Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation

Jun Guo 1, Lloyd T Lam 1, Kenton L Longenecker 1, Mai H Bui 1, Kenneth B Idler 1, Keith B Glaser 1, Julie L Wilsbacher 1, Chris Tse 1, William N Pappano 1, Tzu-Hsuan Huang 2

Highlights
•Upregulation of QPRT in cancer cell is a novel resistance mechanism to NAMPT inhibitor.
•Single amino acid mutation of NAMPT (Y18C) confers resistance to NAMPT inhibitor.
•A novel NAMPT inhibitor (A-1293201) overcomes the NAMPT(Y18C) resistance mechanism.

Abstract
Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD+), and NAD+ depletion ultimately results in cell death. The rate limiting step within the NAD+ salvage pathway required for converting nicotinamide to NAD+ is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD+ depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD+ synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD+ de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic.

Introduction
One of the key metabolites essential for sustaining cellular energy metabolism is nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential cofactor of oxidation-reduction reactions required during glycolysis and oxidative phosphorylation to produce ATP [1]. NAD+ is also used as a substrate by poly-ADP-ribose polymerases, sirtuins, and ADP-ribosyl transferases, which respectively play important roles in DNA damage repair, deacetylation, and generation of second messengers required for intracellular calcium release [1], [2]. Three NAD+ biosynthetic pathways have been identified in human, and each pathway depends on the expression of pathway-specific enzymes [1], [2]. The rate limiting step in the salvage of NAD+ from nicotinamide (NAM) is catalyzed by NAMPT. Changes in NAMPT activity can impact NAD+ metabolism and NAD+ dependent cellular processes. In the Priess-Handler pathway [3], NAD+ is salvaged from nicotinic acid (NA), and the rate limiting enzyme is nicotinic acid phosphoribosyltransferase (NAPRT), which converts NA to nicotinic acid mononucleatide (NaMN). The de novo pathway, also referred to as the kynurenine pathway, utilizes the amino acid tryptophan as a substrate through a series of eight enzymatic steps to generate quinolinic acid (QA), a substrate of QPRT. QPRT phosphoribosylates QA to generate nicotinic acid mononucleatide (NaMN), which is further converted to NAD+. It has been reported that QPRT is a key enzyme in de novo NAD+ synthesis and QPRT-depleted cells are highly sensitive to spontaneous cell death compared to wild-type cells [4].

Tumor cells are more reliant on the NAMPT salvage pathway for NAD+ regeneration [5]; thus, blocking the function of NAMPT is an attractive therapeutic strategy for the cancer treatment. Two distinct structural classes of NAMPT inhibitors (e.g. GMX1778 and APO866) advanced into clinical trials for oncology indications [5]. Acquired resistance towards NAMPT inhibitors has been reported previously to be caused by increased expression of NAMPT as well as mutations in NAMPT [6], [7]. A GMX1778-resistant clone of HCT116 colon cancer cell line was reported to have an IC50 value for GMX1778-induced cell killing over 1000 fold greater than that determined for the parental cell line. Sequencing analysis revealed a single mutation of amino acid residue 217 from glycine to arginine, which was hypothesized to result in steric clash for binding of GMX1778 within the tunnel to the NAMPT active site [8]. NAMPT mutations that confer resistance of HCT116 and NYH, and PC-3 cells to GMX1778, APO866 and TP201565, an analog of APO866, have also been mapped to G217R, H191R, D93del, and Q388R [6]. Recently, Wang et al. have reported six NAMPT mutations, which include G217R, D93del, G217A, G217V, S165F and S165Y in NAPRT1 deficient tumor cell lines that became resistant to a second generation NAMPT inhibitor, GNE-618, after continuous treatment with
this compound [5], [7].

To further understand mechanisms of NAMPT inhibition resistance and aid design of next-generation small molecule inhibitors, we generated a human fibrosarcoma cell line, HT1080, which is resistant to GMX1778. We identified and characterized a novel point mutation in NAMPT that confers resistance to GMX1778. Interestingly, HT1080 cells expressing this point mutation of amino acid residue 18 from tyrosine to cysteine in NAMPT are completely resistant to GMX1778, but are sensitive to A-1293201, a non-substrate NAMPT inhibitor that we recently disclosed [9]. Furthermore, we found that activation of the de novo NAD+ biosynthesis pathway via upregulation of QPRT could also be a contributor towards resistance to GMX1778. Targeting QPRT may be a promising strategy for the treatment of the tumors that developed resistance to current NAMPT inhibitors through increased utilization of the de novo NAD+ synthesis pathway.

Section snippets
Cell culture and cell assays
HT1080 and NCI-H441 cells were obtained from the ATCC. Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). GMX1778 and APO866 (Sigma) were dissolved in DMSO. An AbbVie novel NAMPT inhibitor (A-1293201) was synthesized using methods described in U.S. Patent 2016/9302989 B2 [10]. Compounds were serially diluted in DMSO then further diluted in cell growth medium, and the final DMSO concentration in the medium was 0.1%. For cell viability assays.

Characterization of the GMX1778 resistant cell line
Previously, resistance to NAMPT small molecule inhibitors in HCT116, NYH, and PC-3 cells has been attributed to NAMPT overexpression and somatic NAMPT coding sequence mutations [6]. To identify other potential mechanisms of resistance to NAMPT inhibitors, we developed a GMX1778 resistant HT1080 cell line (HT1080-GMX) by culturing the cells in gradually increasing concentrations of GMX1778 from 30 nM up to 1000 nM over time. The IC50 value for GMX1778-induced cell killing of the resistant cell.

Discussion
Although many molecularly-targeted therapies show initial efficacy in patients, a significant issue that often arises is the emergence of resistance in tumors, ultimately resulting in treatment failure. Published studies with compounds GMX1778 and APO866 identified increased NAMPT protein levels and point mutations in NAMPT which conferred cellular resistance and suggest that acquired resistance to NAMPT inhibition could be a potential concern [6], [8].

Disclosures
J.G., L.T.L., K.L.L., M.H.B., K.B.I., K.B.G., J.L.W., C.T., W.N.P., and T.H.H. are employees of AbbVie. The design, research, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and CHS828 approval of the manuscript. This does not alter our adherence to BBRC policies on sharing data and materials. Authors have declared that no competing interests exist.