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 preactivated nucleoside released. 3′-Deoxyadenosine (3′-dA) is a naturally occurring adenosine analogue with established anticancer 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 IC50 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 patients with cancer. 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 tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.

Figure S2 related to figure 3: Validation of top hits from genome wide haploid screen A) Genes of interest were deleted using an all-in-one gRNA-CRISPR/Cas9 construct and following Puromycin selection. Dose response curves of polyclonal HAP1 knockout cells (black) and wildtype control cells (red) from selected gene hits of the genome wide haploid screen. Cells were treated with NUC-7738 or 3'-dA for 48 hours. B) NUDT2 was deleted in HAP1 cells using all-in-one gRNA-CRISPR/CAS9, as shown by Western blot analysis of NUDT2 knockout cells using specific antibodies to NUDT2. Polyclonal wildtype control and knockout cells were treated with either NUC-7738 or 3'-dA. IC50 values were determined and the fold changes between knockout and wildtype cells were calculated. C) Single cell-derived clonal knockouts for NUDT2 (KO1 and KO2) and two wildtype isogenic cell lines. Western blot analysis of NUDT2 knockout cells using specific antibodies to NUDT2 is shown. IC50 values were determined following treatment with NUC-7738 or 3'-dA and the fold changes between knockout and wildtype cells were calculated. D) HINT1 knockout HAP1 cells were treated with 3'-dA or NUC-7738 and levels of cleaved PARP were analysed on a Western blot using specific antibodies.

Figure S5 related to figure 5: NUC-7738 down-regulates cell survival pathways and induces apoptosis. A) A) Renal 786-O and UM-RC-2 cells were treated with NUC-7738 for 24 hours and stained for NF-kB p65 and nuclear dsDNA (DAPI). P65 was seen in the nucleus of controls but disappeared after treatment with NUC-7738. B) Haematoxylin and Eosin (H&E) staining of ex vivo tissue treated with NUC-7738 for 24 hours. H&E examination showed increased numbers of shrunken pyknotic nuclei corresponding to caspase 3 positive cells.

Table S2 related to Figure 2: Genome wide haploid genetic screen identifies genes necessary for the activity of 3'-dA and NUC-7738

NUC-7738 and 3′-dA affect the NF-κB pathway. A, Enrichment plots for NF-κB pathway for all 4 conditions. ES, Enrichment score. B, Expression levels of genes found in the leading edge of NF-κB pathway enrichment. Blue indicates downregulation and brown indicates upregulation of transcript. Significance plot indicates whether the gene was significantly differentially expressed. Red stands for Padj > 0.05 and green Padj < 0.05. Order of columns corresponds with expression heatmap. C, NF-κB activity was measured using the SEAP reporter gene assay in THP-1 cells. NF-κB activity was induced with lipopolysaccharide (LPS) in the presence or absence of NUC-7738. SEAP production as result of NF-κB activity was measured using the QuantiBlue colorimetric enzyme assay. Values were normalized to untreated LPS stimulated cells. D, NF-κB activity was induced with LPS in the presence or absence of NUC-7738 in NF-κB THP1 reporter cell line. Cells were harvested and fractionated into nuclear and cytosolic fraction. Western blot analysis using specific antibodies was employed to detect NF-κB p65 (RELA), nuclear marker Lamin B1, and cytosolic marker GAPDH. E, NF-κB p65 and Caspase 3 in ex vivo tissue treated with NUC-7738 for 24 hours. NF-κB p65 was seen in the nucleus of controls but disappeared after treatment with NUC-7738, and an increase in Caspase 3 was observed after 24 hours. H&E, Hematoxylin and eosin.