Abstract A062: Preclinical study of the fungal derivative cordycepin as an anti-cancer agent

Abstract

Introduction: Cordycepin or 3′-deoxyadenosine is an extract from the fungus Cordycepssinensis, a popular traditional medicine in the Far East. Although its active metabolite 3′-deoxydenosine 5′-triphosphate (3’dATP) has potent in vitro anti-cancer activity, it has shown limited efficacy in clinical trials. This is due to its poor bioavailability and rapid degradation by the ubiquitous catabolic enzyme adenosine deaminase (ADA). Here, using genetic and cellular cancer model systems, we investigated the biological impact of cordycepin in cancer cells. Experimental Procedures: Cell proliferation was measured in the presence of cordycepin and antagonists to determine its IC50. A haploid leukaemia-derived cell line, Hap1, was mutagenized using retroviral gene trapping and the mutant pool was selected for clones resistant to cordycepin which were sequenced by NGS. CRISP/Cas9 knock out combined with pharmacokinetic determination of the IC50 enabled us to validate the top hits. RNA deep sequencing-based transcriptomic analysis and gene set enrichment were used to identify the signalling pathways most affected by cordycepin and to define possible mechanisms of action. Results: We found 106 significant gene hits using the haploid genetic screen. The intracellular antiproliferative activity of cordycepin depended on both its uptake by human equilibrative nucleoside transporter 1 (hENT1) and its rate-limiting phosphorylation by the intracellular enzyme adenosine kinase (ADK). CRISPR/Cas9 knock out or selective inhibition of hENT1 or ADK led to a greater than 10-fold reduction in the activity of cordycepin. When extracellular degradation by ADA was inhibited, cordycepin showed up to 78x greater anti-proliferative potency in cancer cells. Furthermore, after 6h of treatment with cordycepin, cells showed significant differential expression of more 1000 genes. Integrating both datasets, we demonstrated by gene set enrichment analysis that, once successfully activated within the cell, 3’dATP induces cell death by a dual mechanism; by both activating the intrinsic apoptosis pathway and suppressing the TNFa-NFkB pro-survival signalling axis. Conclusions: We demonstrate here that, in the absence of ADA and in the presence of abundant hENT1 and ADK, the active metabolite of cordycepin, 3’ dATP, works as a dual driver of apoptosis in cancer cells. This suggests that a strategy of bypassing ADA and hENT1 while administering cordycepin or a chemically-modified version of cordycepin (that overcomes these resistance mechanisms) is essential to elicit any clinical anti-cancer effect. We are currently conducting a phase I trial of NUC-7738, a ProTide form of cordycepin designed to overcome these resistance mechanisms. Agents like NUC-7738 can enhance the stability and bioavailability of cordycepin, enabling it to effectively deliver its dualistic anticancer effect in patients. Citation Format: Hagen Schwenzer, Erica De Zan, Ruud van Stiphout, Cornelia H de Moor, Sebastian Nijman, Gareth Bond, Sarah P Blagden. Preclinical study of the fungal derivative cordycepin as an anti-cancer agent [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 A062. doi:10.1158/1535-7163.TARG-19-A062