Review

NADþ Kinase as a Therapeutic Target in CancerPhilip M. Tedeschi, Nitu Bansal, John E. Kerrigan, Emine E. Abali,Kathleen W. Scotto, and Joseph R. Bertino

Abstract

NADþ kinase (NADK) catalyzes the phosphorylation of nico-tinamide adenine dinucleotide (NADþ) to nicotinamide adeninedinucleotide phosphate (NADPþ) using ATP as the phosphatedonor. NADPþ is then reduced to NADPH by dehydrogenases, inparticular glucose-6-phosphate dehydrogenase and the malicenzymes. NADPH functions as an important cofactor in a varietyofmetabolic and biosynthetic pathways. The demand forNADPHis particularly high in proliferating cancer cells, where it acts as acofactor for the synthesis of nucleotides, proteins, and fatty acids.Moreover, NADPH is essential for the neutralization of the

dangerously high levels of reactive oxygen species (ROS) gener-ated by increased metabolic activity. Given its key role in metab-olism and regulation of ROS, it is not surprising that several recentstudies, including in vitro and in vivo assays of tumor growth andquerying of patient samples, have identified NADK as a potentialtherapeutic target for the treatment of cancer. In this review, wewill discuss the experimental evidence justifying further explora-tion of NADK as a clinically relevant drug target and describe ourstudies with a lead compound, thionicotinamide, an NADKinhibitor prodrug. Clin Cancer Res; 22(21); 5189–95. �2016 AACR.

IntroductionHighly proliferating cancer cells require sufficient amounts of

NADH and NADPH to act as reducing agents for reductivesynthesis of nucleic acids, protein, and lipid biosynthesis(Fig. 1A). A lack of these precursors can lead to a halt in cellgrowth and eventual cell death. NADPH is also key to themaintenance of a healthy redox status in cells (1, 2), as it isinvolved in neutralizing the reactive oxygen species (ROS) asso-ciated with rapid growth. To achieve the metabolic and ROS-mediating requirements associated with rapid proliferation, can-cer cells can alter the expression and regulation of metabolicgenes. Indeed, several of these genes are under the regulation ofoncogenes and tumor suppressor proteins. For example, thetumor suppressor p53 exerts control of NADPH levels by inhibit-ing glucose-6-phosphate dehydrogenase (G6PD) activity and byrepressing activity of the malate dehydrogenase enzymes M1and M2, both of which contribute to the cellular NADPH pool(Fig. 1A; refs. 3, 4). When p53 is mutated or rendered nonfunc-tional through interaction with inhibitory proteins, this control islost; as a result, cancer cells generate more NADPH for protectionfrom ROS and macromolecular synthesis to allow rapid prolif-eration. Another example is the M2 splice variant of pyruvatekinase (PKM2), which is subjected to complex regulation by bothoncogenes and tumor suppressors; overexpression of PKM2 leads

to the increased production of NADPH by diverting glucosemetabolism into the pentose phosphate pathway (5).

All cells harbor some level of ROS. Indeed, moderate levelsof ROS are beneficial for cancer cells, as they can contributeto increased cell proliferation and genomic instability (6–8).However, excessive ROS can cause oxidative damage to proteins,lipids, and DNA, leading to cell death. This fine line betweenROS-enhanced and -decreased proliferation makes cancer cellsvulnerable to stressors that alter this balance, including manychemotherapeutic drugs as well as radiation (9–13). The primarydefense against ROS in cancer cells is the upregulation of super-oxide dismutase and the NADPH-dependent glutathione (GSH)and thioredoxin redox cycles (14). The GSH system functions viaglutathione peroxidase enzymes that inactivate H2O2 and otherperoxides by conversion of GSH to reduced glutathione disulfide(GSSG). GSSG is then cycled back to GSH by glutathione reduc-tase using NADPH. Myc, by increasing glutamine uptake, gluta-minolysis, and, thereforeGSHproduction aswell as bypromotingthe expression of PKM2, also contributes to redox control incancer cells (15). Thus, the same proteins that when mutateddrive the cancer phenotype are also responsible for the alteredmetabolism and redox status that allow cancer cells to survive andproliferate.

Decreased levels ofNADPHarepredicted tohave a selective andtwo-pronged effect on tumor survival: inhibition of critical bio-synthetic pathways and reduction in the ability of cancer cells toneutralize high ROS levels. A key player in the regulation ofNADPH is NADþ kinase (NADK), which generates NADP thatis then converted to NADPH by dehydrogenases. NADK was firstidentified by Arthur Kornberg in 1950 following isolation fromSaccharomyces cerevisiae (16) and has since been purified from avariety of organisms (17–20). Human cytoplasmic NADK(cNADK) is an obligate dimer with a monomer size of about50 kDa. Unlike several prokaryotic NADKs, human cNADK isunable to use inorganic phosphate; instead, the phosphorylationof NAD to NADP requires ATP and magnesium ion as a cofactor(21). Of note, the active site is composed of portions of both

Departments of Medicine and Pharmacology, Rutgers Robert WoodJohnson Medical School, the Rutgers Cancer Institute of New Jersey,and the Rutgers Graduate School of Biomedical Sciences, NewBruns-wick, New Jersey.

Corresponding Authors: Joseph R. Bertino, Rutgers Cancer Institute of NewJersey, 195 Little Albany Street, Room 3033, New Brunswick, NJ 08903. Phone:732-235-8510; Fax: 173-2235-8181; E-mail: bertinoj@cinj.rutgers.edu; andKathleen W. Scotto, kathleen.scotto@rutgers.edu

doi: 10.1158/1078-0432.CCR-16-1129

�2016 American Association for Cancer Research.

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monomers, a distinctive feature of this enzyme (Fig. 1B).Although the structure and function of the human cNADK havebeen known for over a decade, a mitochondrial NADK (mNADK)has been discovered recently and might play a predominant rolein controlling the ROS generated in the mitochondria (vide infra;refs. 22, 23).

Most of the studies of NADK have focused on the enzyme inplants and bacteria. For example, NADK has been shown toplay diverse roles in Arabidopsis thaliana, participating in nitro-gen/carbon metabolism, photosynthetic efficiency, and protec-tion from oxidative stress (24–26). The key role of NADK inincreasing NADPH levels and macromolecular synthesis hasbeen exploited by overexpressing this enzyme to increase theoutput of a desired product (27–30), with less attention givento approaches to inhibit NADK activity. To date, most efforts todevelop NADK inhibitors have focused primarily on pathogenic

bacteria, due to differences in active site structure and substratesbetween prokaryotic and mammalian NADK (27); in thisregard, a special effort has been made to identify NADKinhibitors for the treatment of multidrug-resistant tuberculosisand malaria (31, 32). Inhibition of human NADK for thetreatment of diseases, such as inflammation and cancer, hasonly recently been proposed (33).

Mitochondrion-Localized NADKDespite the key role of NADPH, and, therefore, NADK in

modulating cellular ROS, our studies showed that knockdownof cNADK only had a modest effect on ROS levels (34), andothers have shown that overexpression of cNADK (cytoplasmicNADK) only resulted in minimal protection from ROS (35).Pollak and colleagues suggested that human cells may dependmore on the NADPþ-dependent dehydrogenases to reduceNADPþ rather than levels of NADPþ for oxidative defense(35). These results may also be explained by the recent iden-tification of a mitochondrial NADK (mNADK). Because NADPis membrane impermeable, the existence of a mitochondrialNADK was long postulated but only recently characterized (21,22). Mitochondrial NADH and NADPH are required for pro-tection from oxidative stress as well as for the biosynthesis ofmacromolecules. Similar to cNADK, mNADK might play amajor role in generating NADH and NADPH for protection ofmitochondria from ROS. As tumor cells have increased levels ofROS, inhibition of mNADK might at first glance seem aneffective strategy. However, this will have to be carefully tested,as the likely critical role of this enzyme for protection againstROS in the heart and brain could prove this approach too toxicfor clinical use. Supporting this concern, a pediatric patientwas recently identified with a deficiency in mNADK due to ahomozygous nonsense mutation in exon 10, leading to apremature stop codon (36). By the time the patient was 4.5years old, he had developed severe encephalopathy, spasticquadriplegia, blindness, and epilepsy and died soon thereafter.Thus, cNADK might be a more feasible target for inhibitordevelopment.

Cytoplasmic NADK expression in cancerUsing a gain-of-function screening platform, a novel cNADK

mutant, NADK-I90F, was isolated from a panel of mutantsoriginating in pancreatic ductal adenocarcinoma cancerpatients (37). Our laboratory characterized the molecular struc-ture and enzymatic activity of this mutant cNADK and foundthat as compared with the wild-type enzyme, cNADK-I90F hada higher Vmax and lower Km for NADþ and ATP, indicating anincrease in activity (37). Consistent with this finding, cellsexpressing this mutant had low ROS levels and elevated levelsof NADPH (Fig. 2). When the effect of NADK-I90F on cellgrowth and metabolism was evaluated in vitro and in vivo, it wasfound to drive transformation of normal pancreatic ductal cellsdue to its increased enzymatic activity. This further supportsthat cNADK, both wild type and certain mutant forms, is aclinically relevant enzyme target for cancer therapy. Notably,both overexpression and several additional NADK mutantshave been identified in a small percentage (<5%) in multipletumor types, including pancreatic cancer in The CancerGenome Atlas database, but more information is required todetermine the significance of the mutations reported.

© 2016 American Association for Cancer Research

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Figure 1.

A, the role of NADþ kinase (NADK) and dehydrogenases (G6PD) and the malicenzymes, M1 and M2, in generating NADPH. NADPH is required for reductivesynthesis (nucleic acids, proteins, and fatty acids) andprotects cells fromROSbygenerating reduced forms of glutathione (GSH) and thioredoxin (Trx). B,modelof NADþ (space-filling model) bound to human NADK with each monomercolored green and cyan created using PyMOL.

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The Search for Potent Inhibitors of HumanNADK: Preclinical and Clinical Studies ofNAD Analogues

Oneof thefirst attempts todevelopNADKinhibitorswasmadebyPankiewicz and colleagues (32, 38), using a novel approach.Although more than 30 clinically approved kinase inhibitors havebeendeveloped that bind to theATP-binding site of various tyrosineprotein kinases, these investigators reasoned that as theATP-bindingsite in NADK is solvent exposed and chelation with Mgþþ isnecessary for ATP binding, targeting the binding of ATP to NADKwouldnotbe aneffective approach.Rather, they focused their effortson inhibitors that targeted theNAD-binding site in the enzyme (33).Although NAD kinases belong to a large family of NAD-utilizingenzymes with conserved NAD-binding sites, the domain in humanNADKwas considered sufficiently different from that ofmost NAD-dependent enzymes to allow for the identification of selectiveinhibitors. For example, the NAD-binding site has been successfullytargeted in other enzymes: tiazofurin, a potent inhibitor of inosinemonophosphate dehydrogenase (IMPDH), is used to treat chronicmyelogenous leukemia, and mycophenolic acid, another IMPDHinhibitor, is used as an immunosuppressant (33).

In collaborationwithPetrelli and colleagues (32),we examinedseveral NAD analogues. Of particular interest was benzamideriboside, whichwas anabolized to benzamide adenine nucleoside(BAD) in cells. We found that in addition to its known potentinhibition of IMPDH, BAD also inhibited NADK and, as aconsequence, markedly lowered NADPH levels, resulting in a

loss of dihydrofolate reductase (DHFR) levels, as this coenzymeprotects DHFR from proteolysis (39). BAD inhibited growth ofCEM lymphocytic cells at a lowmicromolar concentration.Unfor-tunately, although the precursor drug, benzamide riboside, hadencouraging preclinical anticancer activity, animal toxicity studiesshowed that treatment with BAD caused skeletal muscle loss andhepatotoxicity, precluding further drug development.

ThionicotinamideDuring a search for a small compound library for novel NAD

analogues, we identified nicotinamide adenine dinucleotide(NADS) and nicotinamide adenine dinucleotide phosphate(NADPS) and showed that the cytotoxic effect of NADS/NADPSwas largely due to inhibition of NADK activity (40). As thioni-cotinamide (TN) is converted to NADPS upon entering the cell,we tested the possible role of TN as a prodrug inhibitor of NADKand found that it was as effective as NADPS. As TN ismore readilyavailable than NADS/NADPS, we pursued our analyses using TNas our lead compound. We examined several cell lines for sensi-tivity to TN. The T-cell lymphoma cell lines CEM-CCRF andMOLT-4, the diffuse large B-cell lymphoma cell line RL, as wellas the C85 colon cancer cell line were all sensitive, with IC50sapproximately 10 mmol/L. Inhibition of NADK activity by TN orshRNA-mediated knockdown decreased colony formation andthe growth of C85 cells to a similar degree (notably, the colonieswere small, suggesting effects on early precursors), supporting thehypothesis that it is the effect of TN onNADK aswell as inhibitionofG6PD (34) that underlies the cytotoxic effects observed (Fig. 3).

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Cells expressing mutant NADK-I90F exhibit low ROS and elevated NADP and NADPH levels. Total NADP and NADPH (NADPt) and NADPH levels were determinedin normal pancreatic ductal cells (HPDE); pancreatic ductal adenocarcinoma cells (BxPC-3 and Panc-1); or cell lines expressing either the vector control,NADK, or NADK-I90F using the NADP/NADPH-Glo assay (Promega; A). ROS levels were assayed in normal pancreatic ductal cells (HPDE) or pancreatic cancercells (BxPC-3 and Panc-1; B). Expression levels of NADK and NADK-I90F were determined (C). Adapted from Tsang and colleagues (37).

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TN decreased the levels of NADPH in C85 cells, resulting in amodest but reproducible increase in steady-state ROS levels,particularly when the oxidative stressor, hydrogen peroxide, waspresent (�3-fold increase). Importantly, other NADPH-reliantpathways, such as fatty acid synthesis and nucleotide synthesis,were also inhibited by TN (34).

TN was also effective in sensitizing cells to several chemother-apeutic agents, including methotrexate, gemcitabine, docetaxel,and irinotecan (34). As there was an increase in the level of ROSas well as reduction in NADPH levels in cells treated with both TNand chemotherapy, the TN-induced loss ofNADK activity/NADPHcontributed to the synergistic effect. Thus, both inhibition ofmacromolecular synthesis and an increase in oxidative stress mayplay an important role in the toxicity of these combinations.

The effectiveness of TN as a single agentwas examined in a pilotstudy of two human xenograft models in mice. TN caused tumorregression and a protracted halt in tumor growth for the durationof observation (Fig. 4). The human C85 colon cancer xenograftwas treated with the same dose and similar schedule of TN as theRL lymphoma xenograft, and tumor response was compared withC85 xenografts in which NADK was downregulated by shRNA(�50%). Importantly, both TN and shRNA inhibited the growthof this tumor without toxicity, validating NADK as a key antican-cer target (34). These promising results justify further develop-ment of NAD analogues for clinical use.

The only NADK inhibitor that has been tested in the clinic is6-amino nicotinamide, which had shown promising preclinicalanticancer activity, although animal studies showed that neu-rologic toxicity was dose limiting (41). Unfortunately, neuro-toxicity prevented dose escalation, and minimal activity wasseen at the maximum tolerated dose in clinical trials (42). Themechanism underlying the antitumor and neurotoxic effects ofthis drug are not clear, and have been ascribed to its abilityto inhibit phosphogluconate dehydrogenase and, hence, thepentose phosphate pathway (43). However, an earlier reportshowed that it potently inhibited the phosphorylation of NADin mitochondria (44), a more compelling explanation for theneurotoxic effects. Nicotinamide was found to reverse theseside effects, as well as its antitumor activity, presumably byinhibiting uptake and/or conversion of 6-amino nicotinamideto 6-amino NAD, its active form. Notably, in contrast, noneurotoxicity was observed with TN, indicating that whateverthe basis for this toxic effect with 6-amino nicotinamide, it isnot a characteristic of all nicotinamide analogues.

Will Inhibitors of NADK Have SelectiveAnticancer Activity?

The most compelling evidence that NADK is a novel target foranticancer drug development comes from our studies discussed

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Exogenous nicotinamide inculture media can abrogatethionicotinamide (ThioNa)toxicity. A, thionicotinamidetoxicity is inversely correlatedwithnicotinamide levels. UntreatedC85 cells and C85 cells in whichcNADK has been stably knockeddown are unaffected.Representative wells are shownfor each condition. B, averagecolony increases in ThioNa-treatedcells as nicotinamide levelsincrease. C, average colonynumber increases as medianicotinamide levels increase.D, the proposed intracellularbiosynthetic pathway fromThioNa to nicotinamide adeninedinucleotide phosphate, reduced(NADPSH). Nam, nicotinamide;n.s., not significant. Reprinted withpermission from Tedeschi andcolleagues (34): Suppression ofcytosolic NADPH pool bythionicotinamide increasesoxidative stress and synergizeswith chemotherapy. MolecularPharmacology 2015;88:720–7.

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above, which showed that knockdown of this enzyme in thehuman colon carcinoma cells inhibited clonogenic tumor cellproliferation in vitro and xenograft growth in vivo. In addition,knockdown studies of cytoplasmic NADPH and inhibition ofcNADK andG6PD by TN have not demonstrated neurotoxicity inmice at doses that inhibited tumor growth, suggesting that drugsthat specifically target the cytoplasmic enzyme might have a safetherapeutic index. We conclude that inhibition of cNADK willhave selective anticancer activity by reducing the NADPH poolavailable for biosynthetic pathways and protection fromROS. TNby decreasing NADPH levels also had the additional effect ofdestabilizing and thereby decreasing levels of DHFR. As thisdecrease inDHFR contributed to the toxicity of TN, a combinationof a DHFR inhibitor and inhibition of NADK by TN had syner-

gistic anticancer activity in vitro (35). Moreover, synergistic cellkilling was also observed when TNwas used in combination withother drugs that inhibit DNA synthesis, likely because of the factthat both treatments led to enhanced ROS and decreased mac-romolecular synthesis (3).

Further studies are needed to clarify the role of mitochondrialNADK in protecting cells from ROS and mitochondrial metab-olism, as well as to compare the effect of NADK inhibitors on thecytoplasmic and mitochondrial enzyme. To identify inhibitorsthat select between cNADK and mNADK, as well as mutantforms of cNADK, two approaches are being used: a library screenand rational design using a crystal structure of the enzyme (44).Although the key binding-site residues are conserved betweenthe mitochondrial and cytoplasmic enzymes (21, 22), there aresignificant structural differences between the two enzymes thatmay be exploited (Fig. 5). The conserved residues are D184,D383, L188, N284, and E285; apart from the potential fordifference in binding-site shape and dynamics (induced fit),there are proximal differences where selectivitymay be achieved.These include the R385 residue in cNADK (glycine in mNADK);the D412 residue in mNADK (while this is D383 in cNADK, theresidue side chain points away from the binding site); F211,which provides a pi-stack interaction for the adenine ring ofNAD in cNADK (replaced by L192 in the mitochondrialenzyme); and the more subtle Y327 residue (tryptophan inmNADK, not shown in Fig. 5), giving a larger pi-stacking groupfor interaction with the nicotinamide ring of NAD. New leadsobtained from the library screen will be modified to enhanceselectivity and efficacy based upon what we learn from in silicodocking to the crystal structure.

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Thionicotinamide (ThioNa) induces tumor regression in RL lymphomaxenografts (A). Six- to 8-week-old NOD/SCID-g male mice were inoculatedsubcutaneously with RL cells. When the tumors were palpable, animals weretreated with 100 mg/kg TN on days indicated by arrows. Tumor volume wasdetermined using calipers. Inhibition of NADK by ThioNa or siRNA-mediatedknockdown of NADK reduces tumor growth in C85 xenografts (B). NOD/SCID-gmale mice were inoculated subcutaneously with 1 � 106 C85 cells or C85 cellsexpressing shRNA directed against NADK. When tumors were palpable (day 3),animals bearing normal C85 xenografts were dosed with 100 mg/kgthionicotinamideondays indicatedwith arrows. Tumor volumewasmeasured asabove. Thisfigure shows days 1 to 12 of the study, for clarity. In both studies, therewas no evidence of toxicity (weight loss, neurotoxicity). Reprinted withpermission from Tedeschi and colleagues (34): Suppression of cytosolic NADPHpool by thionicotinamide increases oxidative stress and synergizes withchemotherapy. Molecular Pharmacology 2015;88:720–7.

© 2016 American Association for Cancer Research

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Structural alignment of the cNADK crystal structure (enzyme depicted asribbons colored blue, with key residues depicted as CPK colored ball-stickmodels and NADþ depicted as a ball-stick model colored magenta), with thehomology model of the mitochondrial enzyme mNADK (depicted as ribbonscolored red). The mNADK residues are ball-stick models colored green, withlabels in italics. (Illustration prepared using VMD).

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ConclusionsThe in vitro and in vivo effects of NADK inhibition on cancer cell

death combinedwith the identificationofanactivatingmutation inNADK that initiates carcinogenesis make a strong case for devel-oping inhibitors that lower NADPH tumor levels and raise thepossibility of finding specific inhibitors of this or other mutantNADK enzymes. The identification of new, more selective inhibi-torswill provide a tool todissect the role of cNADK andmNADK in

the cancer phenotype and provide a new class of clinically relevant,broad-spectrum anticancer therapeutics for further development.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Received May 3, 2016; revised June 16, 2016; accepted June 20, 2016;published OnlineFirst August 31, 2016.

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2016;22:5189-5195. Published OnlineFirst August 31, 2016.Clin Cancer Res   Philip M. Tedeschi, Nitu Bansal, John E. Kerrigan, et al.  

Kinase as a Therapeutic Target in Cancer+NAD

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