NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth

Abstract

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy's role as an adjunctive modality.

Keywords: Glycolysis; N-MYC; NAD; NAMPT (Nicotinamide Phosphoribosyltransferase); metabolism; neuroblastoma; precision medicine.

Figure 1

NAMPT and NAPRT expression in NB cell lines. (A) Pathway of NAD+ generation. De novo pathway denoted with green arrows depicting tryptophan breakdown. Three salvage pathways denoted with orange arrows. Nicotinic acid used as a substrate for Nicotinate phosphoribosyltransferase (NAPRT) and subsequent combination with products downstream of tryptophan prior to final conversion to NAD+ via NAD synthase (NADS). Nicotinamide as a substrate utilized by Nicotinamide phosphoribosyltransferase (NAMPT). Nicotinamide riboside is used as a substrate by nicotinamide riboside kinase (NRK). Each pathway requires Nicotinamide-nucleotide adenylyltransferase (NMNAT) prior to conversion to (NAD+). (B) Expression of NAMPT, NAPRT and N-MYC were evaluated by western blot assay. (C) Immunofluorescence of NAMPT and NAPRT (both in red) in NB1691 cells. Nuclei are stained with DAPI (blue).

Figure 2

NAMPT inhibition reduces NB1691 cell viability via induction of apoptosis. (A) NB1691 cells were exposed to the NAMPT inhibitor STF-118804 for 24 and 48 hours at concentrations shown and viability determined using trypan blue assay. (B) NB1691 cells were exposed to ST-118804 and caspase 3/7 activity visualized with immunofluorescence. (C) Cleaved (active) caspase 3 and cleaved PARP were evaluated by western blot analysis. Alpha-tubulin is used as a loading control. (D) Quantification of cleaved PARP and cleaved caspase 3 as compared to non-treated (nM). Experiments repeated in triplicate. *p < 0.05 compared to non-treated controls.

Figure 3

NAMPT inhibition reduces NB1691 cancer stem cell self-renewal. (A) NB1691 stem cells were plated at 25 cells/well in 96-well plates and treated with STF-118804 at concentrations shown. Number of neurospheres formed were counted 10 days later. (B) Representative light micrographs of NB1691 neurospheres at 10 days. *p < 0.05 compared to non-treated controls.

Figure 4

NAMPT inhibition reduces ATP and increases p-ACC levels. (A) NB1691 cells were exposed to STF-118804 at concentrations shown and ATP determined using ATP assay. (B) Mechanism by which reduction in ATP results in increased phosphorylation of Acetyl Coenzyme-A Carboxylase (p-ACC). (C) Levels of p-ACC in response to 24-hour NAMPT inhibition were evaluated by western blot. Alpha-tubulin is used as a loading control. *p < 0.05 compared to non-treated controls.

Figure 5

NAMPT inhibition reduces N-MYC levels and AKT pathway activation, and results in greater than additive cytotoxicity when combined with PI3K pathway and glycolytic pathway inhibitors. (A) NB1691 cells were treated with NAMPT inhibitors; STF-118804, CHS 828 and FK 866 at concentrations shown, and levels of phospho-AKTs, and N-MYC were evaluated by western blot. (B) Growth factors activate PI3K, causing AKT activation, GSK3β sequestration, subsequent stabilization of N-MYC leading to tumor cell proliferation. (C) NB1691 cells were pre- treated with10nM FK-866 then exposed to either NVP-BEZ235 (NVP), OSU-03012 (OSU), KU-0063794 (KU), everolimus (Evero), GDC-0941 (GDC), 2-deoxy glucose (2-DG), lonidamine (Lon) or 3-bromopyruvate (3-BrpA) at concentrations shown. Cytotoxicity determined using LDH assay. *p < 0.05 compared to non-treated controls, **p < 0.05 combination drug treatment and FK-866 compared to drug treatment alone.

Figure 6

NAMPT inhibition blocks tumor growth in NB1691 xenograft model. NB1691 cell were injected into the flank of nude mice and when tumors reached approximately 200mm³ (treatment day 0), mice were treated with either vehicle, 25mg/kg STF-118804 (STF), 500mg/kg 2-DG (2-DG) or 25mg/kg STF-118804 + 500mg/kg 2-DG (STF+2-DG) daily. All mice were euthanized on day 13 of treatment. (A) Tumor volume over treatment period. (B) Fold change in tumor volume relative to initial tumor volume. (C) Average weight of each experimental group over course of treatment. *p<0.05 compared to vehicle-treated controls.