Supplementation of nicotinic acid with NAMPT inhibitors results in loss of in vivo efficacy in NAPRT1-deficient tumor models.

Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy.

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I.

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.

Orotate in milk and urine of dairy cows with a partial deficiency of uridine monophosphate synthase.

Cows with a partial deficiency of uridine monophosphate synthase have elevated orotate in their milk and urine. Variability of orotate was assessed in two such cows monitored biweekly for two complete lactations. Concentrations of milk and urinary orotate showed extensive variation during lactation, but coefficients of variation were similar for deficient and normal cows. Orotate averaged 500 micrograms/ml milk for deficient cows (versus a normal of 80 micrograms/ml), and a threshold of 200 micrograms/ml distinguished normal from deficient cows. Deficient cows had low milk orotate upon initiation of lactation and exhibited a latency of 1 to 7 wk to attain that threshold. The deficiency also resulted in a lactation-induced orotic aciduria. Orotate averaged 32.2 micrograms/ml urine for deficient cows (versus a normal of 7.3 micrograms/ml), and a threshold of 15 micrograms/ml urine differentiated the animals. Latency was 3 to 18 wk for deficient cows to exceed that threshold. Total orotate output and orotate concentration were elevated in milk and urine of lactating cows deficient for uridine monophosphate synthase. The output of orotate was predominantly in milk rather than urine for both deficient and normal cows. Additionally, orotate was elevated in blood of deficient cows when they were lactating.