Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

Autoři: Said A. Goueli aff001;  Kevin Hsiao aff001
Působiště autorů: Department of Cell Signaling, Research and Development, Promega Corp. Madison, WI, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increase significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressive and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and produces AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known inhibitors as well as monoclonal antibodies. This should speed up screening for novel inhibitors that might lead to more effective cancer therapy.

Klíčová slova:

Cancer treatment – Cell membranes – Enzyme inhibitors – Enzymes – Luminescence – T cells – Enzyme purification – Adenosine


1. Sharma P, Hu-Lieskovan S, Wargo J.A, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 2017; 168: 707–723 doi: 10.1016/j.cell.2017.01.017 28187290

2. Blay J, White T.D, Hoskin D.W. The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res. 1997;57: 2602–2605 9205063

3. Spychala J. Tumor-promoting functions of adenosine. Pharmacology &Therapeutics 2000; 87:161–173

4. Antonioli L, Yegutkin GG, Pacher P, Blandizzi C, Hasko G. Anti-CD73 in cancer immunotherapy: Awakening new opportunities. Trends in Cancer 2016; 2: 95–109 doi: 10.1016/j.trecan.2016.01.003 27014745

5. Beavis P.A, Stagg J, Darcy P.K, Smyth M.J. CD73: a potent suppressor of antitumor immune responses Trends in Immunology 2012; 33: 231–237 doi: 10.1016/ 22487321

6. Di Virgilio F, Adinolfi E. Extracellular purines, purinergic receptors, and tumor growth. Oncogene 2017; 36: 293–303 doi: 10.1038/onc.2016.206 27321181

7. Yang J, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China Liao X, Department of Oncology, Ganzhou People`s Hospital, Ganzhou, 341000, China Yu J, Department of Medicine, University of Louisville, Louisville, KY 40292, United States Zhou P. Role of CD73 in Disease: Promising Prognostic Indicator and Therapeutic Target. Cur. Med. Chem. 2018; 25: 2260–2271

8. Bastid J, Regairaz A, Bonnefoy N, Cecile D, Giustiniani J, Laheurte C, et al. Inhibition of CD39 enzymatic function at the surface of tumor Cells alleviates their immunosuppressive activity. Cancer Immunol Res. 2015; 3:254–264 doi: 10.1158/2326-6066.CIR-14-0018 25403716

9. Hunsucker S.A, Mitchell BS, Spychala J. The 5’-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacology and Therapeutics 2005;107: 1–30 doi: 10.1016/j.pharmthera.2005.01.003 15963349

10. Turcotte M, Spring K, Pommey S, Chouinard G, Cousineau I, George J, et al. CD73 is associated with poor prognosis in high-grade serous ovarian cancer. Cancer Res. 2015; 75: 4494–4503 doi: 10.1158/0008-5472.CAN-14-3569 26363007

11. Inoue Y, Yoshimura K, Kurabe N, Kahyo T, Kawase A, Tanahashi M et al. Prognostic impact of CD73 and A2A adenosine receptor expression in non-small-cell lung cancer. Oncotarget. 2017; 8:8738–8751 doi: 10.18632/oncotarget.14434 28060732

12. Xu S, Shao QQ, Sun JT, Yang N, Xie Q, Wang DH et al. Synergy between the ectoenzymes CD39 and CD73 contributes to adenosinergic immunosuppression in human malignant gliomas. Neuro Oncology 2013;15: 1160–1172 doi: 10.1093/neuonc/not067 23737488

13. Hausler SF, Del Barrio IM, Diessner J, Stein RG, Strohschein J, Honig A et al. Anti-CD39 and anti-CD73 antibodies A1 and 7G2 improve targeted therapy in ovarian cancer by blocking adenosine-dependent immune evasion. Am J Transl. Res. 2014; 6: 129–139 24489992

14. Hilmenyuk T, Kallert S, Klar R, Hinterwimmer L, Schell M, Van Ark M et al. Inhibition of Cd39 and Cd73 by 3rd-generation antisense oligonucleotides to improve immunity against tumors. Proceedings: AACR Annual Meeting 2018;78, Issue 13 Supplement Chicago, IL

15. Hay CM, Sult E, Huang Q, Mulgrew K, Fuhrmann SR, McGlinchey KA. et al. Targeting CD73 in the tumor microenvironment with MED19447. Oncoimmunology 2016; 5: e1208875 doi: 10.1080/2162402X.2016.1208875 27622077

16. Geoghegan JC, Diedrich G, Lu X, Rosenthal K, Sachsenmeier LF, Wu H, et al. Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action. MABS 2016; 8: 454–467 doi: 10.1080/19420862.2016.1143182 26854859

17. Yang X, Pei S, Wang H, Jin Y, Yu F, Zhou B, et al. D. Tiamulin inhibits breast cancer growth and pulmonary metastasis by decreasing the activity of CD73. BMC Cancer 2017; 17: 255–266 doi: 10.1186/s12885-017-3250-4 28399915

18. Perrot I, Michaud HA, Giraudon-Paoli M, Vivier E, Pature lC, Bonnefoy N. Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies. Cell Reports 2019; 27: 2411–2425 doi: 10.1016/j.celrep.2019.04.091 31116985

19. Young A, Ngiow SF, Barkauskas DS, Suit E, Hay C, Blake SJ et al. Co-inhibition of CD73 and A2AR Adenosine signaling improves anti-tumor immune responses. Cancer Cell 2016; 30: 391–403 doi: 10.1016/j.ccell.2016.06.025 27622332

20. Borodovsky A, Wang Y, Ye M, Shaw JC, Sachsenmeier K; Deng N et al. Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models AACR Annual Meeting 2018; DOI: 10.1158/1538-7445.AM2018-3751

21. Allard B, Pommey S, Smyth MJ, Stagg J. Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs. Clinical Cancer Res, 2013; 19: 5626–5635

22. Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nature Commun.2018; 9:1–13

23. Mondal S, Hsiao K, Goueli S. Utility of adenine monophosphate detection system for monitoring the activities of diverse enzyme reactions. Assay and Drug Development Technologies 2017; 15: 330–341 doi: 10.1089/adt.2017.815 29120675

24. Bono MR, Fernandez D; Flores-Santibanez F, Rosemblatt M, Sauma D. CD73 and CD39 ectonucleotidaes in T cell differentiation: Beyond immunosuppression. FEBS Letters 2015; 589: 3454–3460 doi: 10.1016/j.febslet.2015.07.027 26226423

25. Yegutkin GG Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signaling cascade. Biochem. Biophys. Acta. 2008; 1783: 673–694 doi: 10.1016/j.bbamcr.2008.01.024 18302942

26. Freundlieb M, Zimmermann H, Müller CE. A new, sensitive ecto-5'-nucleotidase assay for compound screening. Anal Biochem 2014; 446: 53–58 doi: 10.1016/j.ab.2013.10.012 24144488

27. Bianchi V, Spychala. Mammalian 5’-nucleotidases. J. Biol. Chem. 2003; 278:46195–46198 doi: 10.1074/jbc.R300032200 12947102

28. Knapp K, Zebisch M, Pippel J, El-Tayeb A, Muller CE, Strater N Crystal structure of the human ecto-5’-nucleotidase (CD73): Insights into the regulation of purinergic signaling. Structure 2012; 20: 2161–217 doi: 10.1016/j.str.2012.10.001 23142347

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden