATP via AK1 and NDPK (lower left).

Fig. 2

Compartmentalization of adenosine biochemistry. Along with the canonical route of sequential ATP and ADP breakdown through CD39, additional ectoenzymatic pathways can contribute to the generation of AMP and its subsequent CD73-mediated hydrolysis to adenosine. Those include the direct conversion of ATP into AMP and PP_i by ENPP1, AK1-mediated transphosphorylation of ADP into ATP and AMP, and alternative adenosine-producing pathways from extracellular NAD and cGAMP, mediated via the CD38-ENPP1-CD73 and ENPP1-CD73 axes, respectively. The extracellularly generated adenosine can further be converted into inosine and hypoxanthine via sequential ADA and PNP reactions or be taken up by the cells via nucleoside-selective transporters. The intracellular adenosine metabolism depends on the concerted action of cytoplasmic forms of purine-inactivating (ADA and PNP) and phosphorylating (ADK-S, AK, and NDPK) enzymes. Mitochondria produce ATP through oxidative phosphorylation (OxPhos). Therefore, there is a tight link between mitochondrial bioenergetics and adenosine homeostasis. In the cell nucleus, adenosine is part of the transmethylation pathway, which adds methyl groups from SAM to DNA (DNA-CH₃) via DNA methyltransferase (DNMT). SAH can also be hydrolyzed to adenosine and L-homocysteine (HCy) by SAHH. Importantly, SAHH catalyzes a bidirectional reaction with the thermodynamic equilibrium favoring the production of SAH from adenosine and HCy. Through this mechanism, nuclear ADK-L drives the flux of methyl groups through the biochemical pathway leading to increased DNA and histone methylation. For other abbreviations, see Table 1.

Fig. 3

Diversity of biologic effects of adenosine and other purinergic agonists in the tumor microenvironment. Adenosine and its precursor metabolites (ATP, ADP, NAD, and cGAMP) trigger diverse protumorigenic and antitumor effects in virtually all components of the TME, mediated via both extracellular and intracellular mechanisms. ATP generally functions as a ‘danger sensor’ and ‘find me’ signal, promoting tumor cell cytotoxicity and phagocytosis by proinflammatory NK cells, cytotoxic T cells, M1-type macrophages, and additional tumor-associated effector immune cells. Furthermore, ATP has a variety of vasoactive, mitogenic, and proangiogenic effects in the TME, whereas its metabolite ADP acts as a potent prothrombotic molecule implicated in cancer-associated thrombosis. ATP-derived adenosine, in turn, attenuates the inflammation and promotes immune evasion by tumor cells and in addition maintains intratumoral blood flow, angiogenesis, lymphangiogenesis, and vascular endothelial and epithelial barrier functions. BEC, blood endothelial cell; CAP, cancer-associated platelet; LEC, lymphatic endothelial cell; MDSC, myeloid-derived suppressor cell; MTC, metastatic tumor cell; NF, normal fibroblast; NLRP3 inflammasome, NOD-like receptor family pyrin domain containing 3 inflammasome; RBC, red blood cell; TAM, tumor-associated macrophage; TC, tumor cell; TIL, tumor-infiltrating lymphocyte.

Fig. 4

Variations in the purinergic signatures between immune hot and immune cold breast tumors. To identify purinergic mechanisms of the tumor escape in the context of the T cell–inflamed and non–T cell–inflamed TME, the gene expression profiles for major purinergic enzymes and ARs were analyzed at single-cell resolution via publicly available scRNA-seq datasets of immune hot (A) and immune cold (B) TNBC samples obtained from two patients (Wu et al., 2020). Raw data were analyzed by Seurat (version 4.0) for graph-based clustering and the analysis of gene expression. Sctransform was used for data normalization and variance stabilization of molecular count data (C. Hafemeister, Genome Biology). Principle component analysis (PCA) and a graph-based clustering approach were used by running the functions FindNeighbors and FindClusters. The clustering was visualized with Uniform Manifold Approximation and Projection (UMAP). Cells were typed based on the expression of known marker genes, as indicated. Dot plots show the expression of key purinergic enzymes and ARs. The average expression levels of the indicated genes are shown on a pseudocolor scale (log2(FC)), with the size of the dot representing the percentage of cells in a subset where the gene is detected relative to other subsets.

Fig. 5

Cell- and tissue-specific distribution of purinergic enzymes in the breast tumor microenvironment. Tumor-draining LNs were obtained from a treatment-naïve breast cancer patient undergoing mastectomy surgery with axillary lymph node dissection. The tumor in this example is a hormone-receptor-negative/Her2-positive infiltrative ductal carcinoma (GIII, Ki-67 25%, LN status 4/13). (A) CD73 activity was assayed by incubating tissue cryosections (6 μm thickness) with AMP in the presence of Pb(NO₃)₂, followed by detection of AMP-derived P_i as a brown precipitate. LNs were also stained with hematoxylin and eosin (H&E) as well as with antibodies against CD73, together with the stromal marker α-smooth muscle actin (SMA-α) and a pan-cytokeratin AE1+AE3 antibody cocktail, which differentiates epithelial tumors from nonepithelial tumors. The bright-field (A) and fluorescence (B) images were captured using Pannoramic Midi and Pannoramic-1000 slide scanners, respectively (3DHistech Ltd., Budapest, Hungary). (C and D) Spatial distribution of purinergic enzymes was also analyzed by high-resolution 3D microscopy. LN tissues were embedded in low melting point agarose, sectioned at 150 μm thickness using a vibrating microtome, and incubated in free-floating staining assays with antibodies against CD73, CD39, and ADK, together with anti-SMA-α and anti-cytokeratin antibodies, as indicated. Z-stacks were captured using a spinning disk confocal microscope and presented as reconstructed 3D images. The right-hand panels display merged images with nuclei counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bars: 2 mm (A), 600 μm (A insets and B), and 60 μm (C and D). For experimental details, refer to Losenkova et al. (2020, 2022). BV, blood vessel; F, fibroblast; GC, germinal center; LV, lymphatic vessel; TC, tumor cell.