PT - JOURNAL ARTICLE AU - Rakesh K Singh AU - Leon van Haandel AU - Daniel P Heruth AU - Shui Q Ye AU - J. Steven Leeder AU - Mara L Becker AU - Ryan S Funk TI - Nicotinamide Phosphoribosyltransferase Deficiency Potentiates the Anti-proliferative Activity of Methotrexate through Enhanced Depletion of Intracellular ATP AID - 10.1124/jpet.117.246199 DP - 2018 Jan 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - jpet.117.246199 4099 - http://jpet.aspetjournals.org/content/early/2018/02/02/jpet.117.246199.short 4100 - http://jpet.aspetjournals.org/content/early/2018/02/02/jpet.117.246199.full AB - Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis (JIA). Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX, however the mechanism of this interaction has yet to be defined. Therefore, in this study we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. The siRNA-based silencing of NAMPT expression resulted in a greater than three-fold increase in the sensitivity to MTX (p<0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not found to be significantly altered. MTX was not found to potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (p<0.005). Supplementation with hypoxanthine and thymidine completely reversed the anti-proliferative activity of MTX in NAMPT-deficient cells, and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the anti-folate activity of MTX, and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.