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. Ó2013 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- Ó2013 American Association for Cancer Research. nary experiments confirmed that AICAr induces apoptosis 1310 Mol Cancer Ther; 12(7) July 2013 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst April 12, 2013; DOI: 10.1158/1535-7163.MCT-12-1042 Pyrimidine Starvation in Multiple Myeloma Cells 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