Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) as a therapeutic strategy in cancer

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Abstract

NAD is a metabolite that is an important cofactor and second messenger for a number of cellular processes such as genomic stability and metabolism that are essential for survival. NAD is generated de novo from tryptophan or recycled from NAM through the NAMPT-dependent salvage pathway. Alternatively, cells can convert NA to NAD through the NAPRT1-dependent salvage pathway. Tumor cells rapidly turn over NAD but do not efficiently utilize the de novo synthesis pathway. Hence, they are more reliant on the NAMPT salvage pathway for NAD regeneration making this enzyme an attractive therapeutic target for cancer. NAMPT is over-expressed in a number of cancer types such as colorectal, ovarian, breast, gastric, prostate, gliomas as well as B-cell lymphomas. A number of novel, potent and selective NAMPT small molecule inhibitors have been synthesized to date that have displayed robust anti-tumor activity in tumor models in vitro and in vivo. These inhibitors efficiently suppress NAD production in a time dependent manner and sustained reduction of NAD levels leads to loss of ATP and ultimately cell death. This review will summarize the chemical properties of these unique NAMPT inhibitors as well as their mechanism of action, pharmacodynamic activity and efficacy in tumor models in vitro and in vivo. An overview of biomarkers that predict response to treatment and mechanisms of resistance to NAMPT inhibitors will also be provided. Additionally, NAMPT inhibitors that have advanced into clinical trials will be reviewed along with experimental strategies tested to potentially increase the therapeutic index of these inhibitors.

Introduction

Dysregulation of cellular metabolism is now recognized as an important contributor to tumor cell growth, as mutations and gene amplifications have been found in numerous enzymes involved in regulating key metabolic pathways (Locasale et al., 2011, Tomlinson et al., 2002, Yan et al., 2009) and known oncogenes can reprogram cellular metabolism (Garcia-Cao et al., 2012, Ying et al., 2012). Additionally, cancer cells shift their reliance on energy generation from the Krebs (tricarboxylic acid) cycle to glycolysis as a means to generate ATP and key metabolites required to sustain robust proliferation: a phenomenon known as the Warburg effect (Ward & Thompson, 2012). This reliance, or addiction, on alternative metabolic pathways potentially provides novel therapeutic approaches that can be utilized to inhibit tumor growth.

One of the key metabolites essential for sustaining cellular energy metabolism is nicotinamide adenine dinucleotide (NAD), an important cofactor that plays a central role in cellular redox reactions and alternates between two states, NAD and NADH. In addition, NAD serves as a second messenger required for a number of cellular processes essential for survival such as: mitochondrial function, calcium homeostasis and mobilization, anti-oxidation, gene expression, immunological functions (including cytokine production) and cell death (reviewed by Ying, 2008). NAD is required for DNA repair and maintenance of genomic stability serving as donor of ADP-ribose, which is generated upon degradation of NAD to nicotinamide (NAM) by poly (ADP-ribose) polymerase I (PARP-1) (Schreiber et al., 2006). Sirtuins (SIRTs) are a family of histone deactylases consisting of 7 enzymes that are important for increasing cellular life span when deprived of nutrients and require NAD as a substrate for SIRT-mediated deacetylation reactions (Fulco et al., 2008, Zhao et al., 2004). While NAD levels do not change during redox reactions, the reduction in NAD pools by NAD degrading enzymes such as PARPs and SIRTs requires the constant resynthesis of NAD in order to maintain sufficient levels for cell survival.

Biochemically, eukaryotic cells utilize three main pathways to generate NAD either by catabolism of tryptophan or by two alternative salvage pathways (Fig. 1). De novo synthesis of NAD from tryptophan, also referred to as the kynurenine pathway, requires a series of 8 enzymatic reactions and occurs predominantly in the liver (Heyes et al., 1997). The primary salvage pathway in which NAD is recycled from NAM is mediated by nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is the rate-limiting enzyme in this pathway and converts NAM to nicotinamide mononucleotide (NMN) by catalyzing the reversible addition of a ribose group from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to NAM (Fig. 2). To facilitate this reaction, NAMPT is autophosphorylated on H247, which enhances the affinity for NAM by 160,000-fold and increases enzymatic activity by 1125-fold (Burgos et al., 2009). Even though autophosphorylation of NAMPT is transient, it stabilizes the interaction between the two monomers of the dimer that form the active site and increases the affinity of the enzyme for PRPP by ~10-fold (Burgos et al., 2009). Moreover, autophosphorylation of NAMPT decreases the Km for NAM binding from 855 nM to 5 nM and when ATP hydrolysis is coupled to NMN synthesis catalytic efficiency increases by 1100-fold (Burgos & Schramm, 2008). Thus, in the presence of sufficient concentrations of ATP in which the Km for NAMPT is 7 mM, NAMPT is extremely efficient at recycling NAM into NMN (Burgos & Schramm, 2008). An alternative salvage pathway is dependent on nicotinic acid (also referred to as niacin or vitamin B3), which is supplied exogenously and metabolized by nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) to nicotinic acid mononucleotide (NAMN) and subsequently converted to NAD through the Preiss-Handler pathway (Preiss and Handler, 1957, Preiss and Handler, 1958) (Fig. 1).

Given that NAMPT is the rate-limiting enzyme in a key NAD recycling pathway, small changes in NAMPT activity can dramatically impact NAD metabolism and NAD dependent cellular processes (Revollo et al., 2004, Rongvaux et al., 2002). Interestingly, NAMPT was originally identified as a secreted cytokine-like factor that synergized with interleukin-7 and stem cell factor to promote pre-B-cell colony formation and, therefore, was initially designated PBEF (pre-B-cell enhancing factor) (Samal et al., 1994). Additional confirmation of growth activity was based on the role of PBEF as a cytokine that was upregulated in activated neutrophils and inhibited apoptosis when cultured neutrophils were grown in the presence of conditioned media containing recombinant PBEF. It was not until 2001 that NAMPT was first cloned from Haemophilus ducreyi in a screen to identify proteins that promoted the conversion of NAM to NAD. Once the DNA sequence was available, it was quickly realized that the closest human homolog of PBEF was NAMPT (Martin et al., 2001). In 2002, the murine version of PBEF was cloned and shown to possess nicotinamide phosphoribosyltransferase activity thereby confirming that PBEF was indeed equivalent to NAMPT (Rongvaux et al., 2002). In humans, the NAMPT gene is found at chromosome locus 7q22, spans 34.7 kb, contains 11 exons and 10 introns generating a cDNA of 2,357 kb. The translated protein contains 491 amino acids and has a molecular weight of 52 kDa (Kitani et al., 2003, Ognjanovic et al., 2001). Three predominant mRNA transcripts of NAMPT have been identified containing 2.0, 2.4, and 4.0 kb (Kitani et al., 2003, Yang et al., 2007). NAMPT is ubiquitously expressed in all tissues and the coding sequence is highly conserved across mammalian species and lower organisms such as insects, sponges and prokaryotes (McGlothlin et al., 2005, Rongvaux et al., 2002).

In most cell types, NAMPT is expressed intracellularly in the cytoplasm, nucleus, and mitochondria and is commonly referred to as iNAMPT (intracellular NAMPT). A second, secreted form of NAMPT has also been observed and is referred to as eNAMPT (extracellular NAMPT) or visfatin. While the role of iNAMPT in NAD regeneration and cellular metabolism has been extensively characterized the function of eNAMPT is less understood. Enzymatic activity of eNAMPT has been demonstrated in NAMPT haplodeficient mice in which expression of eNAMPT is decreased resulting in lower plasma NMN levels and defective insulin secretion in pancreatic β –cells upon glucose stimulation (Revollo et al., 2007). The latter defect was rescued by administration of NMN in vivo (Revollo et al., 2007) suggesting that eNAMPT plays an essential role in maintaining systemic NAD biosynthesis required for normal pancreatic β-cell function. Adipocytes also secrete eNAMPT and plasma levels of circulating eNAMPT have been shown to be elevated in obese humans (Berndt et al., 2005, Fukuhara et al., 2005). Given that macrophages influence obesity-related pathophysiology and inflammation, Zhang et al. studied the function of eNAMPT and its role in maintaining macrophage survival. Indeed, eNAMPT promoted macrophage survival following endoplasmic stressors or apoptotic inducers that are observed in obesity-associated diseases by stimulating interleukin-6 secretion and subsequent STAT3 activation (Zhang et al., 2008). However, enzymatically inactive mutated versions of eNAMPT remained biologically active compared to wild-type eNAMPT. Moreover, the pro-survival activity of nonenzymatic version of eNAMPT was not recapitulated by the addition of exogenous NMN or reversed by FK866 (a potent and selective NAMPT inhibitor that will be further described in subsequent sections). Given the low ATP pools that exist in the extracellular mileu, synthesis of NAD by eNAMPT may not be its primary function as other investigators have discovered the catalytic activity of eNAMPT to be reduced under normal physiologic conditions (Hara et al., 2011). Therefore, it is unclear whether eNAMPT also plays an essential biochemical role in the regeneration of NAD required for the cellular functions and survival described earlier. For the remainder of this review we will focus on the biology, biochemistry and relevant pharmacology of iNAMPT in cancer and will simply be referred to as NAMPT in the following sections.

Appropriate regulation of cell metabolism is required to support robust proliferation of all cells and especially cancer cells given their high metabolic demands. Additionally, as NAD is rapidly turned over by degrading enzymes such as PARPs and SIRTs, tumor cells are more dependent on salvage pathways to rapidly restore NAD pools that are required for survival (Schreiber et al., 2006). Moreover, it has been shown that some cancer cell lines are unable to utilize the de novo pathway as they lack at least one of the enzymes responsible for the conversion of tryptophan to NAD (Heyes et al., 1997, Xiao et al., 2013). The Preiss-Handler pathway is frequently found to be inactive in cancer cells due to lack of expression of the rate-limiting enzyme NAPRT1 (O’Brien et al., 2013, Shames et al., 2013, Watson et al., 2009, Xiao et al., 2013). However, cancer cells that do express NAPRT1 cannot efficiently utilize the NA dependent salvage pathway since systemic NA levels are generally insufficient to drive NAD generation (Kirkland, 2009). Therefore, tumor cells that lack NAPRT1 depend on NAMPT for NAD generation and cell survival making them more susceptible to the cytotoxic effects of NAMPT inhibitors (Olesen, Thougaard, Jensen and Sehested, 2010, Shames et al., 2013, Watson et al., 2009, Xiao et al., 2013).

Overexpression of NAMPT has been observed across a broad range of solid tumors including colorectal, ovarian, breast, gastric, prostate, well-differentiated thyroid cancers, endometrial carcinomas, melanoma, gliomas and astrocytomas (reviewed in Schackelford et al., 2013). Clinically, higher NAMPT expression is associated with worse prognosis in astrocytoma/glioblastoma and correlates with increased tumor growth, metastases and cellular dedifferentiation in melanoma (Maldi et al., 2013, Reddy et al., 2008). Increased NAMPT expression has also been observed in hematological malignancies particularly in lymphomas such as: diffuse large B-cell lymphoma, follicular B-cell lymphoma, Hodgkin’s lymphoma and peripheral T-cell lymphoma and is associated with a more aggressive malignant lymphoma phenotype (Olesen et al., 2011). In addition, the increased levels of NAMPT observed in malignant versus benign tissues are associated with alterations in tumorigenic activity. For example, Wang et al. discovered that inhibition of elevated NAMPT expression in prostate cancer cells suppressed growth and invasion in vitro and growth of tumor xenografts in vivo (Wang et al., 2011). NAMPT overexpression has been shown to promote acquired resistance to chemotherapeutic agents, including fluorouracil, doxorubicin, paclitaxel, etoposide, and phenylethyl isothiocyanate (Bi et al., 2011, Folgueira et al., 2005, Wang et al., 2011).

Given the essential role that NAD plays in tumor cell metabolism, growth and survival, targeting NAMPT with small molecule inhibitors may prove efficacious as a cancer therapeutic. In this review, we aim to provide a comprehensive overview focusing on small molecule inhibitors of NAMPT synthesized to date including their chemical structures/scaffolds, binding modes, and biochemical and cellular potencies. This review will also focus on the pharmacology of NAMPT inhibitors, including their mechanism of action in tumor cell lines; preclinical efficacy and pharmacodynamics in tumor xenografts. Biomarkers that predict response to NAMPT inhibition in these models and mechanisms of drug resistance will also be discussed. Additionally, an overview of those inhibitors that have progressed into clinical trials as well as safety liabilities observed clinically and nonclinically will be provided. Lastly, in an attempt to improve the therapeutic index of the next generation of small molecule NAMPT inhibitors, experimental strategies that have been tested preclinically to mitigate on-target toxicities will also be discussed.

Section snippets

Clinical stage NAMPT inhibitors

NAMPT inhibitors that advanced into human clinical trials are presented in Fig. 3. These compounds include GMX1778 (also known as CHS-828) (Olesen et al., 2008, Schou et al., 1997, Watson et al., 2009), its more water-soluble prodrug GMX1777 (Beauparlant et al., 2009, Binderup et al., 2005), and APO866 (also known as FK866 and WK175) (Hasmann and Schemainda, 2003, Wosikowski et al., 2002). Both GMX1778 and APO866 were originally identified as potent antiproliferative agents in cell-based

Mechanism of action

NAMPT inhibitors synthesized to date display exquisite biochemical and cellular potency demonstrating the essential role NAD plays in tumor cell survival. Pharmacological inhibition of NAMPT leads to a rapid decrease in NAD levels in tumor cells in vitro and in vivo (Beauparlant et al., 2009, Cerna et al., 2012, Hasmann and Schemainda, 2003, O’Brien et al., 2013, Olesen et al., 2008, Tan et al., 2013, Watson et al., 2009, Xiao et al., 2013). The t1/2 for NAD depletion in the presence of NAMPT

Clinical trials of NAMPT small molecule inhibitors

As previously described, three NAMPT inhibitors (APO866, GMX1778 and, its prodrug, GMX1777) have entered clinical trials and completed phase I clinical trials but further evaluation was discontinued primarily due to dose-limiting toxicities (summarized in Table 2). APO866/FK866 was administered as a 96 hour intravenous infusion every 28 days and the recommended phase II dose was determined to be 0.126 mg/m2/hr. Objective responses, however, were not observed and thrombocytopenia was the

Toxicities of NAMPT inhibitors and mitigation strategies

The co-existence of the NAPRT1 dependent NAD salvage pathway, which converts NA to NAD, has led to the proposal that NA co-treatment could be utilized as a mitigation strategy to increase the therapeutic index of NAMPT inhibitors in patients. Unlike many tumor types that lack NAPRT1, normal tissue cells contain functional NAPRT1 and are able to generate NAD when provided NA. Thus, co-administration of NA with a NAMPT inhibitor may preferentially protect normal tissue but not NAPRT1 deficient

Summary and future considerations

Given the essential role NAD plays in a number of cellular processes that are required to sustain tumor cell growth and survival, regeneration of NAD by NAMPT makes this metabolic enzyme an attractive therapeutic target for the treatment of cancer. Indeed, a number of novel, potent and selective NAMPT small molecule inhibitors effectively reduce NAD levels in tumor cells and demonstrate impressive cytotoxic activity across a large panel of tumor cell lines representing solid and hematological

Conflicts of interest statement

All authors are employees of Genentech, a member of the Roche Group.

Acknowledgment

We thank Dr. Nicholas Skelton at Genentech for expert advice on structural modeling of NAMPT inhibitors and preparation of Fig. 4, Fig. 5.

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