Abstract 414: Biochemical and cellular monitoring of the activity of the ecto-5’-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology

Abstract

Abstract The ecto-5’-nucleotidase (CD73) is glycosylphosphatidylinositol (GPI) anchored cell surface protein that is involved in switching on adenosinergic signaling. Its enzymatic and nonenzymatic activities (via its interaction with extracellular matrix components) are involved in cancer associated processes and not completely independent of each other. It catalyzes the hydrolysis of AMP into adenosine and phosphate, where adenosine plays important role in tumor immune escape. It is overexpressed in many types of cancer cell lines and patient's biopsies including breast, colon, ovarian, gastric, ovarian, etc. In addition to being important as clinical and prognostic marker in cancer patients, overexpression of CD73 is associated with resistance to antitumor agents, and inhibition of CD73 activity or knocking it down by SiRNA reversed chemoresistant phenotype of glioblastoma multiforme cells. Because of the significant role of CD73, it is of importance to develop an assay that monitor the activity of CD73 in order to develop modulators of its activity. Towards this goal, we have developed a bioluminescent assay to monitor the activity of the enzyme in biochemically pure as well cellular anchored enzyme forms. The assay is homogenous and formatted for HTS screening research, very sensitive, and robust as indicated by the high Z’. We also show that the use of inhibitors such as adenosine 5’-(α-β-methylene) diphosphate (APCP) generates data with biochemically pure enzyme similar to the cellular bound form. We also demonstrate that cells that are enriched in CD73 can be easily identified from those that have low CD73. Thus, the current assay is robust, sensitive, and easy to use making it an ideal assay for HTS screening for new modulators of CD73 and generation of potential novel cancer therapeutics Citation Format: Said A. Goueli, Kevin hsiao. Biochemical and cellular monitoring of the activity of the ecto-5’-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 414.