525 Adenosine uptake through equilibrative nucleoside transporter-1 suppresses antitumor immunity by pyrimidine starvation

<h3>Background</h3> Immunosuppression by adenosine is an important cancer immune checkpoint. Extracellular adenosine can signal through specific receptors or be transported across the cell membrane through nucleoside transporters. While adenosine receptors are known to regulate tumor immunity, the impact of adenosine transporters remains unknown. In this study, we investigated the effect on tumor immunity of equilibrative nucleoside transporter-1 (ENT1), the major regulator of extracellular adenosine concentrations. <h3>Methods</h3> We used selective inhibitors and gene targeting approaches to study the role of ENT1 on tumor growth in vitro and in vivo. We performed flow cytometry and quantitative PCR to profile the immune tumor microenvironment. To study the effect of adenosine uptake via ENT1, we performed coculture assays and metabolomic analysis of activated human T cells in presence of adenosine and a selective ENT1 inhibitor. <h3>Results</h3> In vivo, we demonstrated that blocking or deleting host ENT1 significantly enhanced CD8⁺ T cell-dependent anti-tumor responses. Tumors inoculated into ENT1-deficient mice showed increased infiltration of effector CD8⁺ T cells and significant upregulation of granzyme B, IFN-γ, IL-2, TNF-α and CXCL10. ENT1-deficiency was further associated with decreased tumor-infiltrating T regulatory cells and CD206^high macrophages, and suppressed CCL17 production. ENT1-deficiency also significantly potentiated the therapeutic activity of PD-1 blockade. T cells upregulated ENT1 following activation and blocking ENT1 enhanced their effector function when co-cultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake in activated T cells inhibited the activity of phosphoribosylpyrophosphate synthetase (PRPS), thereby suppressing production of uridine 5′-monophosphate (UMP) and its derivatives required for DNA and RNA synthesis. <h3>Conclusions</h3> In summary, our study identified ENT1-mediated adenosine uptake as an important and previously unappreciated mechanism of adenosine-mediated immunosuppression via pyrimidine starvation that can be targeted to enhance antitumor T cell responses. Our findings have important implications on our interpretation of current adenosine-targeting agents in clinical trial and suggest ENT1 is a potential therapeutic target to enhance the therapeutic activity of immune checkpoint blockade. <h3>Ethics Approval</h3> Animal experimentations were performed in accordance with guidelines from the Canadian Council on Animal Care (CCAC) and were approved by an Institutional Animal Care and Use Committee (Protocol #C22051JSs).

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