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Therapeutics, Targets, and Chemical Biology

The Alkylating Chemotherapeutic TemozolomideInduces Metabolic Stress in IDH1-Mutant Cancersand Potentiates NADþ Depletion–MediatedCytotoxicityKensuke Tateishi1,2,3, Fumi Higuchi1,3, Julie J. Miller3,4,5, Mara V.A. Koerner1,3,Nina Lelic1,3,GaneshM. Shankar1,3, ShotaTanaka3,4,5, DavidE. Fisher6,Tracy T. Batchelor4,5,A. John Iafrate3,7, Hiroaki Wakimoto1,3, Andrew S. Chi3,8, and Daniel P. Cahill1,3

Abstract

IDH1-mutant gliomas are dependent upon the canonicalcoenzyme NADþ for survival. It is known that PARP activationconsumes NADþ during base excision repair (BER) of chemo-therapy-induced DNA damage. We therefore hypothesized thata strategy combining NADþ biosynthesis inhibitors with thealkylating chemotherapeutic agent temozolomide could poten-tiate NADþ depletion–mediated cytotoxicity in mutant IDH1cancer cells. To investigate the impact of temozolomide onNADþ metabolism, patient-derived xenografts and engineeredmutant IDH1-expressing cell lines were exposed to temozolo-mide, in vitro and in vivo, both alone and in combination withnicotinamide phosphoribosyltransferase (NAMPT) inhibitors,which block NADþ biosynthesis. The acute time period (<3hours) after temozolomide treatment displayed a burst ofNADþ consumption driven by PARP activation. In IDH1-mutant–expressing cells, this consumption reduced further the

abnormally lowered basal steady-state levels of NADþ, intro-ducing a window of hypervulnerability to NADþ biosynthesisinhibitors. This effect was selective for IDH1-mutant cells andindependent of methylguanine methyltransferase or mismatchrepair status, which are known rate-limiting mediators ofadjuvant temozolomide genotoxic sensitivity. Combinedtemozolomide and NAMPT inhibition in an in vivo IDH1-mutant cancer model exhibited enhanced efficacy comparedwith each agent alone. Thus, we find IDH1-mutant cancers havedistinct metabolic stress responses to chemotherapy-inducedDNA damage and that combination regimens targeting nonre-dundant NADþ pathways yield potent anticancer efficacy invivo. Such targeting of convergent metabolic pathways in genet-ically selected cancers could minimize treatment toxicity andimprove durability of response to therapy. Cancer Res; 77(15);4102–15. �2017 AACR.

IntroductionSomatic mutations in the isocitrate dehydrogenase genes

IDH1/2 define a class of adult diffuse gliomas with a distinctetiology and natural history (1–6). Molecular correlative analysesof international randomized trial cohorts have suggested thatpatients with IDH-mutant glioma, including both those with andwithout chromosome 1p/19q codeletion, gain a survival benefitfrom treatment with DNA-alkylating chemotherapy (7, 8). As aresult of this emerging evidence, chemotherapy is now frequentlyintegrated into the treatment regimen of these patients, eventhough they typically present with lower grade histology whencompared with IDH wild-type gliomas. The oral alkylating agenttemozolomide is commonly utilized by clinicians for this treat-ment, due to its tolerability in the adjuvant setting.

Unfortunately, the vast majority of these cancers still recur afteradjuvant or salvage temozolomide treatment. The activities ofthe O-6 methylguanine DNA methyltransferase (MGMT) repairenzyme (9) and the mismatch repair (MMR) pathway (10) arecriticalmechanistic determinants of temozolomide-induced cancercell cytotoxicity (11) and subsequent evasion and resistance totherapy (12–14). Salvage therapeutic strategies for post-temozolo-mide glioma recurrences are complicated by acquired mutationsinactivating theMMRpathway,with the resultingalkylator-induced

1Department of Neurosurgery, Massachusetts General Hospital Cancer Center,Harvard Medical School, Boston, Massachusetts. 2Department of Neurosur-gery, Yokohama City University, Yokohama, Kanagawa, Japan. 3TranslationalNeuro-Oncology Laboratory, Massachusetts General Hospital Cancer Center,Harvard Medical School, Boston, Massachusetts. 4Division of Hematology/Oncology, Massachusetts General Hospital Cancer Center, Harvard MedicalSchool, Boston, Massachusetts. 5Stephen E. and Catherine Pappas Center forNeuro-Oncology, Department of Neurology, Massachusetts General HospitalCancer Center, Harvard Medical School, Boston, Massachusetts. 6Departmentof Dermatology, Massachusetts General Hospital Cancer Center, HarvardMedical School, Boston, Massachusetts. 7Department of Pathology, Massa-chusetts General Hospital Cancer Center, Harvard Medical School, Boston,Massachusetts. 8Laura and Isaac Perlmutter Cancer Center, NYU LangoneMedical Center, New York, New York.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Daniel P. Cahill and Hiroaki Wakimoto, MassachusettsGeneral Hospital, MGH Brain Tumor Center, 55 Fruit Street, Boston, MA 02114.Phone: 617-724-0884; Fax: 617-724-0887; E-mail: [email protected];[email protected]; and Andrew S. Chi, Laura and Isaac PerlmutterCancer Center, NYU Langone Medical Center, New York, NY 10016. Phone: 212-731-6267; Fax: 646-754-9696; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2263

�2017 American Association for Cancer Research.

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hypermutation driving a treatment-resistant malignant phenotype(15, 16). Improved chemotherapeutic strategies are needed tosecure durable clinical responses in patients with IDH-mutantgliomas.

In addition to gliomas, mutations in IDH1 are found in adiverse spectrum of histopathologic tumor types, including leu-kemia, chondrosarcoma, cholangiocarcinoma, and a minor frac-tion ofmelanomas and breast cancers. Across each of these cancertypes, IDH1 mutation is typically found in different backgroundgenetic contexts. As a common feature, however, mutant IDH1driveswidespreadmetabolic alterations in cancer cells (17). Theseinclude the production of 2-hydroxyglutarate (2HG; ref. 18),modulation of HIF1a (19), pyruvate dehydrogenase (20), andlactate dehydrogenase (21), as well as altered citric acid cycle flux(22), and depleted steady-state pools of several canonical meta-bolites, including glutathione (23) and NADþ (24). This alteredbaseline metabolism results in the exposure of distinct enzymatictargets, including glutaminase (25) and the NADþ biosyntheticenzyme nicotinamide phosphoribosyltransferase (NAMPT;ref. 24), to selective inhibition with small molecules, resultingin genotype-specific metabolic vulnerabilities in IDH-mutantcancer cells.

We hypothesized that study of the metabolic consequences oftemozolomide exposure in IDH1-mutant cancers could uncovernovel opportunities for therapeutic targeting. Despite the impor-tant role of O6-methylguanine adducts in mediating adjuvanttemozolomide sensitivity, the majority (>80%) of temozolo-mide-induced DNA lesions are actually N3-methyladenine andN7-methylguanine adducts. These lesions are rapidly processedby the base excision repair (BER) machinery (26), as opposed tothe O6-methylguanine–dependent MGMT and MMR systems.Importantly, the dynamic capacity of BER does not becomesaturated with these lesions (27), which is why they are notrate-limiting determinants of cytotoxicity in adjuvant temozolo-mide-treated cancers. Their abundance nevertheless does induce asignificant stress response, through PARP, which polymerizesNADþ intopoly(ADP-ribose) (PAR) as themolecular repair signalactivating recruitment of downstream BER proteins. Recognizingthis activated PARP pathway, alongside the sirtuin (SIRT) path-way, is a primary mediator of NADþ consumption in cells (28),we assessed whether chemotherapeutic targeting of these nonre-dundant NADþ pathways could be exploited in IDH1-mutantcancer cells.

In experiments we describe here, we observed a burst of NADþ

consumption associated with PARP activation during the initialtime period immediately following temozolomide treatment. InIDH1-mutant cancer cells, this consumption resulted in a tran-sient but critical reduction of the already abnormally loweredbasal steady-state levels of NADþ, introducing a window ofhypervulnerability to NADþ biosynthesis inhibitors. This findingprovided a rationale for the therapeutic combination of temozo-lomide and NAMPT inhibitors, which resulted in improvedefficacywhen comparedwith their administration as single agentsin an in vivo IDH1-mutant cancer model.

Materials and MethodsCreation of glioma tumorsphere lines

Under IRB-approved protocols, the patient-derived gliomalines used in this study (MGG18, MGG23, MGG85, MGG91,MGG119, MGG152, and MGG171) were obtained from 2008 to

2014 and were cultured in serum-free neural stem cell medium asdescribed previously (29–31). BT142 (IDH1R132H-mutant ana-plastic oligoastrocytoma) line was obtained from ATCC in 2014and was not further authenticated. UACC257 line (IDH1/2 wild-type melanoma), HT1080 (IDH1R132C), and U87 (IDH1 wildtype) lines were authenticated in 2017 by comparison of STRprofiles to the ATCC public dataset. They were cryopreserved atpassage number 3 or less prior to use for in vitro experiments.Normal human astrocytes (NHA) were obtained from ScienCellin 2014 and cultured in Astrocyte Medium (ScienCell) and werenot further authenticated. All standard cell line media weresupplemented with 10% FBS and penicillin–streptomycin–amphotericin B.

IDH1 genotyping and MGMT promoter methylation analysisIDH1 genomic DNA PCR products (Platinum Taq polymer-

ase) spanning coding exons were Sanger sequenced (BeckmanCoulter Genomics). To assess MGMT promoter methylationstatus, methylation-specific PCR on genomic and bisulfite-modified DNA (Qiagen DNeasy Blood & Tissue Kit and EpiTectBisulfite Kit) was performed in a two-step approach asdescribed previously (32).

IDH1-R132H cell line generationTo generate IDH1R132H overexpressing lines, UACC257 cells

were transduced with IDH1R132H lentivirus (ViraPower HiPer-form T-Rex Gateway Expression System, Invitrogen), with thedetails of generation in Supplementary Methods. MGG18-IDH1-R132H cells have been described previously (24). Afterincubation with tetracycline (1 mg/mL) for 2 months, cells wereused for experiments.

shRNA and control shRNA cell line generationTo knockdown MSH6, MGG152 or HT1080 cells were

infected with lentivirus containing human MSH6 shRNA (#1,TRCN0000286578, Sigma Aldrich, #2 V3LHS_318784, GEDharmacon), with the details of generation described in theSupplementary Methods. To knock down NAPRT1, UACC257cells were infected with GIPZ Lentiviral Human NAPRT shRNA(V3LHS_359032, GE Dharmacon). GIPZ nonsilencing lenti-viral shRNA control (RHS4348, GE Dharmacon) was used as amatched control.

Cell viability, cytotoxicity, PARP activity, and apoptosisanalyses

To assess cell viability, under treatment conditions, CellTiter-Glo (Promega) assays were performed at the indicated timepoints, and the IC50 values were determined. PARP activitywas quantified by HT Colorimetric PARP/Apoptosis Assay Kit(Trevigen) according to themanufacturer's recommendations. Toevaluate caspase-3/7 activities, 7,000 to 8,000 cells were treatedwith DMSO or NAMPT inhibitors (12.5 nmol/L) and/or temo-zolomide (200 mmol/L), or Z-VAD-FMK (50 mmol/L) for 6 or 24hours and were tested by Caspase-Glo 3/7 Assay (Promega)according to the manufacturer's recommendations. Details aredescribed in Supplementary Methods.

NADþ quantitationTo evaluate qualitative values of NADþ, NADH, NADPþ, and

NADPH, NAD/NADH-Glo Assay and NADP/NADPH-Glo Assay

TMZ Induces NADþ Depletion in IDH1-Mutant Cancers

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(Promega) were used according to themanufacturer's recommen-dations. To measure NADþ quantitatively, NADþ/NADH Quan-tification Colorimetric Kit (BioVision Incorporated) was usedaccording to the manufacturer's recommendations. Protein con-centrations were measured and used for normalization. NADt(NADH and NAD) and NADH signals were measured at OD450 nm (BIOTEK). NADþ concentrations were calculated by sub-tracting NADH fromNADt. Data were expressed as pmol/1� 106

cells or pmol/mg protein.

Animal studiesAll mouse experiments were approved by the Institutional

Animal Care and Use Committee at Massachusetts General Hos-pital (Boston, MA). HT1080 cells (2.5 � 106) were subcutane-ously implanted into the right flank of 7- to 10-week-old femaleSCID mice. When maximum tumor diameter reached 5 mm,cohorts were randomized to vehicle (oral gavage, n ¼ 6), FK866(15mg/kg, i.p, n¼ 6), temozolomide (50mg/kg, oral gavage, n¼6), or FK866 (15 mg/kg, i.p) plus temozolomide (50 mg/kg, oralgavage, n¼ 6). Distilledwater with 20%Captisol (Cydex) and 5%dextrose (Sigma-Aldrich) was used as vehicle control. Treatmentwas given four times a week for 2 weeks. Tumor diameters weremeasured three times a week using a digital caliper. Calculatedvolume (mm3) ¼ length (mm) � width (mm)2 � 0.5. Tumorvolumewas normalized at day 0 and expressed as percent change.

IHCTumor tissue sections were incubated with anti-Ki-67 (1:125,

Wako), LC3A/B (1:100, Cell Signaling Technology), cleaved-PARP (1:100, Cell Signaling Technology), or cleaved-caspase-3(1:100, Cell Signaling Technology) primary antibody.

Statistical analysesAll of the experiments were performed in three replicates per

condition. For parametric analyses, two-tailed Student t tests orone-way ANOVA were used, and for analysis of frequencies ofnominal data, two-tailed Fisher exact test was used, withoutBonferroni correction. Data were expressed as mean � SE.P values less than 0.05 were considered statistically significant.

ResultsTemozolomide induces NADþ consumption via PARPactivation

The PARP-activated BER pathway consumes NADþ duringrepair of the DNA lesions induced by temozolomide exposure.Because of decreased NADþ biosynthesis, IDH1-mutant cancercells contain a lower baseline NADþ level compared with IDHwild-type cancer cells. We therefore investigated the effect oftemozolomide onNADþ levels inmutant IDH1 cells. We initiallytested the cytotoxic andPARP-inducing effect of temozolomide ina panel of endogenous IDH1-mutant and wild-type gliomatumorspheres (Fig. 1A; Supplementary Fig. S1A; SupplementaryTable S1).

Overall, sensitivity to temozolomide monotherapy variedacross the panel, irrespective of IDH1 mutation or MGMT pro-moter methylation status (Supplementary Table S1). InMGG152cells (IDH1 mutant, MGMT promoter methylated), temozolo-mide cytotoxicity was observed after day 4 (Fig. 1B). We observedPARP activation after temozolomide treatment, resulting in amarked induction of PAR in IDH1-mutant cancers in a time- and

dose-dependent manner (Fig. 1C and D; Supplementary Fig.S1B). Notably, PARylation was induced rapidly, within 1 hour,and significantly decreased thereafter, consistent with its role inBER signaling response. Accordingly, temozolomide decreasedNADþ levels in a dose-dependent manner in multiple IDH1-mutant cell lines (Fig.1E; Supplementary Fig. S1C).

To investigate whether this effect was PARP specific, we repeat-ed these experiments with the addition of the PARP inhibitorolaparib. Olaparib significantly suppressed PARP activation andPAR expression observed with temozolomide treatment (Fig. 1F).Olaparib also reversed the NADþ reduction induced by temozo-lomide (Fig. 1G; Supplementary Fig. S1D). Thus, temozolomidepromotes acute NADþ consumption via PARP activation inIDH-mutant cancer cells.

NAMPT inhibitors and temozolomide have additivecytotoxicity in endogenous IDH1-mutant cancer cells

As noted above, IDH1-mutant cancer cells contain a lowerbaseline NADþ level compared with IDH wild-type cancer cells,rendering IDH1-mutant cells selectively sensitive to NADþ bio-synthesis blockade by NAMPT inhibition (24). We hypothesizedthat temozolomide-induced NADþ reduction could thereforepotentiate the metabolic cytotoxicity of NAMPT inhibitors inIDH-mutant cells. To investigate this further, we first tested thetemozolomide sensitivity of several endogenous IDH1-mutantcancer cell lines. Exposure to temozolomide alone for 24 to 36hours had no or marginal impact on viability of IDH1-mutantcells, consistent with the effective BER ofDNA damage during thistime interval.

However, the same exposure to temozolomide significantlyaugmented the acute cytotoxic effect of the NAMPT inhibitorsFK866 and GMX1778 (Fig. 2A and B; Supplementary Fig. S2A).Testing a range of NAMPT inhibitor doses revealed that low doseNAMPT inhibitor (2.5 nmol/L) combined with temozolomidehad more potent cytotoxicity at this early time point (24 hours)than high dose (250 nmol/L) NAMPT inhibitor monotherapy inIDH1-mutant cancer cells (Fig. 2C). Importantly, this metaboliccombination effect seen in IDH1-mutant cancer cells was distinctfrom, and not dependent upon the rate-limiting genotoxic dam-age of single-agent temozolomide itself, nor its well-establishedmodifier, MGMT promoter methylation status (SupplementaryTable S1). In contrast, we did not detect any effect on cell viabilitywith temozolomide, NAMPT inhibitors, and their combinationin several IDH1 wild-type glioma tumorsphere cells at 24 hours(Fig. 2D; Supplementary Fig. S2B). At 72 hours, temozolomidecombination with a higher dose range of NAMPT inhibitorsshowed some cytotoxic effects in endogenous IDH1 wild-typeglioma tumorsphere lines (Supplementary Fig. S2C and S2D).In addition, combination treatment was not detectably toxic toNHA (Fig. 2E). Thus, combined treatment with NAMPT inhi-bitors and temozolomide had potent and additive effects onacute cytotoxicity, in a manner that had greater selectivity forIDH1-mutant cells.

Temozolomide and NAMPT inhibitor combination inducedadditive NADþ depletion in endogenous IDH1-mutant cancers

To further explore the mechanism of this genotype-selectivecombination effect, we tested whether the combination ofNAMPT inhibitor and temozolomide, each of which reducesintracellular NADþ levels as single agents, would have an additiveeffect on NADþ levels in IDH1mutant cancer cells. We found that

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combined NAMPT inhibition and temozolomide exposure sig-nificantly decreased NADþ and NADH levels compared withsingle-agent treatment, and the effect was dose dependent (Fig.3A; Supplementary Fig. S3A). We also found that NADPþ andNADPH levels were only moderately decreased by the combina-tion treatment (Supplementary Fig. S3A). Measurement of NADþ

levels at different time points revealed that, in contrast to thesustained NADþ reduction induced by NAMPT inhibitors, thetemozolomide-induced decrease in NADþ was transient andrecovered to baseline at 6 to 12 hours posttreatment (Fig. 3B;Supplementary Fig. S3A and S3B). Interestingly, preincubationwith temozolomide for 36 hours prior to addition of NAMPTinhibitor attenuated combination benefit (Supplementary Fig.S3C), underscoring the role of the transient temozolomide-

induced NADþ decrease in the additive effect on NADþ levelsand efficacy. Nevertheless, the combination of NAMPT inhibitorsand temozolomide progressively decreased NADþ levels overtime when given concurrently (Fig. 3B).

To investigate whether the additive effect of concurrent NAMPTinhibitor and temozolomide treatment onNADþ levels inmutantIDH1 cells were mediated through PARP, we tested the combi-nation with and without PARP inhibition with small-moleculeinhibitors. Indeed, olaparib and veliparib were each sufficient tonear-completely rescue the decrease in NADþ levels as well as cellviability when given concurrently with NAMPT inhibitor andtemozolomide in multiple IDH1-mutant cancer cells (Fig. 3CandD; Supplementary Fig. S3D and S3E). In addition, exogenoussupplementation with NADþ or nicotinamide mononucleotide,

Figure 1.

Temozolomide (TMZ)-induced PARP activation deregulates NADþ metabolism. A, CellTiter-Glo cell viability assay after 6-day temozolomide treatment of GBMtumorsphere lines, HT1080, and NHA. Left, IDH1-mutant lines; right, IDH1/2 wild-type lines with temozolomide at indicated doses compared with DMSOcontrol. B, Relative cell viability of MGG152 cells treated with 200 mmol/L of temozolomide compared with DMSO control at the indicated time. C, Left, PARPactivity after temozolomide treatment (200 mmol/L) at indicated time; right, Western blot analysis of PAR expression in temozolomide (200 mmol/L)-treatedMGG152 cells at different time points. Actin, loading control. D, Left, PARP activity after 1-hour treatment with temozolomide at indicated dose; right,Western blot analysis of PAR expression in MGG152 cells with temozolomide at indicated dose for 1 hour. Actin, loading control. E, Relative NADþ levels inMGG152 cells with temozolomide treatment at indicated dose for 3 hours. � , P < 0.05 for difference between DMSO versus temozolomide. F, PARP activity (left)and PAR expression (right, Western blot analysis) in MGG152 cells treated with DMSO, temozolomide (200 mmol/L), olaparib (5 mmol/L), or temozolomideplus olaparib for 1 hour. � , P < 0.05 for difference from DMSO. Actin, loading control. G, Relative NADþ levels in MGG152 cells with olaparib (5 mmol/L),temozolomide (200 mmol/L), or temozolomide plus olaparib for 3 hours. � , P < 0.05 for difference between DMSO versus temozolomide. �� , P < 0.05 fordifference between temozolomide and temozolomide plus olaparib. Scale bars, SE.






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the product of the NAMPT reaction and the immediate precur-sor of NADþ, restored NADþ levels and near-completely abro-gated the cytotoxic effect of temozolomide and NAMPT inhib-itor combination on IDH1-mutant cell viability (Fig. 3E and F;Supplementary Fig. S3F and S3G). These experiments indicatethe effects of combined temozolomide and NAMPT inhibitorwere both NADþ- and NAMPT specific. Together, these dataindicate that combined treatment with NAMPT inhibitor andtemozolomide results in additive NADþ depletion in IDH1-mutant cancer cells.

Combination therapy with NAMPT inhibitor andtemozolomide is selectively toxic to IDH1-mutant cancer cells

To experimentally confirm whether combined temozolomideand NAMPT inhibitor treatment is selective for IDH1 mutationitself, we engineered an isogenic system using the UACC257melanoma line to stably express mutant IDH1 (UACC257-IDH1R132H) or control GFP (UACC257-GFP; Fig. 4A). There wasno significant difference in proliferation between these lines(Supplementary Fig. S4A). Consistent with previous observationsin a different model system (24), expression of mutant IDH1lowered NAPRT1 expression (Fig. 4A) and steady-state NADþ

levels (Fig. 4B).Next, we tested the combination on cell viability in the engi-

neered lines. Consistent with our previous findings, expression ofmutant IDH1 induced sensitivity toNAMPT inhibitors. Although aminor additive cytotoxic effect was seen in control GFP-expressingcells, combination treatment potently decreased cell viability inthe mutant IDH1–expressing cell line (Fig. 4C). To confirm thisIDH1 genotypic selectivity in a second line, we used a tetracycline-inducible IDH1-mutant glioblastoma tumorsphere line, MGG18-IDH1-R132H (Fig. 4D; ref. 24). As a control, tetracycline did notaffect MGG18 response to NAMPT inhibitor or temozolomide(Supplementary Fig. S4B). Again, when mutant IDH1 was induc-ed, no significant difference in cell proliferation was observed(Supplementary Fig. S4C). Consistent with our aforementionedfindings with the UACC257 engineered lines, MGG18-IDH1-R132H became more acutely sensitive to NAMPT inhibitor treat-ment and demonstrated marked additive sensitivity to combina-tion treatment with temozolomide, while combined therapy wasminimally cytotoxic without mutant IDH1 induction (Fig. 4E).

We hypothesized that the mechanism of enhanced cytotox-icity in IDH1-mutant cells, compared with wild-type cells, wasmediated through the reset of basal steady-state NADþ levels byinhibition of NAPRT1, a rate-limiting enzyme of NADþ bio-synthesis. To validate this threshold effect, we established astable NAPRT1 knockdown line of UACC257 (Fig. 4F). Con-sistent with our results observed in the endogenous andenforced IDH1-mutant lines, NAPRT1 knockdown decreased

steady-state levels of NADþ (Fig. 4G). Importantly, single-agentNAMPT inhibitor and combination treatment decreased cellviability of theNAPRT1 knockdown line to a greater extent thanthat of controls (Fig. 4H). Altogether, these findings indicatethat mutant IDH1 decreases basal NADþ through NAPRT1inhibition, mediating increased vulnerability to combinationtreatment with NAMPT inhibitor and temozolomide in agenotype selective manner.

Combined temozolomide and NAMPT inhibitor treatmentinduces autophagy and apoptosis, and results in cell death inIDH1-mutant cells

In parallel with ATP-based cell viability assay, we confirmedcell death from combined NAMPT inhibitor and temozolo-mide treatment by counting viable cells after 48-hour treat-ment (Fig. 5A; Supplementary Fig. S5A and S5B). We furtherfound that combination treatment significantly inhibited cloneformation of HT1080 cells (Fig. 5B), even with lower doses oftemozolomide (Supplementary Fig. S5C). We observed induc-tion of LC3-II expression, a marker of autophagy, at 48 hoursafter NAMPT inhibitor treatment (Supplementary Fig. S5D).Combined treatment with temozolomide more stronglyinduced LC3-II expression than single-agent exposure witheither NAMPT inhibitor or temozolomide, and LC3-II expres-sion was detectable as early as 6 hours posttreatment (Fig. 5C),indicative of an earlier engagement of a metabolic stressresponse. Furthermore, exogenous 3-methyladenine (3-MA),an autophagy inhibitor, partially rescued the cytotoxicity of thecombination treatment (Fig. 5D; Supplementary Fig. S5E). Inaddition to autophagy, we found combination treatmentinduced PARP cleavage (Supplementary Fig. S5F) andincreased caspase-3/7 activity (Supplementary Fig. S5G), sug-gesting a contribution of apoptosis to cell death. However, thepan-caspase inhibitor Z-VAD-FMK, which strongly suppressedcaspase-3/7 activity (Supplementary Fig. S5H), only mildlyrescued cell viability after combined temozolomide andNAMPT inhibitor treatment (Supplementary Fig. S5I). Thesedata provide evidence that the additive cytotoxicity of temo-zolomide when combined with an NAMPT inhibitor in IDH-mutant cells is primarily mediated through augmentation of ametabolic stress response.

Therapeutic effect of combined NAMPT inhibitor andtemozolomide is independent of MMR pathway activity

The clonal emergence of resistance to alkylating chemotherapyin a population of cancer cells is known to be mediated byinactivation of genes in the MMR system (33), and an MMRdeficiency is typically acquired in post-temozolomide recurrentgliomas (13–16). To assess the independence of our observed

Figure 2.Cytotoxic effect of temozolomide (TMZ) and NAMPT inhibitors in IDH1-mutant cancers. A, CellTiter-Glo cell viability assay after 24-hour treatment of IDH1-mutantcancer lineswith no inhibitor (white bars), FK866 (graybars), orGMX1778 (black bars) and indicateddoses of temozolomide. � ,P<0.05 for differencebetween FK866and FK866 plus temozolomide. �� , P < 0.05 for difference between GMX1778 and GMX1778 plus temozolomide. B, CellTiter-Glo cell viability assay of IDH1-mutantcancer lines. Top, treatmentwith temozolomide (200mmol/L;white bars), FK866 (12.5 nmol/L; graybars), or FK866 plus temozolomide (black bars) at indicated timepoint. � , P < 0.05 for difference between DMSO and FK866. �� , P < 0.05 for difference between FK866 and FK866 plus temozolomide. Bottom, treatmentwith temozolomide (200 mmol/L; white bars), GMX1778 (12.5 nmol/L; gray bars), or GMX1778 plus temozolomide (black bars) at indicated time point. � , P < 0.05 fordifference between DMSO and GMX1778. �� , P < 0.05 for difference between GMX1778 and GMX1778 plus temozolomide. Data were expressed as % DMSO.C and D, Relative cell viability of IDH1-mutant cancer lines (C) and IDH1 wild-type lines (D) after 24-hour exposure to DMSO (blue lines) and temozolomide(200 mmol/L; red lines) with/without FK866 (top) or GMX1778 (bottom) at indicated concentrations. E, Relative cell viability of NHA treated with DMSO(white bars) or temozolomide (200 mmol/L; black bars) and/or NAMPT inhibitors at 72 hours. Scale bars, SE.






combination effect from known mediators of temozolomidegenotoxic cytotoxicity, we examined whether temozolomidepotentiation of NADþ depletion in IDH1-mutant cancer cellsvaried in relation to MMR status. To this end, we establishedstable MSH6 knockdown lines of HT1080, which is MGMTunmethylated (Supplementary Table S1; Fig. 6A and B). As

expected, knockdown of MSH6 (MSH6 #1 and MSH6 #2)induced resistance to temozolomide, confirming the critical roleof MSH6 in MMR-mediated temozolomide sensitivity (Fig. 6C).However, MSH6 knockdown did not alter NAMPT inhibitorsensitivity in HT1080 cells (Fig. 6D). Importantly, and strikinglyindicative of this second independentmechanism, temozolomide

Figure 3.

NADþ depletionwith temozolomide (TMZ) and NAMPT inhibitors in endogenous IDH1-mutant cancers.A, Relative NADþ levels in IDH1-mutant lines with no inhibitor(white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with/without temozolomide for 6 hours. P < 0.05 for difference betweenDMSO and temozolomide in no inhibitor–treated cells (�), FK866-treated cells (��), and GMX1778-treated cells (��). B, Relative NADþ levels in IDH1-mutant linestreated with FK866 (12.5 nmol/L; red lines), GMX1778 (12.5 nmol/L; orange lines), temozolomide (200 mmol/L; blue lines), FK866 plus temozolomide (black lines),and GMX1778 plus temozolomide (green lines) at indicated time point. DMSO-treated cells were used as control. C,Relative NADþ levels in IDH1-mutant lines treatedwith no inhibitor (white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with temozolomide (200 mmol/L), olaparib (5 mmol/L), ortemozolomideplus olaparib for 6 hours. � ,P<0.05 for difference betweenNAMPT inhibitors versusNAMPT inhibitors andolaparib. �� ,P<0.05 for difference betweenNAMPT inhibitors plus temozolomide versus NAMPT inhibitors, temozolomide, plus olaparib. D, Relative cell viability of IDH1-mutant lines treated with noinhibitor (white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with DMSO, temozolomide (200 mmol/L), olaparib (5 mmol/L), ortemozolomide plus olaparib for 24 hours. � , P < 0.05 for difference between NAMPT inhibitors versus NAMPT inhibitors and olaparib. �� , P < 0.05 fordifference betweenNAMPT inhibitors plus temozolomide versus NAMPT inhibitors, temozolomide, plus olaparib.E,RelativeNADþ levels in IDH1-mutant lines treatedwith no inhibitor (white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with temozolomide (200 mmol/L), NMN (1 mmol/L), ortemozolomide plus NMN for 6 hours. � , P < 0.05 for difference between NAMPT inhibitors versus NAMPT inhibitors plus NMN. �� , P < 0.05 for difference betweenNAMPT inhibitors plus temozolomide versus NAMPT inhibitors, temozolomide, plus NMN treatment. F, Relative cell viability of IDH1-mutant lines treated withno inhibitor (white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with DMSO, temozolomide (200 mmol/L), NMN (1 mmol/L), ortemozolomide plus NMN for 24 hours. � , P < 0.05 for difference between NAMPT inhibitors versus NAMPT inhibitors plus NMN. �� , P < 0.05 for differencebetween NAMPT inhibitors plus temozolomide versus NAMPT inhibitors, temozolomide, plus NMN treatment.

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Figure 4.

Selective vulnerability of IDH1-mutant cancer cells to combination therapy with NAMPT inhibitor and temozolomide. A, Western blot analysis of IDH1R132H (left)and NAPRT1 (right) in UACC257-GFP and -IDH1R132H cells. Vinculin, loading control.B,Mean baseline absolute NADþ levels in UACC257-GFP (1,916.8� 19.9 pmol/1� 106

cells) and UACC257-IDH1R132H (1,494.2 � 71.8 pmol/1 � 106 cells) � , P < 0.05 for difference between UACC257-GFP and-IDH1R132H cells. C, Relative cell viability ofUACC257-GFP treated with DMSO (black bars) or temozolomide (200 mmol/L; orange bars), and UACC257-IDH1R132H with DMSO (blue bars) or temozolomide(200 mmol/L; red bars) with indicated doses of FK866 (left) or GMX1778 (right) for 96 hours. P < 0.05 for difference between temozolomide versus temozolomide plusNAMPT inhibitors in UACC257-GFP cells (�), DMSO versus NAMPT inhibitors in UACC257-IDH1R132H cells (��), and temozolomide versus temozolomide plusNAMPT inhibitors in UACC257-IDH1R132H cells (��). D,Western blot analysis of IDH1R132H in MGG18-IDH1-R132H cells with or without 1 mg/mL tetracycline (Tet). Vinculin,loading control. E, Relative cell viability of MGG18-IDH1-R132H (tet�) cells with DMSO (black bars) or temozolomide (200 mmol/L; orange bars), and MGG18-IDH1-R132H(tetþ) cells with DMSO (blue bars), or temozolomide (200 mmol/L; red bars) with FK866 (left) or GMX1778 (right) treatment for 96 hours. P < 0.05 for differencebetween temozolomide versus temozolomide plus NAMPT inhibitors inMGG18-IDH1-R132H (tet�) cells (�), DMSO versus NAMPT inhibitors in MGG18-IDH1-R132H (tetþ)cells (��), and temozolomide versus temozolomide plus NAMPT inhibitors in MGG18-IDH1-R132H (tetþ) cells (��). F, Western blot analysis of NAPRT1 in control(nonsilencing) and NAPRT1 shRNA–transduced UACC257 cells. Vinculin, loading control. G, Mean baseline absolute NADþ levels in nonsilencing shRNA–transducedUACC257 (1,342.5 � 72.2 pmol/1 � 106 cells) and NAPRT1 shRNA–transduced UACC257 cells (724.8 � 58.8 pmol/1 � 106 cells; � , P < 0.05). H, Relative cell viability ofnonsilencing (NS) shRNA–transduced UACC257 with DMSO (black bars) or temozolomide (200 mmol/L; orange bars), and NAPRT1 shRNA–transduced UACC257 cellswith DMSO (blue bars), or temozolomide (200 mmol/L; red bars) with FK866 (left) or GMX1778 (right) treatment for 96 hours. P < 0.05 for difference betweentemozolomide versus temozolomide plus NAMPT inhibitors in nonsilencing shRNA–transduced cells (�), DMSO versus NAMPT inhibitors in NAPRT1 shRNA–transducedUACC257 cells (��), temozolomide versus temozolomide plus NAMPT inhibitors in NAPRT1 shRNA–transduced UACC257 cells (��).






equally potentiated the effect of NAMPT inhibitor treatment inHT1080 cells with and without MSH6 knockdown (Fig. 6D).Similarly, in MGMT methylated MGG152 cells (Fig. 6A), weestablished stable MSH6 knockdown cells and confirmed MSH6knockdown increased resistance to temozolomide (Fig. 6E and F).Again, irrespective of MSH6 status, NAMPT inhibitors displayedequal efficacy (Fig. 6G), and temozolomide treatment equiva-lently augmented NAMPT inhibitor cytotoxicity in MGG152 cells(Fig. 6G). Thesefindings indicate the independent effectiveness oftemozolomide metabolic augmentation targeting NADþ inIDH1-mutant cells, regardless of the status of resistance determi-nants to temozolomide genotoxic monotherapy effect.

Combined temozolomide and NAMPT inhibitor therapy hasadditive activity against IDH1-mutant xenograft tumors

The NAMPT inhibitor FK866 potently inhibits the growth ofHT1080 (IDH1R132C) xenograft tumors implanted in the flank

of immunocompromised mice (24). We tested whether temo-zolomide alteration of NADþ metabolism could potentiate thein vivo growth inhibiting effects of NAMPT inhibitor in thisIDH-mutant xenograft model. We first measured intratumoralNADþ levels with temozolomide treatment, two doses ofNAMPT inhibitor (15 or 30 mg/kg per day), and combinedtherapy with temozolomide and FK866 at 15 mg/kg per day.Monotherapy with temozolomide and both doses of NAMPTinhibitor significantly reduced intratumoral NADþ levels com-pared with vehicle-treated tumor tissue, and there was a dose-dependent decrease in NADþ levels with NAMPT inhibitortreatment (Fig. 7A). Notably, reduced-dose FK866 (15 mg/kgper day) plus temozolomide treatment reduced intratumoralNADþ to the levels equivalent to that of high dose FK866 (30mg/kg per day).

On the basis of these observations, we tested whetherthe combination of reduced-dose FK866 with temozolomide

Figure 5.

Cell deathmechanism in combined temozolomide (TMZ) andNAMPT inhibitor treatment of IDH1-mutant cells.A,Cell viability of IDH1-mutantMGG152 after treatmentwith FK866 (12.5 nmol/L), temozolomide (200 mmol/L), or FK866 plus temozolomide (top) and GMX1778 (12.5 nmol/L), temozolomide (200 mmol/L) orGMX1778 plus temozolomide (bottom) measured by ATP-based CellTiter-Glo assay (white bars) and viable cell count (Trypan blue exclusion) assay (black bars) atindicated time points. P < 0.05 for comparing NAMPT inhibitors versus NAMPT inhibitors plus temozolomide with the CellTiter-Glo assay (�), and the Trypanblue exclusion assay (��). DMSO-treated cells were used as control at each time point. B, Clonogenic assay of HT1080 cells treated for 27 hours with DMSO,temozolomide (200 mmol/L), FK866 (12.5 nmol/L), or FK866 (12.5 nmol/L) plus temozolomide (200 mmol/L) showing surviving fraction after treatment. Data areexpressed as % DMSO. � , P < 0.05 for difference between DMSO versus temozolomide plus FK866. C, Western blot analysis of LC3-I and LC3-II expressionsin MGG152 cells treated with DMSO, FK866 (12.5 nmol/L), temozolomide (200 mmol/L), or FK866 plus temozolomide for 6 hours. Actin, loading control. D, Relativecell viability of IDH1-mutant lines treated with no inhibitor (white bars), FK866 (12.5 nmol/L; gray bars), or GMX1778 (12.5 nmol/L; black bars) with DMSO,temozolomide (200 mmol/L), 3-MA (5mmol/L), or temozolomide plus 3-MA for 24 hours. � , P <0.05 for difference between NAMPT inhibitors and NAMPT inhibitorsplus 3-MA. �� , P < 0.05 for difference between NAMPT inhibitors plus temozolomide versus NAMPT inhibitors, temozolomide, plus 3-MA. Scale bars, SE.

Tateishi et al.





Figure 6.

Efficacy of combined temozolomide and NAMPT inhibitortreatment for MMR-deficient IDH1-mutant cells. A, MGMTpromoter methylation status in HT1080 andMGG152 cells.MSP, methylation-specific PCR; U, unmethylated; M,methylated. B, Western blot analysis of MSH6 innonsilencing (NS) and MSH6 shRNA–transduced HT1080cells (MSH6 #1 and #2). Actin, loading control. C, Cellviability assay of nonsilencing (NS) and MSH6 shRNA–transduced HT1080 cells (MGMT promoter unmethylated;MSH6 #1, left; MSH6 #2, right) treated with temozolomidefor 6 days. �, P < 0.05 for difference betweennonsilencing versus MSH6 shRNA–transduced cells.D, Relative cell viability of nonsilencing and MSH6 shRNA(MSH6 #1 and #2)–transduced HT1080 cells withNAMPT inhibitors with or without temozolomide(200 mmol/L) for 48 hours. P < 0.05 for differencebetween NAMPT inhibitors versus temozolomide plusNAMPT inhibitors in nonsilencing shRNA–transduced cells(�), NAMPT inhibitors versus temozolomide plus NAMPTinhibitors in MSH6 shRNA–transduced cells (�� , MSH6 #1;�� , MSH6 #2). E, Western blot analysis of MSH6 innonsilencing shRNA and MSH6 shRNA (MSH6 #1)–transduced MGG152 cells. Actin, loading control. F, Cellviability assay of nonsilencing and MSH6 shRNA (MSH6#1)–transduced MGG152 cells (MGMT promotermethylated) with temozolomide treatment for 6 days.� , P < 0.05 for difference between nonsilencing andMSH6 shRNA–transduced cells. G, Relative cell viabilityof nonsilencing and MSH6 shRNA (MSH6 #1)–transducedMGG152 cells treated with NAMPT inhibitors with orwithout temozolomide (200 mmol/L) for 24 hours.Scale bars, SE.






Figure 7.

In vivo therapeutic effects of combined temozolomide (TMZ) and NAMPT inhibitor treatment in an endogenous IDH1-mutant cancer model. A, Mean absoluteNADþ levels in HT1080 xenograft specimens treatedwith vehicle, temozolomide (50mg/kg), FK866 (15 or 30mg/kg), or FK866 (15mg/kg) plus temozolomide. Datawere expressed as pmol/mg protein. � , P < 0.05 for comparison between FK866 (15 mg/kg) and FK866 (30 mg/kg). �� , P < 0.05 for comparison betweenFK866 (15 mg/kg) and FK866 (15 mg/kg) plus temozolomide (50 mg/kg). B and C, Therapeutic effects of combined temozolomide and NAMPT inhibitor treatmentin subcutaneous HT1080 (B) and U87 (C) xenografts. Plot depicts tumor volume changes over time, with values being normalized to tumor volumes at day 0when treatments began. Each point represents the average of tumor volumes in a group treatedwith vehicle (black line; n¼6), temozolomide (50mg/kg; purple line;n¼ 6), FK866 (15 mg/kg; blue line; n¼ 6), or FK866 (15 mg/kg) plus temozolomide (red line; n¼ 6). � , P < 0.05 comparison between FK866 (15 mg/kg) and vehicle.�� , P < 0.05 comparison between FK866 (15 mg/kg) plus temozolomide (50 mg/kg) and vehicle. �� , P < 0.05 comparison between FK866 (15 mg/kg) plustemozolomide (50 mg/kg) and FK866 (15 mg/kg). D, Hematoxylin and eosin (H&E), Ki-67, and LC3A/B staining of HT1080 xenograft tissues after treatmentwith vehicle (top) and FK866 and temozolomide (bottom). Scale bar, 100 mm. E, Bar graphs showing percent positive staining cells of Ki67 (71.0% � 9.3% invehicle vs. 31.0%� 10.2% in FK866 plus temozolomide; P¼0.003), and LC3A/B (21.5%� 1.6% in vehicle vs. 90.7%� 2.4% in FK866 plus temozolomide; P¼0.0009).F, Bodyweight changes of animals bearing subcutaneous HT1080 xenografts during the treatment with vehicle (black line; n ¼ 6; oral gavage), temozolomide(50 mg/kg; purple line; n ¼ 6; oral gavage), FK866 (15 mg/kg; blue line; n ¼ 6; i.p), or FK866 (15 mg/kg) plus temozolomide (red line; n ¼ 6). Arrows, the timepoints when treatment doses were given.

Tateishi et al.





has antitumor effect in HT1080 xenografts. Compared withvehicle treatment, FK866 (15 mg/kg) alone, but not temozo-lomide alone, significantly suppressed tumor growth. Indeed,combination treatment was significantly superior in inhib-iting tumor growth compared with monotherapy with eitheragent or vehicle (Fig. 7B). In contrast, we did not observe thecombination effect in IDH wild-type U87 xenografts (Fig. 7C).Histopathologic analysis demonstrated the presence of necrot-ic regions in tumor tissues receiving combination treatment,but not in those receiving vehicle treatment. IHC analysisrevealed that combination treatment significantly decreasedKi-67 expression (Fig. 7D and E) and induced the autophagymarker LC3-A/B (Fig. 7D and E), similar to patterns previouslyobserved in single-agent NAMPT inhibitor treatment (24). Incontrast, we did not find significant differences in cleavedPARP and cleaved caspase-3 expression between combinationand control groups (Supplementary Fig. S6). Importantly, wedid not observe significant adverse toxicity during treatment,and no differences in body weight were detected between allthe treatment groups (Fig. 7F).

DiscussionSteady-state levels of cellular NADþ are established by the

balance of metabolite influx from biosynthetic enzymes, such asNAPRT1 and NAMPT, and efflux via NADþ consumption byenzyme complexes including the PARP and SIRT proteins (28).In IDH1-mutant cancer cells, the baseline pool of NADþ isdiminished compared with that in IDH1 wild-type cells, due toreduced expression of the NADþ biosynthetic enzyme NAPRT1(24). In our findings herein, we observed a burst of NADþ

consumption associated with PARP activation during the initialtime period after temozolomide treatment. This NADþ consump-tion critically depleted NADþ levels and introduced a window ofhypervulnerability to NAMPT inhibitors. This finding provided arationale for the therapeutic combination of temozolomide andNAMPT inhibitors, which resulted in improved efficacy, whencomparedwith their use as single agents, in both in vitro and in vivoIDH1-mutant cancer models.

Importantly, our evidence indicates the effect is genotypeselective, as we observe greater potency against IDH1-mutantcells compared with wild-type (including noncancerous) cells.Whether our findings would extend across the diverse spectrumof IDH1-mutant cancers would require further validation.Nonetheless, the efficacy of combining these two agents mayallow for optimized dosing tomaintain therapeutic indexwithoutcompromising anticancer activity; for instance, by enablingreduced doses of agents that induce significant toxicity at dosesneeded for efficacy in monotherapy (34). This is particularlyrelevant to NAMPT inhibitors, which have been observed to havedose-limiting thrombocytopenia and gastrointestinal toxicities(35). Also, given the time dependence of temozolomide-inducedPARP activation observed in vitro, future studies would berequired to delineate the optimal timing window for combinedadministration of these agents. Analogously, we can envisionstrategies that combine systemic administration of one agentwith local administration of the partner drug (or prodrug withCNS penetration before activation), to maximize intratumoralpharmacodynamic effect.

Furthermore, our findings may offer a new mechanistic per-spective on the accumulating evidence that IDH1-mutant gliomas

can display clinical responsiveness to alkylating chemotherapy.Our data raise the possibility thatmetabolic vulnerabilities duringthe acute DNA damage response could, in part, mediate thiseffectiveness. The discovery of an alternative mechanism of che-motherapy-induced cytotoxicity in IDH-mutant cancer cellswould also provide an explanation for the paradox of whyMGMTpromoter methylation is not a predictive biomarker of chemo-therapy response in IDH-mutant gliomas (36), which is in con-trast to the evidence base derived from IDH wild-type glioblas-tomas (9, 37).

Finally, and notably from a clinical standpoint, our findingssuggest that exploiting thismechanismof temozolomide-inducedmetabolic stress may allow for the recovery of an effective ther-apeutic index even in recurrent cancers with MMR-mediatedalkylator resistance. Indeed, Sobol and colleagues have previouslyhighlighted this ability to shift the spectrum of rate-limiting DNAdamage response by modulating PARP/NADþ consumption torestore cytotoxicity in MMR-deficient IDH wild-type glioma cells(38). On the basis of our findings here in IDH1-mutant tumors,we propose that upfront combination treatment regimens couldpotentially minimize or even avoid the development of thisescape pathway of MMR deficiency in recurrences, improvingthe durability of treatment response. Further studies of otherchemotherapeutic agents in clinical use, such as carmustine andlomustine, are needed to fully understand how they may exploitmetabolic vulnerability for IDH1-mutant cancers. Notably,these alkylating agents generate a spectrum of DNA adductsthat differ compared with temozolomide, especially with regardto N3-methyladenine and N7-methylguanine lesions (27), andtherefore, their effects in combination with NADþ-reducingagents may differ as well.

In conclusion, we demonstrate the rational conception of acombined therapy with the alkylating agent temozolomide andtargeted metabolic inhibition for IDH1-mutant tumors. Thesefindings offer an evidence base for treatment strategies with thepotential to minimize toxicity, allow for salvage therapy, andultimately provide a more effective treatment for patients withthese cancers.

Disclosure of Potential Conflicts of InterestT. Batchelor is a consultant/advisory board member for Amgen, Merck,

NXDC, Proximagen, and Roche. A.J. Iafrate has ownership interest (includingpatents) in Amgen and is a consultant/advisory board member for Roche. D.P.Cahill has received speakers bureau honoraria from Merck. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K. Tateishi, J.J. Miller, T.T. Batchelor, H. Wakimoto,A.S. Chi, D.P. CahillDevelopment of methodology: K. Tateishi, F. HiguchiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Tateishi, M.V.A. Koerner, N. Lelic, A.J. IafrateAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Tateishi, J.J. Miller, N. Lelic, G.M. Shankar,A.J. Iafrate, H. Wakimoto, D.P. CahillWriting, review, and/or revision of the manuscript: K. Tateishi, J.J. Miller,G.M. Shankar, S. Tanaka, D.E. Fisher, T.T. Batchelor, A.J. Iafrate, H. Wakimoto,A.S. Chi, D.P. CahillAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Tateishi, M.V.A. Koerner, S. Tanaka,T.T. Batchelor, D.P. CahillStudy supervision: H. Wakimoto, A.S. Chi, D.P. CahillOther (extending study to melanomas with IDH1 mutation): D.E. Fisher






AcknowledgmentsThe investigators thank Drs. Hang Lee and Alona Muzikansky for biostatis-

tical input and review. The investigators would additionally like to thank theircolleagues within the DFHCC for helpful discussions, and timely and carefulreview of the manuscript.

Grant SupportThis work was supported by NIH P50CA165962-01A1 to T.T. Batchelor

(principal investigator), D.P. Cahill, H. Wakimoto, and A.S. Chi, K24CA125440-06 to T.T. Batchelor, a Burroughs Wellcome Fund Career Award to

D.P. Cahill, Grant-Aid for Scientific Research (16K10765 to K. Tateishi),Yokohama Academic Foundation (K. Tateishi), The Yasuda Medical Founda-tion (K. Tateishi), and a Society of Nuclear Medicine and Molecular ImagingWagner-Torizuka Fellowship (K. Tateishi).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 21, 2016; revised January 31, 2017; accepted June 6, 2017;published OnlineFirst June 16, 2017.

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2017;77:4102-4115. Published OnlineFirst June 16, 2017.Cancer Res Kensuke Tateishi, Fumi Higuchi, Julie J. Miller, et al. Mediated Cytotoxicity

− Depletion+-Mutant Cancers and Potentiates NADIDH1Stress in The Alkylating Chemotherapeutic Temozolomide Induces Metabolic

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