Abstract C122: NUC-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro

Abstract

Background: Cordycepin or 3′-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. The ProTide NUC-7738, a phosphoramidate transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesized that it might activate AMP-activated protein kinase (AMPK), the key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Clear cell renal cell carcinoma (ccRCC) is characterized by lipid accumulation due to dysregulation of the genes fundamental in metabolic pathways and so this disease might be a promising target for metabolic treatment. Phosphorylation of AMPK (pAMPK) is associated with downregulation of mTOR signalling, highlighting the potential of NUC-7738 to regulate this important cancer pathway. Methods: The expression of pAMPK and AMPK in ccRCC from 293 patients was analyzed by immunofluorescence, images were captured digitally and were analyzed using QuPath software. The effect of NUC-7738 on the growth and confluence of nine renal cancer cell lines was assessed using SRB assay and Celigo scanner, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in the ratio of pAMPK:AMPK caused by NUC-7738 and results read using LiCor Odyssey. Effect of NUC-7738 on AMPK activation in ex vivo ccRCC tissue slices was analyzed using immunofluorescence and QuPath software. Results: Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. NUC-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment, with inhibition of mTOR activity predominantly observed after 48 hours. NUC-7738 also increased pAMPK levels in ex vivo ccRCC tissue slices. Conclusion: Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. NUC-7738 caused activation of AMPK in ex vivo ccRCC tissue slices and in cell lines, and demonstrated efficacy against cell lines in both normoxic and hypoxic conditions. Renal cancer tissue typically has low expression of pAMPK, raising the prospect that AMPK modulation may offer a therapeutic option for ccRCC. These results suggest that inhibition of the mTOR pathway may be one of the anti-cancer mechanisms through which NUC-7738 exerts its activity. Citation Format: Mary Kudsy, Mustafa Elshani, Sarah Puthur, In Hwa Um, Grant D. Stewart, Maeve Rahilly, Alex Chapman, Michelle Myers, David J. Harrison. NUC-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C122. doi:10.1158/1535-7163.TARG-19-C122