Metabolomics Identifies Pyrimidine Starvation As the Mechanism of 5-Aminoimidazole-4-Carboxamide-1- Β-Riboside-Induced Apoptosis in Multiple Myeloma Cells

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Published OnlineFirst April 12, 2013; DOI: 10.1158/1535-7163.MCT-12-1042

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Metabolomics Identiﬁes Pyrimidine Starvation as the Mechanism of 5-Aminoimidazole-4-Carboxamide-1- b-Riboside-Induced Apoptosis in Multiple Myeloma Cells

Carolyne Bardeleben1, Sanjai Sharma1, Joseph R. Reeve3, Sara Bassilian3, Patrick Frost1, Bao Hoang1, Yijiang Shi1, and Alan Lichtenstein1,2

Abstract To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-b-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl- a-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identiﬁed by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce multiple myeloma cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in multiple myeloma cells. Mol Cancer Ther; 12(7); 1310–21. 2013 AACR.

Introduction tumor cytoreductive effects of AICAr can be mediated AICAr (5-aminoimidazole-4-carboxamide-1-b-riboside) through AMPK activation and mTOR inhibition (2–4). is a nucleoside that is taken up by cells by adenosine However, cytotoxic affects do not always correlate with transporters and upon phosphorylation by adenosine AMPK and mTOR activity. For example, AICAr- induced kinase becomes AICA ribotide (AICAR or ZMP). It can apoptosis of EGFRvIII-expressing glioblastomas does function as an AMP mimic and activate AMP-activated not correlate with the degree of inhibition of mTORC1 kinase (AMPK; ref. 1). Because TORC1 activity can be signaling. Instead, it is due to AMPK-mediated down- inhibited by activated AMPK, AICAr has been studied as regulation of lipogenesis (5). In addition, AICAr can an antitumor agent. Several studies have shown that the induce apoptosis in some models by AMPK-independent mechanisms (6–9). mTOR is a potential target in multiple myeloma. Rapa- logs, which primarily inhibit TORC1, induce G arrest in Authors' Afﬁliations: 1The Division of Hematology-Oncology, Greater Los 1 Angeles VA Healthcare Center; and 2Jonsson Comprehensive Cancer multiple myeloma cells, but not apoptosis in vitro (10). Center and 3CURE: Digestive Diseases Research Center, University of AICAr has been reported to inhibit growth in myeloma California Los Angeles (UCLA) Division of Digestive Disease, UCLA, Los Angeles, California cells through activation of AMPK (11). However, a sub- stantial amount of apoptosis was only shown in the 8226 Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). cell line. AMPK activation in 8226 cells was not shown in this report and, thus, the mechanism of AICAr-induced Corresponding Author: Alan Lichtenstein, Division of Hematology-Oncol- ogy, Greater Los Angeles VA Healthcare Center, 111H, VA West LA Med apoptosis in multiple myeloma remains unclear. In addi- Ctr., 11301 Wilshire BLVD, Los Angeles, CA 90073. Phone: 310-268-3622; tion,asrapalogsonlyinduceG1 arrest in multiple myeloma Fax: 310-268-4508; E-mail: [email protected] cells, the ability of AICAr to induce apoptosis is unlikely to doi: 10.1158/1535-7163.MCT-12-1042 be explained simply by mTORC1 inhibition. Our prelimi- 2013 American Association for Cancer Research. nary experiments conﬁrmed that AICAr induces apoptosis

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in multiple myeloma cell lines but, as previously reported Immunoblots (12), rapamycin only induced G1 arrest. As AICAr poten- Whole-cell lysates were prepared using cell lysis tially induces several metabolic alterations that adversely buffer (Cell Signaling) supplemented with 1 mmol/L affect cells, we then conducted a metabolomics screen in phenylmethylsulfonylfluoride immediately before use. an attempt to pinpoint the mechanism of AICAr-induced Western blots were conducted as previously described apoptosis. We found that apoptosis was due to the inhib- (13). ited activity of uridine monophosphate (UMP) synthetase (UMPS) with subsequent pyrimidine starvation. Apoptosis assay Apoptosis was assayed by staining for activated cas- Materials and Methods pase-3 (BD Biosciences) and assessed using flow cytome- Reagents try as previously described (13). AICA riboside was purchased from Calbiochem. Met- formin, nucleosides, dithiothreitol (DTT), 5-phosphoribo- Cell-cycle analysis syl-a-pyrophosphate (PRPP), orotate, orotidine mono- Cells were incubated in 50 mg/mL propidium iodide in phosphate (OMP), and ZMP were purchased from Sig- 0.1% sodium citrate solution for 5 minutes before running ma-Aldrich. N-(phosphonacetyl)-L-aspartate (PALA) was on the flow cytometer (Accrui C6). Cell-cycle profiles were obtained from the Developmental Therapeutics Program analyzed using ModFitLT 3.2. (NCI/NIH, Bethesda, MD). All antibodies were purchased from Cell Signaling except for anti-UMP synthetase, pur- UMPS functional assay chased from Abcam. Recombinant UMP synthetase was The UMPS assay was as described (14). The reaction purchased from Origene and recombinant adenosine mixture contained 20 mmol/L Tris-HCl, pH 7.5, 2 phosphoribosyltransferase (APRTase) was purchased mmol/L DTT, 5 mmol/L MgCl ,300mmol/L PRPP, 14 14 2 from Prospec. [6- C] orotate (50 mCi/mmol) and [8- C] 5 mmol/L [6-14C] orotate (50 mCi/mmol), and 0.4–0.8 adenine (50 mCi/mmol) was purchased from MP Biome- mg recombinant UMPS. Reactions were incubated at dicals. Chemical structures for bortezomib, melphalan, 37C for 30 minutes. Products were separated using and metformin are shown in Supplementary Fig. S1. thinlayerchromatography(TLC;ref.15).Thereaction was stopped by directly spotting 5 mLofthereaction Cell lines mixture onto a 20 cm 20 cm PEI-cellulose plate and All cell lines were obtained from American Type Cul- drying the sample with hot air. Plates were developed ture Collection (ATCC). 8226, OPM2, U266, and MM1S by ascending chromatography in 0.25 mol/L LiCl2 cells were maintained in RPMI supplemented with 10% /0.1% formic acid. Cold standards of orotate, OMP, FBS, glutamine, nonessential amino acids, penicillin- and UMP were used to identify spots. Plates were streptomycin, and fungazone. H929 cells were main- visualized using a PhosphoImager (GE Storm 840) tained in RPMI media with the same supplements and spots were quantified using ImageQuant software. described above, except the media was supplemented Orotate phosphoribosyl transferase and OMP decar- with 0.05 mmol/L B-mercaptoethanol. HeLA cells were boxylase activity was calculated following Traut and maintained in Dulbecco’s Modified Eagle Medium with Jones (14). the same supplements as 8226 cells. Cell lines were ver- ified with short tandem repeat analysis by ATCC. Nucleotide extraction and high-performance liquid chromatography analysis Screening for metabolites Cells were harvested, washed once in PBS, resus- Treated cells were harvested, washed once in PBS, pended in 5% tricarboxylic acid, sonicated for 10 sec- frozen in liquid nitrogen, and sent to Metabolon Inc. onds, and incubated on ice for 5 minutes. Precipitated Samples were prepared in quadruple. At the time of protein was removed by centrifugation (5,000 g,10 analysis, samples were extracted and prepared for anal- minutes) and the supernatant was neutralized by 5 ysis using Metabolon’s standard solvent extraction meth- extractions with 5 volumes of water-saturated ether. od. The extracted samples were split into equal parts for pH was adjusted to 5. Nucleotides were separated on analysis on the gas chromatography-mass spectrometry a Phenomenex Partisil 10 SAX column (0.45 25 cm) and liquid chromatography/tandem mass spectrometry equilibrated in 5 mmol/L K phosphate (pH 5) at a flow platforms. Following log transformation and imputation rate of 2 mL/min. Samples were eluted isocratically for with minimum observed values for each compound, 20 minutes with 5 mmol/L K phosphate buffer, fol- Welch 2-sample t tests were used to identify biochemicals lowed by a linear gradient to 500 mmol/L K phosphate that differed significantly between treated and control for 45 minutes, followed by 500 mmol/L K phosphate groups. In all, 194 biochemicals were identified. When for an additional 15 minutes. Separation was on a Gilson analyzing 194 compounds, it is expected that 10 com- high-performance liquid chromatography (HPLC) sys- pounds meeting the cutoff for statistical significance (P tem with a Model 117 ultraviolet detector set at 260 and ¼ 0.05) would occur by random chance. 280 nm. Areas of peaks were quantified using UniPoint

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O A N

NH2 O N HO Figure 1. A, chemical structure of NH2 AICAr. B, multiple myeloma lines HO OH were treated with increasing AICAr concentrations of AICAr (0, 100, 250, 500 or 1,000 mmol/L) for 48 hours and assessed for apoptosis BC70 50 24 h by ﬂow cytometry. Black bars, 45 60 48 h 40 control. C, cells were treated with 50 72 h 35 96 h 250 mmol/L AICAr (8226, U266) or 30 40 m 25 500 mol/L (OPM2, MM1S, H929) 30 20 for the indicated times and 15 20 10 apoptosis assessed as in A. Data 10 5 are mean SD apoptosis above % Caspase-3 positive % Caspase-3 positive 0 0 control, n ¼ 3. D, cell-cycle 8226 OPM2 MM1S U226 H929 8226 OPM2 U266 MM1S H929 analysis of cells AICAr 24 hours before apoptosis is induced. D 70 Apoptosis was <11% above control in all samples. The 60 G1 S percentage of cells in G1,S,andG2 G 50 2 phase are indicated. Data are n ¼ 40 mean SD, 2 for 8226, OPM2, and H929; n ¼ 3 for U266 and 30 % Cells MM1S. E, cells were treated with

20 rapamycin (0.1 nmol/L for OPM2, 10 nmol/L all other cell lines) or 10 vehicle and analyzed for cell-cycle

0 distribution at 24 to 72 hours or 8226 8226 OPM2 OPM2 U266 U266 MM1S MM1S H929 H929 apoptosis between 48 to 96 hours. AIC AIC AIC AIC AIC Data are mean SD, n ¼ 2. F, 8226 E 100 F cells were pretreated with 100 G1 90 S nmol/L iodotubercidin or vehicle 80 Apoptosis 50 for 30 minutes followed by addition 70 45 m AICAr of 250 mol/L AICAr or vehicle, 60 40 35 Iodo incubated for 48 hours, and 50 30 AIC + Iodo assessed for apoptosis. Data are

% Cells 40 25 Vehicle n ¼ 30 20 mean SD, 2. 15 20 10 10 5 0 % Caspase-3 positive 0 8226 8226 OPM2 OPM2 U266 U266 MM1S MM1S H929 H929 Treatment Rap Rap Rap Rap Rap

software and converted to molar quantities by compar- APRTase. Reactions were incubated for 1 hour at 37C. TLC ison to ZMP standards. was conducted as described above to analyze products of the reaction. 5-Phosphoribosyl-a-pyrophosphate assay Measurement of intracellular 5-phosphoribosyl-a-pyro- Colorimetric orotate assay phosphate (PRPP) was as described (16). Brieﬂy, cells were Orotate accumulation was measured using a colorimet- treated with 250 mmol/L AICAr for 8 hours, harvested, ric assay as described (17) with modiﬁcations described by washed oncewith PBS, and the pellet frozenat 80 C. Cells Harris and Oberholzer (18). were thawed and resuspended in 50 100 mL 30 mmol/L Tris-HCl, pH 7.5, 0.5 mmol/L EDTA followed by heating in a boiling water bath for 45 seconds and centrifugation at Statistical (ANOVA) analysis 10,000 rpm for 10 minutes at 4C. The supernatant was Proc Mixed (SAS 9.2) was used to construct a repeated removed and used immediately. The amount of PRPP was measures mixed effects model predicting the main out- determined on the basis of the synthesis of [8-14C]AMP come-percent apoptosis. Fixed effects entered in the mod- from [8-14C] adenine in the presence of APRTase. The el were cell line type, treatment, and the interaction of standard reaction mixture contained 30 mmol/L Tris-HCl, treatment by cell line type. Compound symmetry covari- m pH7.5, 6 mmol/L MgCl2,2mmol/LDTT,50 mol/L ance structure was used. Marginal means of cell lines and [8-14C] adenine (50 mCi/mmol), and 0.1 mg recombinant treatments were estimated using LSmeans statement and

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AB 4Hours: 8 48 8226 P-AMPK

AMPK

Actin + + + + – – – – AIC (250 µmol/L) 1 2 4 8 1 2 4 8 Time (h) Lipid

MM1S Amino Acid P-AMPK

AMPK

Actin Peptide + + + + – – – – AIC (500 µmol/L) 8 16 24 48 8 16 24 48 Time (h) Nucleotide

Cofactors Carbohydrate & vitamins

Energy Xenobiotics CD CLL 8226 MM1S 8226 P-AMPK P-p70 (Thr 389) Actin Actin Con Rap AIC AIC Con Met Con Met Con MM1S P-p70 (Thr 389)

Actin

P-p70 (Ser/Thr 421/424)

Actin Con Rap AIC

Figure 2. A, simplified heatmap of 8226 cells treated with 250 mmol/L AICAr (AIC) for 4 and 8 hours compared with vehicle. Red indicates an increase and green a decrease in a given metabolite over vehicle. Darker colors indicate metabolites that achieved statistically significant (P < 0.05) differences between treated and untreated group and light colors indicate metabolites that approach statistical significance (0.05 < P < 0.1). B, 8226 and MM1.S cells were treated with the indicated concentration of AICAr for the indicated amount of time and Western blot probed for AMPK phosphorylation (P-AMPK), total AMPK or actin. C, primary CLL cells were incubated for 16 hours 500 mmol/L AICAR for similar Western blot assay. 8226 and MM1S cells were incubated with 5 mmol/L metformin for 16 (8226) or 72 hours (MM1S) for similar Western blot assay. D, cells were treated either with DMSO (Con), 10 nmol/L rapamycin (R),or 500 mmol/L AICAr (A) for 8 (8226) or 24 hours (MM1S). Western blots were probed for P-p70 (Thr 389 or Ser/Thr 421/424) and actin to assess TORC1 activity. differences between individual pairs of cell lines was based on cell counts (data not shown). AICAr also determined using posthoc t tests where overall main effects induced apoptosis in a concentration-dependent fashion were significant. in all 5 multiple myeloma lines identified by caspase-3 activation (Fig. 1B). On the basis of these preliminary results, further time-course experiments were carried out Results with either 250 mmol/L AICAr (for 8226 and U266 cells) or AICAr induces apoptosis in myeloma cells in vitro 500 mmol/L (for OPM2, MM1S, and H929 cells). AICAr- AICAr (Fig. 1A) had cytoreductive effects on multiple induced apoptosis in a time-dependent fashion (Fig. 1C). myeloma lines (8226, OPM2, U266, MM1S and H929) Apoptosis was first detected consistently by 48 hours. The

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Table 1. AICAr-induced alteration in metabolite apoptosis, cells were treated simultaneously with AICAr and iodotubercidin, an inhibitor of adenosine kinase (Fig. levels in 8226 cells 1F). Treatment with iodotubercidin prevented apoptosis, indicating that AICAr must be converted to ZMP to Pathway AICAr (fold change) induce apoptosis. Purine Xanthine 1.31 Metabolomics screen Hypoxanthine 1.24 Because AICAr can induce metabolic alterations inde- Adenosine 1.90 pendent of mTOR inhibition (5) and because rapamycin AMP 2.20 treatment (i.e., mTORC1 inhibition) could not reproduce ADP 1.38 the apoptosis resulting from AICAr, we conducted a GMP 1.72 high-throughput screen for alterations in metabolites Pyrimidine selectively affected by AICAr. Preliminary time-course m Cytidine 0.3 experiments in 8226 cells indicated that 250 mol/L Orotate 26.12 AICAr induced apoptosis between 8 and 16 hours. We, Uracil 0.41 thus, profiled AICAr-treated 8226 cells at 4 and 8 hours of Uridine 0.34 exposure to lessen the possibility of finding alterations UMP 0.44 that were nonspecific effects of apoptosis. After 4 or 8 Nucleotide coenzymes hours, AICAr only altered the levels of 7 and 29 metabo- CoenzymeA 1.50 lites, respectively (Fig. 2A). The number of metabolites Flavin adenine dinucleotide (FAD) 1.16 affected by AICAr exposure at the 4-hour time point was Glycerolipid not above the threshold of random chance, indicating this CDP-choline 0.57 time point was too early to produce useful data (see Glycerol 3-phosphate 1.48 Materials and Methods for metabolomics data analysis). Choline phosphate 1.23 In concurrently run samples of 8226 cells exposed to 10 Krebs cycle nmol/L rapamycin for 4 or 8 hours, a larger metabolic Citrate 0.71 effect was seen with significant alterations in 33 and 51 cis-aconitate 0.66 metabolites respectively (rapamycin data in Supplemen- Glycolysis tary Fig. S3). As shown in the heatmap in Fig. 2A, AICAr Isobar: fructose 1,6-diphosphate, 2.5 treatment had little effect on carbohydrate, energy, amino glucose 1,6-diphosphate acid, and lipid metabolism with the exception of a few Lactate 0.77 metabolites shown in Table 1. Of particular interest was Cysteine, methionine, SAM, taurine metabolism the increase in phosphocholine and glycerol 3-phosphate C 0.71 and decrease in CDP-choline (Table 1), which suggested N-formylmethionine 1.35 an inhibition of the reaction catalyzed by CTP:phospho- S-adenosylhomocysteine 1.55 choline cytidyltransferase, the rate-limiting step in the Assymetric dimethylarginine 1.48 major pathway of phosphatidylcholine synthesis (see Discussion; ref. 20). In contrast, rapamycin treatment P < NOTE: Bold numbers, 0.05. (Supplementary Fig. S3) resulted in robust effects on carbohydrate, energy, amino acid, and lipid metabolism. Most of these alterations were consistent with prior liter- 8226 cell line was the most sensitive target with rapid ature such as decreased levels of glycolytic intermediates induction of apoptosis. Cell-cycle analysis showed that (21) and inhibited flux through the tricarboxylic acid cycle AICAr also induced an accumulation of cells in S-phase in (22). In contrast, the largest effect of AICAr was on nucle- all 5 cell lines (Fig. 1D) consistent with a previous report otide metabolism (Fig. 2A). Of the 29 metabolites signifi- (11). The increase in S-phase distribution ranged from 10% cantly altered by AICAr at 8 hours, 11 are within nucleotide to 25% in the cell lines. In contrast, rapamycin induced a metabolic pathways (Fig. 2A). The general trend was an G1–S arrest without apoptosis in 4 of 5 cell lines studied increase in purine metabolites and a decrease in pyrimi- (Fig. 1E). Only in OPM-2 cells was there a minimal degree dine metabolites (Fig. 2A and Table 1). The most striking of apoptosis following exposure to rapamycin. Although alteration induced by AICAr was in orotate, an interme- IGF-I is a known survival factor for multiple myeloma diate in de novo pyrimidine synthesis, increasing 26-fold cells (19), it did not confer protection against AICAr- over vehicle control (Table 1). induced apoptosis while protecting against dexametha- Although AICAr must be converted to ZMP for apo- sone (Supplementary Fig. S2) suggesting a distinct mech- ptosis (Fig. 1F) and ZMP is an AMP mimic, the finding that anism of AICAr apoptosis. many of the metabolic effects of rapamycin-induced AICAr becomes phosphorylated by adenosine kinase to mTORC1 inhibition were not detected in AICAr-treated become ZMP, which acts as a mimic of AMP. To deter- cells raised the question of whether AICAr-induced apo- mine whether AICAr must be converted to ZMP to induce ptosis was associated with AMP kinase activation and

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Figure 3. Key steps involved in de novo pyrimidine synthesis and purine synthesis. Biochemicals in boxes are metabolites identiﬁed in the screen, with white arrows indicating statistically signiﬁcant increases or decreases in AICAr-treated cells.

mTORC1 inhibition. Unexpectedly, we could not show AICAr for 8 hours increased orotate 4.5-fold (mean of 3 activation of AMPK in multiple myeloma cells. Focusing experiments). Orotate is a substrate of UMPS, the last on the most AICAr-sensitive 8226 cell line, experiments enzyme in the de novo pyrimidine biosynthetic pathway using a range of AICAr concentrations (100 mmol/L–2 (Fig. 3). UMPS is a multifunctional enzyme that catalyzes 2 mmol/L) and incubation times (30 minutes–24 hours) enzymatic reactions. The first reaction catalyzes the con- failed to detect AMPK activation as monitored by phos- version of orotate to OMP using orotate phosphoribosyl phorylation of Thr 172 (Fig. 2B and Supplementary Fig. transferase (OPRTase) that requires PRPP and releases S4). As positive controls, our AICAr preparation success- PPi (reaction "1" in Fig. 3). The second reaction is catalyzed fully induced AMPK activation in primary chronic lym- by OMP decarboxylase and converts OMP to UMP (reac- phocytic leukemia (CLL) cells as previously reported (2) tion "2" in Fig. 3). The marked increase in orotate levels and metformin, which indirectly activates AMPK by suggests that UMPS is inhibited by AICAr exposure. Con- inhibiting mitochondrial respiration and increasing the sistent with this data is the observed associated decrease in AMP:ATP ratio (23), was capable of activating AMPK in UMP levels (Table 1), the product of the reaction (Fig. 3). All multiple myeloma cells (Fig. 2C). Comparable attempts to pyrimidine nucleotides can be synthesized from UMP (24), show AMPK activation in AICAR-treated OPM-2 and suggesting that the decrease in UMP could result in pyrim- MM1.S cells were likewise unsuccessful (Fig. 2B and idine starvation. Supplementary Fig. S4) while metformin was successful Tumor cells can salvage uridine from their environ- (Fig. 2C). These data show that although AICAr must be ment to circumvent the blockage of de novo pyrimidine converted to ZMP, its mechanism of apoptosis-induction biosynthesis (25, 26). Thus, to determine whether pyrim- does not involve AMPK activation. idine starvation through the inhibition of UMPS induces To assay effects on mTORC1, we treated cells with apoptosis, multiple myeloma cells were treated with either rapamycin or AICAr and probed Western blots for increasing concentrations of uridine in conjunction with phosphorylation of p70S6 kinase. As shown in Fig. 2D and AICAr. The addition of uridine prevented apoptosis and Supplementary Fig. S4 (for OPM-2 cells), while rapamycin prevention was related to the concentration of uridine clearly inhibited phosphorylation of p70, apoptotic con- (Fig.4A).Uridineconferredcompleteprotectionasthere centrations of AICAr did not. These data collectively show is no statistical difference between control cells and that the ability of AICAr to induce multiple myeloma AICAr treated cells þ 100 mmol/Luridine.Asexpected, apoptosis and cell-cycle arrest is not mediated by AMPK neither of the purine nucleosides, guanosine or adeno- activation or mTORC1 inhibition. As our metabolomics sine, could prevent apoptosis (data not shown). The screen identified orotate accumulation as the most strik- rescue by uridine is not due to a nonspecific increase in ing abnormality, we further investigated its significance resistance to apoptosis as the nucleoside is incapable of as described below. preventing apoptosis induced by bortezomib, melphalan, or metformin (Fig. 4B). The finding that uridine prevents Exogenous pyrimidines prevent apoptosis apoptosis in multiple myeloma cells treated with AICAr The AICAr-induced accumulation of orotate identified but not with metformin exposure is especially notewor- in the screen was first confirmed by using a colorimetric thy, as metformin activates AMPK (Fig. 2C) and AMPK is assay (17). Incubation of 8226 cells with 250 mmol/L a known mTOR inhibitor (27). These data provide

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AB70 AIC AIC+ 5 µmol/L U 60 AIC+ 10 µmol/L U 80 Drug AIC+ 25 µmol/L U 70 50 Drug+U µ AIC+ 50 mol/L U 60 40 AIC+ 100 µmol/L U 50 Control 30 40 30 20

% Caspase-3 positive 20

10 % Caspase-3 positive 10 0 0 8226 OPM2 U266 MMIS H929 AICAr Bortezomib Melphalan Metformin Vehicle

AIC C D 60 AIC+ 5 µmol/L C AIC

AIC+ 10 µmol/L C AIC+U 80 AIC+ 25 µmol/L C 50 AIC+T AIC+dC 70 AIC+ 50 µmol/L C AIC+ 100 µmol/L C 40 Control 60 Control

50 30 40

30 20 % Caspase-3 positive 20 % Caspase-3 positive 10 10

0 0 8226 OPM2 U266 MMIS H929 8226 U266 OPM2 MMIS H929

E 8226 P-H2A.X

Actin 8 16 24 8 16 24 Time (h) + + + – – – AIC

MMIS

P-H2A.X

Actin +++ + + – AIC U C T dC nucleoside

P-H2A.X

Actin ++ + + – – – – AIC U C C U C C nucleoside (L) (H) (L) (H)

Figure 4. A, cells were incubated with AICAr (250 mmol/L for 8226 and U266, 500 mmol/L for OPM2, MM1S, and H929) or vehicle in the presence of increasing concentrations of uridine (U) for 48 hours (8226, OPM2) or 96 hours (U266, MM1S, H929) and apoptosis assessed. Data are mean SD, n ¼ 2, except for 8226,

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AB 100 250 µmol/L PALA O H 90 PALA + U N H H O 80 PALA + C O PALA + T P H 70 O PALA + dC O O O 60 Control 50 O H PALA 40 C 30 70 % Caspase-3 positive 20 PALA 60 10 PALA + 5 µmol/L C 0 50 Control 8226 OPM2 U266 MMIS H929 40 D 30 MMIS

20 P-H2A.X % Caspase-3 positive 10 Actin

0 +++ + + – PALA OPM2 MMIS U C T dC

Figure 5. A, chemical structure of PALA. B, cells were incubated with 250 mmol/L PALA or vehicle 100 mmol/L uridine, 100 mmol/L cytidine, 100 mmol/L thymidine, or 100 mmol/L deoxycytidine for 48 to 96 hours. Apoptosis was assessed as described in Fig. 1. Data are mean SD, n ¼ 2–4 replicates. C, OPM2 and MM1S cells were incubated with 250 mmol/L PALA or vehicle 5 mmol/L cytidine. Data are mean SD, n ¼ 2. D, MM1S cells were treated with 250 mmol/L PALA 100 mmol/L uridine, 5 mmol/L cytidine, 100 mmol/L thymidine, or 100 mmol/L deoxycytidine for 48 hours. Extracts were probed for P-H2A.X (Ser 139) or actin as described above. dC, deoxycytidine. additional evidence that AICAr-induced apoptosis is tions of cytidine suggests that at least some cytidine independent of effects on AMPK and mTOR. deaminase is present, allowing conversion of cytidine to Exogenous cytidine also prevents apoptosis although uridine and some protection against pyrimidine starva- the concentration-dependence of protection is more com- tion and apoptosis. plicated (Fig. 4C). Protection is present at low concentra- Exogenous thymidine also reduced apoptosis in all cells tions of cytidine (5–10 mmol/L) but concentrations of 50 lines (white bars, Fig. 4D), suggesting apoptosis was mmol/L and above actually enhance AICAr-induced apo- induced by limiting DNA synthesis rather than RNA ptosis in most of the cell lines (Fig. 4C). Enhanced apo- synthesis. Consistent with the hypothesis that AICAr ptosis with high concentrations of cytidine did not occur induces DNA replicative stress due to limiting deoxyri- in the absence of AICAr. The ability of high concentrations bonucleotide triphosphates (dNTP) is the increase in of cytidine to enhance apoptosis in the presence of inhib- phosphorylation of H2A.X, a marker for replicative stress ited de novo pyrimidine biosynthesis has previously been (Fig. 4E; ref. 31). AICAr-induced phosphorylation of H2A. reported and occurs when there is low cytidine deaminase X was prevented by uridine or thymidine add-back rescue activity (28–30). Cytidine deaminase converts cytidine as well as by a low (5 mmol/L) concentration of cytidine. to uridine and the presence of excessive cytidine cou- The lowest panel in Fig. 4E shows the difference between pled with inhibition of de novo pyrimidine synthesis adding back the apoptotic high concentration of cytidine enhances cell death because of unbalanced CTP/UTP [100 mmol/L (H)] versus low concentration [5 mmol/L (L)] ratios and aborted attempts at proliferation (30). It is with the former inducing an increased amount of H2A.X possible that some multiple myeloma cell lines have a phosphorylation. As activation of caspases can also result deﬁciency in cytidine deaminase. Nevertheless, the sig- in H2A.X phosphorylation, we repeated experiments niﬁcant prevention of apoptosis with lower concentra- described in Fig. 4E with the addition of the caspase n ¼ 3. B, 8226 was incubated with AICAr (250 mmol/L), bortezomib (5 nmol/L), melphalan (10 mmol/L), or metformin (5 mmol/L) 100 mmol/L uridine for 48 hours and apoptosis assessed. Data are mean SD, n ¼ 2. C, cells were incubated with AICAr as described above or vehicle in the presence of increasing concentrations of cytidine (C). Data are mean SD, n ¼ 2 (8226,U266, MM1S) or n ¼ 3 (OPM2, H929). D, cells were incubated with AICAr as described above or vehicle in the presence of 100 mmol/L uridine (U), 100 mmol/L thymidine (T), or 100 mmol/L deoxycytidine (dC). Mean apoptosis and error bars were determined from 3 to 5 replicates. E, 8226 cells were treated with 250 mmol/L AICAr or vehicle for the indicated time and Western blots probed for P-H2A.X (Ser 139) or actin. MM1S cells were treated with 500 mmol/L AICAr and 100 mmol/L of the indicated nucleoside for 72 hours, except in the lowest panel where C(L) is 5 mmol/L and C(H) is 100 mmol/L. Western blot analysis was conducted as described above.

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also similar, with PALA-induced apoptosis being pre- A vented by uridine (Fig. 5B) and low concentrations of UMPS cytidine (Fig. 5C), whereas high cytidine concentrations enhance apoptosis (Fig. 5B). As mentioned above, since Actin some H2A.X phosphorylation could be downstream of + + + – – – AIC apoptosis induction, we also assayed cell transit. Sup- 10 2 4 1 2 4 Time (h) plementary Figure S6 shows the PALA-induced S-phase accumulation was prevented by uridine add back. As UMPS with AICAr, deoxycytidine does not prevent apoptosis Actin (Fig. 5B). Thymidine does not seem to prevent apoptosis as + + + – – – AIC effectively in PALA-treated cells as previously shown in 8 16 24 8 16 24 Time (h) AICAr-treated cells (Fig. 5B). However, this seems to be B time dependent, with thymidine more effectively prevent- 1Lane:2 3 4 5 6 7 ing apoptosis at earlier timepoints (data not shown). As expected, PALA-induced H2A.X phosphorylation is Adenine prevented by uridine and low concentrations of cytidine (Fig. 5D). AMP

Mechanism of UMPS inhibition and pyrimidine Origin starvation Although unlikely, based on the long half-life of UMPS Cell extract A A C C – – – (32), one possibility for the inhibition of UMPS activity is PRPP (exo) – – – – – + – downregulated enzyme expression. However, a Western APRTase + – + – + + – blot analysis conducted with extracts from cells treated with AICAr for varying times showed no decrease in Figure 6. A, Western blot analysis for UMPS expression in 8226 cells protein level (Fig. 6A). A second possibility is that intra- 250 mmol/L AICAr for the indicated time. B, TLC assay for measuring cellularly generated ZMP competitively inhibits the OMP PRPP in cell extracts. A, AICAr treated; C, control. The position of decarboxylase activity (reaction ‘2’ of UMPS in Fig. 3). adenine, AMP migration, and the origin is indicated. Monophosphonucleosides are effective inhibitors of decarboxylase activity with XMP, UMP, and AMP being inhibitor, ZVAD. There was a reduction in H2A.X phos- the most effective (14). ZMP, the phosphorylated form of phorylation, indicating at least some of this effect was a AICAr, can be viewed as an analog of AMP or GMP (1). result of AICAr-induced apoptosis. However, cell-cycle Inhibition of the decarboxylase activity can appear as analysis of AICAr-treated cells (Supplementary Fig. S5) inhibition of the transferase activity (accumulation of showed that uridine rescue was associated with a pre- orotate) because accumulation of OMP will push the vention of S-phase arrest. equilibrium toward orotate (33). To determine whether Addition of deoxycytidine over a wide range of con- ZMP can inhibit UMPS activity, we conducted a func- centrations (5–100 mmol/L) was not able to prevent tional assay using a TLC assay. ZMP inhibited recombi- AICAr-induced apoptosis (Fig. 4D) or replicative stress nant UMPS activity at concentrations above 0.5 mmol/L, (Fig. 4E). The observation that thymidine can rescue cells with the decarboxylase activity inhibited to a greater while deoxycytidine is incapable suggests a defect in one extent than the transferase activity (Table 2). As ZMP branch of the deoxyribonucleoside salvage pathway. Mul- tiple myeloma cells may be deﬁcient in deoxycytidine kinase, which is required to salvage deoxycytidine into Table 2. Activity of the transferase and pyrimidine pools (see Discussion). decarboxylase activities of recombinant UMPS in the presence of varying concentrations of Effects of PALA ZMP To strengthen the conclusion that pyrimidine starvation induces apoptosis in myeloma cells, cells were Transferase Decarboxylase treated with another inhibitor of de novo pyrimidine activity activity synthesis, PALA (Fig. 5A). PALA inhibits aspartate transcarbamylase, the ﬁrst catalytic reaction in the de 0.1 mmol/L ZMP 1.03 (0.06) 0.79 (0.31) novo pyrimidine pathway, and induces apoptosis in 0.5 mmol/L ZMP 0.89 (0.22) 0.41 (0.15) myeloma cells (Fig. 5B and C). In similar fashion to 1.0 mmol/L ZMP 0.79 (0.20) 0.27 (0.09) AICAr, PALA-induced apoptosis is also associated with NOTE: Activity (mean of 4 experiments SD) is compared replicative stress, as shown by induction of H2A.X phos- with activity in the absence of ZMP. phorylation (Fig. 5D). The pattern of pyrimidine rescue is

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Pyrimidine Starvation in Multiple Myeloma Cells

can accumulate in cells to the millimolar range (1, 2, 4, 34), ptosis in that study (as well as in ours), were not assayed this suggested the possibility that AICAr converted to for AMPK activation. In contrast, our focus on apoptosis ZMP could inhibit UMPS inside multiple myeloma and the most sensitive target, 8226 cells, clearly showed cells. An HPLC analysis was, thus, conducted to measure absence of AMPK phosphorylation/activation although ZMP accumulation. Although our assay was able to significant apoptosis ensued. Quite possibly, the lack of detect significant accumulation of ZMP in HeLa cells ZMP accumulation in 8226 cells as indicated by our HPLC treated with thymidine and pemetrexed (as reported in assay, accounts for the inability of AICAr to activate ref. 34) to the level of 4.6 mmol/L, no ZMP accumulation AMPK. The significant rescue afforded by uridine in the was detected in 8226 cells treated with AICAr for 1 to 16 other 4 multiple myeloma cell lines also supports that hours. their apoptotic response was mediated by an identical Another possible reason for UMPS inhibition is PRPP mechanism to that of 8226 cells. In contrast, metformin substrate limitation, first proposed by Thomas and col- could activate AMPK in 8226 multiple myeloma cells by leagues (35). PRPP is generated at the first step of de novo an independent mechanism and this was associated with purine synthesis and is a substrate for UMPS (Fig. 3). The apoptosis, which was unaffected by attempted uridine increased levels of purine metabolites, (i.e., ADP), in rescue. This suggests that AMPK activation may also AICAr-treated cells could potentially inhibit PRPP syn- mediate multiple myeloma cell apoptosis although thetase and the generation of PRPP from D-ribose as AMPK-independent effects of metformin have been hypothesized in Fig. 3. We assessed levels of PRPP in reported (36, 37). Nevertheless, it is clear that AICAr- AICAr-treated cells (8-hour exposure) by the ability of cell induced apoptosis, which can be rescued by exogenous extracts to generate (8-14C) AMP from (8-14C) adenine in pyrimidines, can proceed without AMPK activation. the presence of APRTase using cell extracts as a source of We ruled out the possibility that AICAr inhibited UMPS PRPP. Figure 6B shows a representative experiment. by downregulating its expression or by an inhibitory When exogenous PRPP is supplied in the reaction mixture effect of ZMP. A more likely explanation for UMPS inhi- along with APRTase and radiolabeled adenine (lane 6), bition is decreased levels of PRPP substrate. PRPP, which radiolabeled AMP is generated. When cell extracts are is used as a substrate by UMPS in de novo pyrimidine used as a source of PRPP (lanes 1–4), a signal is generated synthesis, was significantly decreased in AICAr-treated at the same site in the TLC plates. In 3 independent cells. Therefore, limiting amounts of PRPP may account experiments, the amount of AMP product was on average for inhibition of UMPS. This is consistent with previous 3.3 times (1.3) higher in the control cells compared with studies in AICAr-treated Chinese hamster fibroblasts (35) AICAr-treated cells. Thus, AICAr decreases the level of or human B lymphoblasts (16). Adenosine, which our PRPP in multiple myeloma cells. This lower level of metabolomics screen identified as significantly higher in substrate could conceivably limit the activity of UMPS AICAr-treated cells, has been shown to induce pyrimidine resulting in pyrimidine starvation. starvation by a similar mechanism in some cells with an increase in orotate accumulation and a decrease in PRPP (38–41). Other purine metabolites may also depress PRPP Discussion levels. Thus, in addition to adenosine described above, Our data indicate that AICAr induces multiple myelo- the enhanced generation of ADP, which is increased by ma cell apoptosis by inhibition of UMPS activity and AICAr in the screen, could inhibit PRPP as previously pyrimidine starvation in an AMPK-independent manner. suggested (42). A metabolomics screen showed that orotate accumulated The fact that addition of thymidine can prevent most, after exposure to AICAr and that UMP levels were but not all of the AICAr-induced apoptosis suggests that decreased, indicating inhibition of UMPS. Apoptosis apoptosis is induced by replicative stress due to limiting induced by AICAr was prevented by addition of uridine. TTP. Consistent with this notion is the finding of AICAr- Furthermore, PALA, which also inhibits de novo pyrimi- induced phosphorylation of H2A.X, a marker of replica- dine synthesis by a different mechanism, also induced tive stress, which was attenuated by uridine, cytidine and apoptosis in multiple myeloma cells that was similarly thymidine rescue. However, the fact that thymidine does prevented by uridine. Finally, activation of AMPK was not fully protect multiple myeloma cells against apoptosis not detected in AICAr-treated cells while AICAr could compared with uridine suggests that limiting DNA rep- activate AMPK in CLL cells and metformin could activate lication may not fully explain all apoptosis. Pyrimidine AMPK in multiple myeloma cells. nucleotides are required in other metabolic pathways to Our results differ from an earlier report (11) on the generate intermediates such as cytidine diphosphate cho- effects of AICAr in multiple myeloma cells. In that study, line (CDP-choline). CDP-choline is critical for the de novo the authors concluded that the effects of AICAr were phosphatidylcholine synthesis pathway and this pathway mediated via AMPK activation. They showed that AMPK is known to be important in plasma cell development (43). became phosphorylated in U266 multiple myeloma cells As CDP-choline was found to be lower in AICAr-treated following AICAr exposure. However, AICAr treatment of cells in our screen (Table 1), we speculate that some U266 cells in that study induced S-phase arrest with very apoptosis may occur due to inhibition of phosphatidyl- little apoptosis and 8226 cells, the most sensitive to apo- choline synthesis.

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When the de novo pyrimidine synthesis pathway is Disclosure of Potential Conflicts of Interest inhibited, tumor cells are critically dependent upon No potential conflicts of interest were disclosed. salvage pathways. One of these enzymes is deoxycytidine kinase (DCK). After internalization, deoxycytidine, de- Authors' Contributions oxyadenosine, and deoxyguanosine are phosphorylated Conception and design: A. Lichtenstein Development of methodology: C. Bardeleben, J.R. Reeve by DCK in the rate-limiting step in deoxyribonucleoside Acquisition of data (provided animals, acquired and managed patients, salvage for generation of these 3 dNTPs. Although highly provided facilities, etc.): C. Bardeleben, S. Sharma, J.R. Reeve Analysis and interpretation of data (e.g., statistical analysis, biostatis- expressed in lymphocytes (44), recent gene expression tics, computational analysis): C. Bardeleben, J.R. Reeve, S. Bassilian, A. profiling (45, 46) suggests DCK is poorly expressed in Lichtenstein normal and myeloma plasma cells. This may explain the Writing, review, and/or revision of the manuscript: C. Bardeleben, A. Lichtenstein absence of protection against AICAr-induced apoptosis Administrative, technical, or material support (i.e., reporting or orga- when deoxycytidine was used in attempted rescue add- nizing data, constructing databases): C. Bardeleben, P.J. Frost, B. Hoang, back experiments. An additional important salvage path- Y. Shi Study supervision: A. Lichtenstein way enzyme is cytidine deaminase (CDA). Cytidine and deoxycytidine are both substrates for this enzyme yield- Acknowledgments ing uridine and deoxyuridine. Deoxyuridine is phos- The authors thank Thomas Traut and George A. Kassavetis for helpful phorylated by thymidine kinase, which subsequently advice and Fiona Whelan (The Semel Institute Biostatistics Core, UCLA) is converted to dTMP by thymidylate synthase for sub- for conducting the statistical analysis. sequent incorporation of dTTP into DNA (26). Therefore, the inability of deoxycytidine to prevent AICAR-induced Grant Support apoptosis may also be due to limited conversion of deox- This work was supported in part by NIH grants CA132778, CA111448, CA168491, Research funds of the Multiple Myeloma Research Foundation ycytidine to deoxyuridine due to limiting CDA activity. and Research Funds of the Veteran’s Administration to A. Lichtenstein and Gene expression profiling suggests that a significant num- in part by NIH Center Grant, CURE: Digestive Diseases Research Center DK-41301 Peptidomic, RIA and Proteomic Core (J.R. Reeve). ber of patients harbor multiple myeloma clones with The costs of publication of this article were defrayed in part by the downregulated CDA expression compared with nonma- payment of page charges. This article must therefore be hereby marked lignant plasma cells (47, 48). A relative deficiency of these advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. salvage pathway enzymes in the multiple myeloma model may render malignant plasma cells particularly sensitive Received October 31, 2012; revised April 1, 2013; accepted April 5, 2013; to pyrimidine starvation. published OnlineFirst April 12, 2013.

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www.aacrjournals.org Mol Cancer Ther; 12(7) July 2013 1321

Metabolomics Identifies Pyrimidine Starvation as the Mechanism of 5-Aminoimidazole-4-Carboxamide-1- β-Riboside-Induced Apoptosis in Multiple Myeloma Cells

Carolyne Bardeleben, Sanjai Sharma, Joseph R. Reeve, et al.

Mol Cancer Ther 2013;12:1310-1321. Published OnlineFirst April 12, 2013.

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