Synergy Between the NAMPT Inhibitor GMX1777(8) and Pemetrexed in Non–Small Cell Lung Cancer Cells Is Mediated by PARP Activation and Enhanced NAD Consumption

Published OnlineFirst August 21, 2014; DOI: 10.1158/0008-5472.CAN-14-0809

Cancer Priority Report Research

Synergy between the NAMPT Inhibitor GMX1777(8) and Pemetrexed in Non–Small Cell Lung Cancer Cells Is Mediated by PARP Activation and Enhanced NAD Consumption

Manuel Chan1, Michel Gravel1, Alexandre Bramoulle1,Gaelle€ Bridon2,3, Daina Avizonis2,3, Gordon C. Shore1, and Anne Roulston1

Abstract GMX1778 and its prodrug GMX1777 represent a new class of cancer drugs that targets nicotinamide phosphoribosyltransferase (NAMPT) as a new strategy to interfere with biosynthesis of the key enzymatic cofactor NAD, which is critical for a number of cell functions, including DNA repair. Using a genome-wide synthetic lethal siRNA screen, we identified the folate pathway–related genes, deoxyuridine triphosphatase and dihydrofolate reductase, the silencing of which sensitized non–small cell lung carcinoma (NSCLC) cells to the cytotoxic effects of GMX. Pemetrexed is an inhibitor of dihydrofolate reductase currently used to treat patients with nonsquamous NSCLC. We found that combining pemetrexed with GMX1777 produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model of lung cancer. Pemetrexed is known to activate PARPs, thereby accelerating NAD consumption. Genetic or pharma- cologic blockade of PARP activity inhibited this effect, impairing cell death by pemetrexed either alone or in combination with GMX1777. Conversely, inhibiting the base excision repair pathway accentuated NAD decline in response to GMX and the cytotoxicity of both agents either alone or in combination. These findings provide a mechanistic rationale for combining GMX1777 with pemetrexed as an effective new therapeutic strategy to treat nonsquamous NSCLC. Cancer Res; 74(21); 5948–54. 2014 AACR.

Introduction cell growth inhibition is in the low nanomolar range (4). Interest in nicotinamide phosphoribosyltransferase (NAMPT) GMX1777 is a soluble prodrug of GMX used for in vivo studies as a potential anticancer target stems from its involvement (5, 6). NAMPT inhibitors, including GMX1778 (GMX) and þ as the major and rate-limiting step in the biosynthesis of NAD FK866, have shown preclinical activity alone and in combina- (1, 2). Tumor cells have elevated NAMPT expression and are tion with several therapeutic DNA-damaging agents, but the þ – dependent on NAD and ATP to support the increased met- mechanism of synergy has not been clearly elucidated (7 10). abolic demands of rapid cell proliferation. As a cellular Here, we report a synthetic lethal screen that revealed response to DNA damage, PARP is activated and consumes dihydrofolate reductase (DHFR) and deoxyuridine triphospha- þ its substrate NAD for the poly-(ADP-ribosylation) (PAR) of tase (DUT) as genes that modulate cell sensitivity to GMX in þ – proteins (3). In this way, it contributes to increased NAD A549 non small cell lung carcinoma (NSCLC) cells. Peme- demands to support the ongoing DNA repair mechanisms in trexed (PTX) is a multitargeted antifolate that inhibits DHFR, cancer cells. GMX1778 (formerly CHS-828) is a competitive thymidylate synthase, and glycinamide ribonucleotide trans- inhibitor of NAMPT whose potency for NAMPT in vitro and ferase, and is currently approved for treatment of nonsquamous NSCLC (11). We demonstrate that GMX synergizes with PTX in vitro and in the A549 lung adenocarcinoma xenograft model. The combination is most efﬁcacious when PTX pre- 1Laboratory for Therapeutic Development, McGill University, Montreal, Quebec, Canada. 2Metabolomics Core Facility, Rosalind and Morris Good- cedes GMX. The mechanism of synergy involves a PARP-1– þ man Cancer Research Centre, McGill University, Montreal, Quebec, mediated accelerated and sustained NAD decline that results 3 Canada. Department of Biochemistry, McGill University, Montreal, in enhanced cytotoxicity. Quebec Canada. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Materials and Methods M. Chan and M. Gravel contributed equally to this article. Cell lines and compounds A549, HeLa, Panc-1, NCI-H2030, and H1299 cells were Corresponding Author: Anne Roulston, Laboratory for Therapeutic Devel- – opment, McGill University, Room 900A, 3655 Promenade Sir William Osler, obtained from ATCC (2004 2009) and maintained in RPMI- Montreal, QC, Canada H3G 1Y6. Phone: 514-398-5226; Fax: 514-398- 1640 (Hyclone) with recommended supplements. The cell lines 7384; E-mail: [email protected] were not authenticated since ﬁrst acquisition. GMX was a gift doi: 10.1158/0008-5472.CAN-14-0809 from Gemin X Pharmaceuticals Inc.; GMX1777 was synthe- 2014 American Association for Cancer Research. sized by NuChem Therapeutics. PTX was from Selleckchem

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(in vitro experiments) and LC Biosciences (in vivo experiments) nitrile, then mixed in a bead beater for 2 minutes. Lipids were and olaparib (AZD2281) from Selleckchem. Methoxyamine partitioned from extracts with chloroform and following cen- (MX), nicotinamide mononucleotide (NMN), and folic acid trifugation (5 minutes, 3,000 g), the aqueous layer was were from Sigma-Aldrich. removed and dried by vacuum centrifuge. Samples were resuspended in 15 mmol/L ammonium formate in 50% ace- Genome-wide synthetic lethal screen tonitrile and injected for LC-MS/MS analysis (6430 Triple A549 cells were reverse transfected with 20 nmol/L of human Quadrupole; Agilent Technologies). See Supplementary Meth- þ þ On-Target Plus Smart pool genome-wide library (Dharmacon) ods for detailed LC-MS/MS parameters. NAD /NADP levels using RNAiMax (Invitrogen) according to the manufacturer's were normalized to total protein in each sample quantified by instructions in replicates of 12 (4 wells 3 plates). Controls the BCA protein assay (Pierce). included siNT (D-001810-10-20), siPLK-1 (L-003290-00), and siGAPDH (L-004253-00) as nontargeting, transfection, and Results and Discussion sensitizing siRNA pools respectively. Forty-eight hours after To identify modulators of NAMPT inhibitor sensitivity, a transfection, cells were treated with vehicle or a dose range of genome-wide functional genomic screen was performed in the GMX in duplicate. Three days after treatment, cell viability was A549 NSCLC cell line. Drug sensitivity was assayed with a dose measured using the CellTiter-Glo Luminescent Cell Viability range of GMX in duplicate for each siRNA pool (4 siRNAs/gene/ Assay (Promega) according to the manufacturer's instructions. well). The IC50 of GMX for each siRNA pool was compared with See Supplementary Methods for data analysis. the mean IC50 of nontargeting (NT) controls on each plate (Fig. 1A). The siRNA pool targeting DHFR was the top sensitizer Transfections, antibodies, and Western blot analyses (after the drug's own target NAMPT) with an 18-fold decrease siRNAs (20 nmol/L) targeting DHFR (pool, L-008799; Dhar- in the GMX IC50 value. Protein knockdown and sensitization macon; individual SI00299992; Qiagen), DUT (pool, L-010258; were confirmed in a secondary assay with two different sources Dharmacon), and PARP-1 (SI02662996; Qiagen) or NAMPT of siRNAs targeting DHFR (Fig. 1B). Specific knockdown of (L-004581; Dharmacon) were transfected into A549 cells using DUT, another folate pathway enzyme, also sensitized A549 to RNAiMax (Invitrogen) as above. Protein was extracted from GMX (Supplementary Fig. S1). cells 48 hours after transfection and detected by Western DHFR is inhibited by the non–small cell lung chemothera- blotting using antibodies to DHFR (EPR5285; Abcam), DUT peutic PTX. As the DHFR screen results would suggest, the (AB137097; Abcam), PARP-1 (9542; Cell Signaling Technology), combination of PTX with GMX treatment is strongly syner- a-tubulin (DM1A; Sigma), g-H2AX (05-636; Millipore), or PAR gistic in A549 and H1299 NSCLC adenocarcinoma cell lines proteins (MAB3192; Millipore). (Fig. 2A and B). Synergy from the sequential drug combination was also seen in additional cell lines (Supplementary Table S1). Drug combination effects and viability assays The synergy is more pronounced in both lines if PTX treatment Cells were treated for 72 hours for simultaneous treatments precedes GMX treatment by 48 hours. The reverse experiment, or 48 hours followed by 72 hours for sequential treatments. knocking down NAMPT, also sensitizes to PTX treatment Serial dilutions of GMX and/or PTX were added to microtiter (Supplementary Fig. S2). In an A549 xenograft model, the plates (final DMSO 0.2%). Cell viability was determined using administration of PTX (daily 5) preceded 48 hours by CellTiter-Glo. The combination index was determined using GMX1777 treatment (daily 5) results in a synergistic anti- CompuSyn v1.0 (Biosoft) according to the method of Chou and tumor effect without significant toxicity (10% weight loss) for Talalay (12). any single agent or combination treatment group (Fig. 2C). This demonstrates the potential clinical impact of this novel Murine xenografts drug combination. Female SCID hairless outbred (Crl:SHO-Prkdcscid Hrhr) Antifolates such as PTX as well as nonfunctional DUT leads to mice (6–8–week-old, Charles River Laboratories) were injected an imbalance in dUTP/dTTP nucleotide pools and dUTP mis- subcutaneously with 107 A549 cells in a 1:1 PBS/HC Matrigel incorporation followed by activation of base excision repair (BD) suspension . At a mean tumor volume of 100 mm3, animals (BER), which leads to single strand DNA damage, that if unre- were administered PTX i.p. daily for 5 days (600 mg/kg/day in paired, results in double-strand DNA breaks (DSB; refs. 13, 14). 0.9% saline) or GMX1777 i.m. daily for 5 days (150 mg/kg/day in Because PARP activation is involved in the BER and DSB repair 5% dextrose), appropriate vehicle, or a combination of these. pathways (15), we sought to examine the activation of PARP in Differences in tumor volumes between treatment groups were response to PTX. Figure 3A demonstrates that robust protein compared with a one-way analysis of variance using Prism 5.0 PAR occurs 44 hours following PTX treatment that lasts until at (GraphPad). least 72 hours. This demonstrates that PARP is activated and is þ consuming NAD at 48 hours following a sublethal dose (150 þ þ þ NAD /NADH, NADP , ATP, and intact cell quantitation nmol/L) of PTX treatment. Of note, the total NAD decline þ NAD /NADH, ATP, and intact cells were quantified using following treatment with PTX is modest over a 72-hour treat- þ þ the NAD /NADH-Glo, CellTiter-Glo, and CellTiter-Fluor assay ment compared with the decline in NAD from either lethal (25 kits respectively (Promega) according to the manufacturer's nmol/L) or sublethal (6 nmol/L) doses of GMX (Fig. 3B). þ þ þ instructions. To quantify NAD /NADP by LC-MS/MS, cells Although NAD levels decline with similar kinetics with both were washed in cold ammonium formate, lysed in 50% aceto- lethal and sublethal doses of GMX, they can recover only at

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Figure 1. A functional genomic screen identiﬁes genes involved in sensitivity to GMX cytotoxic effects. A, A549 cells were transfected with a genome-wide siRNA library and treated 48 hours later with a dose range of GMX. Viability was measured after 72 hours of GMX treatment by CellTiter-Glo. The IC50 for siRNAs screened are expressed relative to the mean of four nontargeting control siRNAs screened on each plate in rank order. B, DHFR siRNA was validated by transfection with a pool of siRNAs, siDHFR pool (D, Dharmacon), or an individual siRNA, si-DHFR (Q, Qiagen), GMX treatment as indicated. Histograms, mean SD of duplicate samples. Protein knockdown was validated by Western blotting, with a-tubulin as loading control.

nonlethal doses. This suggests that cell fate is determined by a tors and rescue GMX-mediated cytotoxicity (4). NMN has þ reduction in NAD beyond a threshold concentration and/or been detected in plasma (17). Although NMN does not affect duration. In support of these hypotheses, GMX has a steep dose– PTX cytotoxicity, it partially rescues cell death caused by response viability curve in vitro, suggesting that a threshold effect the GMX alone or in combination with PTX (Supplementary (4) and sustained exposure to GMX is required for maximal Fig. S3A). Similarly, the addition of folate completely cytotoxic effect (5). We, therefore, reasoned that the combined abolishes the cytotoxic effects of PTX and the PTX þ GMX þ effects of increased NAD utilization through simultaneous combination (Supplementary Fig. S3B). Therefore, regula- þ PARP activation and inhibition of NAD biosynthesis would be tors of intracellular and extracellular NMN levels, as well as enhanced in PTX þ GMX–treated cells. Indeed, when the two folate supplementation, could affect the efficacy of NAMPT agents are combined in sequence, with PTX followed by GMX, inhibitor–containing therapies. þ þ the steady-state NAD and NADP decline is greater than To determine if the PTX/GMX synergy is mediated through þ additive (Fig. 3C) and the rate of NAD decline is enhanced PARP, cells were treated with PTX and GMX in the presence or (Fig. 3D). Similar effects have been observed for the combination absence of olaparib, a small-molecule PARP inhibitor (Fig. 4A of FK866 with MNNG, or temozolomide (7, 16). Overall, the and B; ref. 18) or following siRNA knockdown of PARP-1 þ combination results in a more sustained NAD inhibition, and (Supplementary Fig. S4A and S4B). Treatment with olaparib þ notably, a more rapid impact on ATP decline and cytotoxic effect. was sufficient to inhibit PTX-induced PAR and NAD deple- þ Nicotinamide mononucleotide (NMN), the product of the tion. It was also sufficient to partially restore NAD levels in the NAMPT enzyme, can bypass the activity of NAMPT inhibi- PTX/GMX-treated cells above the threshold level such that

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Figure 2. The combination of GMX and PTX is synergistic in vitro and in tumor xenografts. A, A549 and H1299 cells were treated simultaneously with PTX alone (red) or in combination with various doses of GMX (blue, green). B, A549 and H1299 cells were pretreated with PTX for 48 hours followed by addition of DMSO (red) or GMX (blue, green) for an additional 72 hours. Cell viability was determined 72 hours after GMX by CellTiter-Glo. IC50 values were determined in Prism 5.0 and combination index (CI) values determined using CompuSyn v1.0. C, SCID hairless outbred mice harboring A549 xenografts were administered vehicles, PTX (5 daily doses) and/or GMX1777 (5 daily doses) starting 2 days following PTX dosing initiation. Error bars, mean SEM; n ¼ 11 for all groups. #, animal sacriﬁced because of tumor ulceration or malaise. Tumor volumes (left) and body weight (right) were measured three times per week. Statistical differences in tumor volumes between the PTX þ GMX treatment group and all other groups were determined by one-way analysis of variance (P < 0.05) Prism 5.0. viability was also restored from 22% to 68%. SiRNA knockdown Figure 4C and D demonstrate that the BER inhibitor þ of PARP-1 completely inhibited PTX-mediated PAR and par- methoxyamine enhances the NAD /NADH-depleting effects tially restored viability to the PTX/GMX-treated cells (Supple- of GMX and the GMX/PTX combination. Methoxyamine mentary Fig. S4A and S4B), demonstrating that the synergy is consequently enhances the cytotoxicity of PTX and GMX mediated in part through PARP-1. Of note, by eliminating the alone, as well as the combination of the two (Fig. 4D). þ PARP substrate, NAD , GMX functionally mimics a PARP Methoxyamine has been previously shown to enhance inhibitor and prevents PTX-induced PAR (Fig. 4A and Supple- PTX-induced accumulation of DSBs and cytotoxicity (19). mentary Fig. S4B); no PAR occurs following GMX treatment This suggests that a BER-inhibiting agent such as methox- alone, as expected (Fig 4B and D). GMX does not prevent the yamine may enhance the cytotoxic effects of the GMX-PTX þ induction of DSB by PTX as measured by g-H2AX levels (Fig. combination, both by increasing NAD depletion and DNA 4B), and, thus, does not interfere with the DNA-damaging damage and may be a useful addition to this combination þ aspects of PTX cytotoxicity or the cell's demand for NAD . strategy.

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þ þ Figure 3. The combination of PTX and GMX causes PARP activation and enhanced NAD depletion beyond the NAD threshold for cell survival in A549 cells. A, cells were treated with 150 nmol/L PTX for the indicated times and the levels of PAR proteins measured by Western blotting. a-Tubulin was used as a þ loading control. B, cells were treated with the indicated concentrations of PTX or GMX. Levels of NAD /NADH, ATP, or intact viable cells relative to þ time-matched DMSO controls were measured using NAD /NADH-Glo, CellTiter-Glo, and CellTiter-Fluor assays, respectively (mean SD of duplicates). C, þ cells were treated with DMSO or 150 nmol/L PTX for 24 hours followed by treatment with DMSO or 12 nmol/L GMX for 48 hours. The intracellular levels of NAD þ þ and NADP were measured from cell extracts and by LC-MS/MS. NAD levels are expressed relative to protein content (mean SD of quadruplicate þ samples). D, cells were pretreated with DMSO or 150 nmol/L PTX for 48 hours before the addition of GMX at 4 or 8 nmol/L. NAD /NADH, ATP, and intact cells were measured as in B. t1/2 (h) for each treatment group was determined in Prism 5.0.

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Figure 4. Role of PARP and BER in the synergy between PTX and GMX in A549 cells. A, cells were pretreated 1 mmol/L olaparib (2 hours) then sequentially þ 150 nmol/L PTX (24 hours), then GMX 12 nmol/L (48 hours). Cells were harvested for NAD quantiﬁcation by LC-MS/MS (left; mean SD of quadruplicates) or viability by CellTiter-Glo (right; mean SD of duplicates) B, PAR modiﬁcation of proteins and g-H2AX levels were measured in extracts þ treated as in A by Western blotting. C, cells were pretreated with 5 mmol/L methoxyamine (MX) 80 nmol/L PTX (24 hours) then 9 nmol/L GMX, and NAD / NADH was assessed after 48 hours using the NAD/NADH-Glo Assay. Results, mean SD of triplicate samples. D, cells were pretreated with 5 mmol/L methoxyamine 80 nmol/L PTX (48 hours) then 9 nmol/L GMX, and cell viability was measured after 72 hours using the CellTiter-Glo Assay. Results, mean SD of triplicate samples.

This is the ﬁrst demonstration that NAMPT inhibitors involves PTX-mediated activation of PARP-1, resulting in an þ synergize with the multitargeted antifolate PTX in vitro and accelerated and sustained NAD and ATP decline followed by in a xenograft model of NSCLC. The mechanism of synergy cell death. PARP-1 is thought to play a role in BER both as a

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sensor and in the repair mechanism. Mortusewicz and collea- Authors' Contributions gues demonstrated that initial PARP recruitment and activa- Conception and design: M. Chan, G.C. Shore, A. Roulston fi Development of methodology: M. Chan, G. Bridon, D. Avizonis, A. Roulston tion generates PAR-modi ed binding sites on PARP-1 and on Acquisition of data (provided animals, acquired and managed patients, histones near the site of DNA damage, recruiting and activating provided facilities, etc.): M. Chan, M. Gravel, A. Bramoulle, G. Bridon, D. Avizonis additional PARP-1 for a second and enhanced wave of PARP-1 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, activation following irradiation DNA damage (20). This sug- computational analysis): M. Chan, M. Gravel, A. Bramoulle, G. Bridon, gests why pretreatment with PTX may result in a more A. Roulston Writing, review, and/or revision of the manuscript: M. Chan, M. Gravel, profound combination effect than when the two agents are G. Bridon, G.C. Shore, A. Roulston added simultaneously or if GMX is added first. This work Administrative, technical, or material support (i.e., reporting or orga- demonstrates the potential for the combination of NAMPT nizing data, constructing databases): M. Gravel Study supervision: A. Roulston inhibitors with various PARP-activating therapeutic agents (in addition to PTX) in clinical settings and that the schedule of administration may influence the treatment efficacy. Grant Support Disclosure of Potential Conflicts of Interest This work was supported by grants from Genome Quebec and CIHR G.C. Shore is a Chief ScientificOfficer in, reports receiving a commercial (MOP-6192) to G.C. Shore. research grant from, and has ownership interest (including patents) in Therillia Development Company. No potential conflicts of interest were disclosed by the Received March 22, 2014; revised July 23, 2014; accepted August 5, 2014; other authors. published OnlineFirst August 21, 2014.

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5954 Cancer Res; 74(21) November 1, 2014 Cancer Research

Synergy between the NAMPT Inhibitor GMX1777(8) and Pemetrexed in Non −Small Cell Lung Cancer Cells Is Mediated by PARP Activation and Enhanced NAD Consumption

Manuel Chan, Michel Gravel, Alexandre Bramoullé, et al.

Cancer Res 2014;74:5948-5954. Published OnlineFirst August 21, 2014.

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