Professor Rajgopal Govindarajan | The Role of CNT1 Nucleoside Transporter in Nucleotide Homeostasis

A supply of nucleotides is necessary to sustain the body’s genetic and metabolic processes. CNT1, a nucleoside transporter, is instrumental in preventing nucleoside excretion. Professor Rajgopal (Raj) Govindarajan at Ohio State University is delving into CNT1’s in vivo activity, utilising mouse models, mass spectrometry, and metabolomics tools. His research has important implications for cancer therapy.

Nucleotide Synthesis – De Novo or Salvaged

Nucleotides are well known for their roles in molecular biology, especially DNA replication, transcription, and translation, as well as cellular bioenergetics. The ‘energy molecule’ adenosine triphosphate (ATP) is itself a nucleotide. The functional repertoire of nucleotides is considerably more vast than this – they play important roles as cofactors, metabolites, and in signalling. Cells need a pool of nucleotides, either synthesised de novo or salvaged (recovered, at least in part).

In de novo nucleotide synthesis, nucleotide bases are assembled from simpler compounds – this process differs between pyrimidines and purines. The pyrimidine bases, cytosine (C), thymine (T), and uracil (U), are assembled separately and attached to ribose to form nucleosides. The purine nucleobases adenine (A) and guanine (G) have more complex structures. To form nucleosides, purine bases are assembled in sections onto a ribose structure.

Alternatively, nucleotides may be salvaged from the breakdown of DNA, RNA or other compounds. This is orchestrated by several proteins – enzymes, as well as solute carrier (SLC) transporters. Nucleic acids are degraded by nucleases into nucleotides, which are degraded further by nucleosidases and phosphatases into nucleosides. SLCs transport nucleosides into cells across the plasma membrane, and kinases attach phosphates to form nucleotides.

An Important Nucleoside Transporter

There are two SLC families – SLC29A equilibrative nucleoside transporters and SLC28A concentrative nucleoside transporters. There is a wealth of in vitro data, but in vivo data on concentrative nucleoside transporters are lacking. Professor Rajgopal (Raj) Govindarajan and his group at Ohio State University, Columbus, are attempting to fill this data gap.

Professor Govindarajan at Ohio State University in the USA has a special interest in concentrative nucleoside transporter 1 (CNT1), encoded by the Slc28a1 gene, which transports pyrimidine nucleosides. CNT1 is a sodium-nucleoside symporter which co-transports Na⁺ with nucleosides in a 1:1 ratio. CNT1 is expressed on epithelial cell surfaces lining major organs, including the kidneys. In the kidney’s nephrons, solutes are filtered out of glomerulus capillaries into the renal tubule, and those needed are selectively reabsorbed into the bloodstream.

In humans, CNT1 is expressed on renal tubular epithelial cell surfaces. Research conducted by the group indicates that CNT1 is involved in the reabsorption of pyrimidine nucleosides, preventing excretion in urine. The group is delving into CNT1’s in vivo activity using mouse models. Mass spectrometry is a major analytical tool in their lab – time-of-flight mass spectrometry with ultra-high performance liquid chromatography (TOFMS-UHPLC) and liquid chromatography-mass spectrometry (LC-MS/MS).

Slc28a1 Gene Knock-Out

The group generated CNT1-knockout (KO) mice for experiments. The Slc28a1 gene was ‘knocked out’ (Slc28a1−/−) in mouse embryos using CRISPR/Cas9 technology. This involved applying a guide RNA (gRNA) to mutate exon 2 of Slc28a1, a single-stranded DNA oligo to produce a deletion in the start codon, and a Cas9 nuclease to enable the edit. This produced a ‘frameshift’, leading to ‘nonsense’ translation, and deletion of a restriction site (for genotyping). KO mice express no functional CNT1, though they were observed to be phenotypically healthy and fertile, except for mild anaemia and kidney impairment. Wild-type (WT) mice with intact Slc28a1 expression (Slc28a1+/+) were used as controls.

Driving Metabolic Alterations

In humans, CNT1 mutations can lead to kidney disease associated with pyrimidine nucleoside excretion in urine. Could something similar be happening in the mice? Immunohistochemical analyses of WT mice by the group revealed CNT1 localisation on proximal tubule cell surfaces – same as humans. Using TOFMS-UHPLC targeted analyses, the group compared nucleoside levels in urine from KO and WT mice starved for 12 hours. KO mice showed increased excretion of pyrimidine (deoxy)nucleosides vs WT mice, whereas purine excretion was similar between them. This suggests a conserved function of CNT1 between mice and humans – reabsorbing pyrimidine nucleosides, preventing their expulsion in urine. 

The group conducted untargeted analyses of KO mice in a normal physiological state. Mice were fed ad libitum (as much as required), and LC-MS/MS analyses were conducted on urine (KO) and blood plasma (KO and WT mice) over 24 hours. In the absence of CNT1, urine analysis showed increased elimination of nucleosides (pyrimidines and purines), pyrimidine nucleobases, amino acids, carnitine, and cortisol. In KO mice, the absence of CNT1 led to significant alterations in the plasma metabolome, including reductions in purines and carnitine derivatives, alongside increases in amino acids, peptides, fatty acids, and corticosterone (vs WT mice).

Untargeted metabolomics data from both urine and plasma were evaluated using a mummichog algorithm and adapted gene set enrichment analysis (MetaboAnalyst 4.0 software). This revealed that both pyrimidine and purine pathways were highly altered by CNT1 knock-out, with 11 differential pyrimidine metabolites and 19 differential purine metabolites present in the urine of KO mice but not WT mice. In the plasma metabolome, CNT1 knock-out led to a major alteration of the purine pathways, with 12 differential purine metabolites present in the plasma of KO mice but not WT mice.

Impact on Cancer Chemotherapy

Many chemotherapy drugs, including gemcitabine (dFdC), cytarabine, clofarabine, fludarabine, capecitabine, 5-fluorouridine, and 5-fluro-2′-deoxyuridine 5-fluro-2′-deoxyuridine are nucleoside analogues. The group investigated whether the absence of CNT1 leads to reduced exposure of the nucleoside drug. They carried out pharmacokinetic studies using dFdC as a probe. dFdC, a fluoro-substituted 2’-deoxycytidine analogue, is commonly used as a first-line or second-line treatment for prostate cancer and other solid tumours.

In vivo, dFdC is deaminated to form its deoxyuracil product, dFdU. dFdC at a dose of 50 mg/kg was administered intravenously to mice, both KO and WT. Plasma samples, collected at seven time points over 4 hours, and pooled urine samples, collected over 2 hours, were analysed with UHPLC-MS/MS. The absence of CNT1 led to reduced drug exposure in the plasma in KO mice vs WT mice. This was due to increased urinary clearance of dFdC and dFdU.

Could the reduced drug exposure impact the therapeutic effect? To find out, the group investigated the efficacy of dFdC in a mouse model of pancreatic cancer. They injected a Kras/p53-mutated mouse PDAC (KPC) cell line, expressing luciferase, into KO or WT mice, inducing pancreatic tumours. A week later, they intravenously administered dFdC at a dose of 25 mg/kg or a vehicle (saline control) into the mice. To measure the tumour burden, they injected the mice with D-luciferin, which emits light when converted by luciferase and imaged the mice in a bioluminescence imaging system. Following dFdC treatment, KO mice had double the tumour burden of WT mice. Overall survival following vehicle treatment was similar between WT and KO mice (about 24 days). Following dFdC treatment, KO mice lived a median of 39 days compared with over 50 days for WT mice, a significantly shorter overall survival (p<0.02).

The group confirmed that reduced drug exposure due to CNT1 absence reduced efficacy. Could this be mitigated by adjusting the dFdC dose in KO mice? To find out, they repeated the experiment, but this time, KO mice received a 25 mg/kg or 37 mg/kg dose of dFdC. Mice treated at a higher dose lived a median of 42 days, compared with 36 days at the standard dose, a significant improvement in overall survival (p<0.02).

Clinical Implications for Human Health

Professor Govindarajan’s group has discovered the instrumental role of CNT1 in renal pyrimidine nucleoside reabsorption. Although based on mouse studies, the insights may be translatable to humans. The findings suggest that functional alterations in CNT1 could reduce the efficacy of chemotherapy drugs, but this may be overcome by adjusting the dosage. Various factors could inform optimal chemotherapy dosing for patients with cancer – Slc28a1 genotype, CNT1 expression, and kidney function. Embracing these new findings could help inform more effective interventions.