Abstract 41: Role of the novel glucose-phosphorylating enzyme ADP-dependent glucokinase in human cancer cell lines featuring knockouts generated by zinc finger nucleases

Abstract

Abstract Increased glucose metabolism under aerobic conditions is a well-known hallmark of cancer, and has wide therapeutic implications. ADP-dependent glucokinase (ADPGK) is a novel mammalian glucose-phosphorylating enzyme (Ronimus & Morgan 2004) with the unique ability to utilize ADP as phosphate donor. Mouse recombinant ADPGK is a monomeric protein of 54 kDa with high specificity for glucose and an apparent Km for glucose of 96 μM, which is about a third of that of hexokinase 2 (HK2). Based on its biochemical properties, we hypothesize that ADPGK has a protective role under stress conditions such as hypoxia or low glucose, utilizing ADP to prime glycolysis when ATP becomes limiting. Given the importance of hypoxia in tumor progression and resistance to therapy, this would make ADPGK a potential therapeutic target. To study the role of this novel enzyme in cancer biology, we determined its expression in 23 human cancer cell lines by mRNA microarray and western blot. Real-time PCR and westerns were used to test changes in ADPGK expression under anoxia. siRNA for ADPGK and HK2, and ADPGK knockouts generated with CompoZrTM (Sigma-Aldrich) zinc finger nucleases (ZFN), were used to investigate the role of ADPGK in glycolysis (lactate production and glucose consumption) and clonogenic survival under short-term anoxia and glucose deprivation. High expression of ADPGK was demonstrated across most tumor cell lines tested at both mRNA and protein levels. In contrast to HK2, ADPGK expression was unchanged under anoxia (up to 18 h), which might indicate its constitutive availability in case of a decrease in ATP. 70-80% ADPGK knockdown in H460 cells, validated by western blot and real-time PCR, did not decrease glycolytic activity during 4 hours of anoxia, while ∼60% HK2 knockdown reduced glycolysis by 30-50%. In the same experiments, clonogenic survival was reduced 30% by ADPGK siRNA and 60% by HK2 siRNA, but 4 hours anoxia did not cause an additional decrease. To provide more complete suppression of ADPGK, we used ZFNs to generate ADPGK knockout clones in the human colon cancer cell line HCT116. These were characterized by western blotting, and bi-allelic gene disruption was validated by sequencing across the ZFN cutting site. Knockouts and wildtype were similar in growth and clonogenic survival under oxic as well as anoxic conditions. Initial experiments with 10 mM 2-deoxyglucose under anoxia resulted in a small but non-significant decrease in clonogenic survival for knockout cells. In summary, suppression (siRNA) or knockout (ZFN) of ADPGK appears to have only minor effects on glycolysis and cell survival in vitro in the cell lines evaluated. This may reflect the elasticity and redundancy of glucose phosphorylation by the ATP-dependent hexokinases under the conditions tested to date. The role of ADPGK in hypoxic cell survival in human tumor xenografts is under evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 41.