Abstract 2127: Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway

Targeting NAD(H) metabolism is being interrogated as a novel anti-cancer strategy. Nicotinamide phoshophoribosyltransferase (NAMPT) was found to be overexpressed in tumor cells, and plays a key role in the replenishment of the NAD(H) pool in cells. In this study we have used a commercially available NAMPT inhibitor, GMX1778, which exerts a cytotoxic effect by decreasing the cellular level of NAD(H). Through serial selection with increasing sub-lethal concentrations of GMX1778, we derived a GMX1778-resistant HT1080 human fibrosarcoma cell line (HT1080-GMX). The IC50 value for GMX1778-induced cell killing of the resistant cell line was 100-fold greater than that determined for the parental cell line. No significant change in the level of expression of NAMPT was observed in HT1080 vs. HT1080-GMX cells. However, a key enzyme in the de novo NAD(H) synthesis pathway known as quinolinate phosphoribosyl transferase (QPRT) was significantly upregulated as validated by Western blot and bDNA analysis. Knockdown of QPRT partially restored the sensitivity to GMX1778 exposure in HT1080-GMX cells, but did not affect the parental cells. The cytotoxicity of GMX1778 can be partially rescued with exogenous quinolinic acid, which permits NAD(H) repletion via QPRT in the de novo pathway, in HT1080-GMX cells but not HT1080 parental cells. Overexpression of QPRT in HT1080 cells did not fully rescue GMX-induced cytotoxicity and strongly suggests that there are additional mechanisms of resistance in the HT1080-GMX cell line beyond QPRT overexpression. Exome sequencing of the NAMPT gene in the HT1080-GMX resistant lines identified a single mutation of amino acid residue 18 from tyrosine to cysteine [NAMPT(Y18C)] located in the first exon of one allele. Tyrosine 18 is located near the active site of NAMPT and associates with NAMPT small molecule inhibitors through a pi stacking interaction, as determined by studies of the enzyme9s crystal structure. Overexpression of the NAMPT mutant Y18C in HT1080 cells significantly reduced the sensitivity to GMX1778 exposure, but overexpression of a NAMPT Y18F mutant had no effects compared to the parental line. Based on our results, activation of an alternate NAD(H) biosynthesis pathway via QPRT or the Y18C mutation within the NAMPT coding sequence can both be contributors to resistance in this line. The characterization of resistance inducing up-regulation of other sources of NAD(H) such as de novo synthesis or mutations in NAMPT may be useful in developing next-generation NAMPT inhibitors that are less affected by known resistance mechanisms. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: jun guo, Chris Tse, William N. Pappano. Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2127.