Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a pre-activated nucleoside released. 3'-deoxyadenosine (3'-dA) is a naturally-occurring adenosine analogue with established anti-cancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design:We describe the synthesis of NUC-7738. We determine the IC 50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in cancer patients. Results:We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide binding protein 1. PK and tumour samples obtained from the ongoing first-in-human Phase 1 clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective pro-apoptotic agent in cancer cells with effects on the NF-B pathway.Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and support its further clinical evaluation as a novel cancer treatment within the growing pantheon of anti-cancer ProTides. Statement of translational relevanceProTide modification of nucleoside analogues is designed to overcome the cellular resistance mechanisms that limit their efficacy. This consists of the chemical addition of a protective phosphoramidate moiety onto the parent nucleoside, in this case the natural nucleoside analogue 3'-deoxyadenosine (3'-dA) to form the ProTide NUC-7738. The mode of action of 3'-dA has been the subject of speculation as its major limitation is its short plasma half-life due to rapid enzymatic deamination by adenosine deaminase (ADA). We demonstrate that NUC-7738 is resistant to deamination by ADA and cleaved by the intracellular phosphoramidase HINT1 into 3'-dAMP for conversion to the active metabolites 3'-dADP and 3'-dATP. We show that NUC-7738 promotes proapoptotic pathways and attenuates NF-B. These findings are validated in tumour samples from patients in an ongoing first-in-human trial of NUC-7738 from which we have identified biomarkers to enrich our understanding of NUC-7738 and patients most likely benefiting from it.

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Background: 3'-deoxyadenosine (3'-dA; also known as cordycepin) is a nucleoside analog that has shown potent anti-cancer activity in non-clinical studies but has not been clinically developed because of its vulnerability to rapid degradation by the circulating enzyme adenosine deaminase (ADA) and its poor uptake into cancer cells. The ProTide NUC-7738 is a pre-activated and protected nucleotide analog (3'-dA 5'monophosphate; 3'-dAMP) specifically designed to overcome the limitations of 3'-dA. NUC-7738's phosphoramidate moiety renders it resistant to ADA degradation. Here we compared NUC-7738 to 3'-dA in several model systems prior to conducting a first-in-class dose-escalation/expansion study of NUC-7738 in patients with advanced cancers. Materials and Methods: To determine the potency of NUC-7738, IC50 values were measured in multiple cancer cell lines and compared to the parent compound, 3'-dA. Chemical inhibitors of ADA and other 3'-dA processing enzymes were applied to assess the relative ability of NUC-7738 to bypass these pathways. Using genome-wide gene-trap screens and RNA sequencing we compared mechanisms of action (MOA) for NUC-7738 and 3'-dA. Results: NUC-7738 demonstrated up to 185x greater anti-cancer potency than 3'-dA across a variety of cancer cells lines. Gene trap experiments showed that the intracellular activating enzyme adenosine kinase (ADK) and the hENT1 transporter were amongst the highest enriched genes for 3'-dA, whilst no enrichments for these genes were observed in NUC-7738 treated cells. In support of this, in vitro inhibition assays showed that unlike 3'-dA, NUC-7738 is resistant to ADA breakdown, is not reliant on hENT1 transport for its cellular uptake, and is independent of ADK for its activity. As expected, RNA sequencing analysis demonstrated overlap between the MOA of NUC-7738 and 3'-dA; both cause cancer cell death via the intrinsic apoptosis pathway and suppression of pro-survival signaling. Further investigation of gene candidates was employed in ex-vivo cancer kidney cancer samples. Conclusion: Phosphoramidate chemistry was used to transform the nucleoside analog 3'-dA into NUC-7738, rendering it resistant to degradation by ADA and enabling it to enter cancer cells independent of nucleoside transporters, both of which contribute to NUC-7738's substantially greater in vitro potency compared to 3'-dA. The gene trap approach allowed a sophisticated comparison of the MOA of NUC-7738 with 3'-dA. By overcoming the resistance mechanisms associated with 3'-dA, NUC-7738 generates higher levels of the active anti-cancer metabolite in cancer cells. These data supported the initiation of NuTide:701, a first-in-human Phase I study assessing the safety, tolerability, pharmacokinetics and pharmacodynamics of NUC-7738 in patients with advanced solid tumors that are resistant to conventional treatment. Citation Format: Hagen Schwenzer, Michaela Serpi, Valentina Ferrari, James Chettle, Josephine Morris, Ruud van Stiphout, Erica de Zan, Sebastian Nijman, Mustafa Elshani, Mary Kudsy, David Harrison, Gareth Bond, Sarah P. Blagden. From bench to bedside: Using ProTide chemistry to transform 3'-deoxyadenosine into the novel anti-cancer agent Nuc-7738 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 931.

Background: Metabolic dysregulation, a hallmark of cancer, allows tumor cells to sustain high rates of growth and proliferation in unfavorable conditions, including hypoxia. Many cancers, including clear cell renal cell carcinoma (ccRCC), demonstrate the Warburg effect with high rates of glycolysis and some are dependent on glutamine. These cancers express high levels of glutaminase (GLS), the enzyme responsible for conversion of glutamine to glutamate. GLS exists as two distinct isoforms, GAC and KGA, which are generated by alternative polyadenylation. The presence of GAC favors more metabolically active cell growth, and the GAC:KGA ratio negatively correlates with survival in ccRCC. Selectively inhibiting the GAC isoform may be a target for therapy. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), has shown encouraging efficacy signals in several solid tumor types in a Phase ½ clinical study (NuTide:701 NCT03829254). It generates 3’-dATP which profoundly affects transcription by altering polyadenylation and has been shown to alter expression of mitochondrial electron transport chain genes. The aim of this study was to investigate the impact of NUC-7738 on GLS alternative polyadenylation and the ratio of GAC:KGA isoforms. Material and Methods: ccRCC (Caki-1 [VHL mutant];786-O [wildtype]) and pancreatic (PANC1; MIAPACA) cancer cell lines were treated with NUC-7738 at IC50 doses in hypoxic and normoxic conditions. NUC-7738 and 3’-dATP were measured by LC-MS. mRNA levels of glutaminase isoforms were determined by RT-qPCR using primers targeting exon 14-15 junction for GAC and exon 16-18 junction for KGA. GAC and KGA protein expression determined by JESS Western analysis. Results: NUC-7738 was converted into 3’-dATP within 6 hours of treatment with an average concentration of 8 pmol/106 cells, which was maintained for ~24 hours and decreased by 50% by 48 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. In ccRCC cells (VHL wildtype and mutant), NUC-7738 reduced transcript expression of GAC isoform, with no change in KGA, indicating a change in polyadenylation site usage. This was reflected in altered protein levels, with decreased GAC in ccRCC and pancreatic cell lines in hypoxic and normoxic conditions after NUC-7738 treatment. Conclusion: NUC-7738 generates sustained levels of 3’-dATP in cells, which is associated with alternative polyadenylation site usage changes. In a variety of cell types, mRNA and protein levels of the more metabolically active GAC isoform were reduced, with an increase in the relative amount of KGA isoform. Increased glutamate production and GAC:KGA ratio are key for survival of some tumors. As alternative polyadenylation is implicated in the control of expression of many genes in cancer, the ability to influence it may lead to new strategies for developing anti-cancer treatments interfering with cellular metabolism. Citation Format: Mustafa Elshani, Ying Zhang, Tia Hawkins, Mary Kudsy, In Hwa Um, Greice Zickuhr, Alison L. Dickson, David J. Harrison. NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 277.

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