cell death and survival - molecular cancer researchflur cells. we propose targeting de novo...

Cell Death and Survival

Targeting mTORC1–Mediated Metabolic AddictionOvercomes Fludarabine Resistance in Malignant B Cells

Arishya Sharma1,2, Allison J. Janocha3, Brian T. Hill4, Mitchell R. Smith4, Serpil C. Erzurum3, andAlexandru Almasan1

AbstractMTOR complex-1 (mTORC1) activation occurs frequently in cancers, yet clinical efficacy of rapalogs is

limited because of the associated activation of upstream survival pathways. An alternative approach is to inhibitdownstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analogue andantimetabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevatedphospho-p70S6K, also known as RPS6KB1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) (T389), anmTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR)derivatives, and primary human CLL cells. Consistent with the anabolic role of mTORC1, FluR cells hadhigher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells. Rapalogs (everolimusand rapamycin) induced moderate cell death in FluR and primary CLL cells, and everolimus significantlyinhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphor-ylation in FluR cells was not coupled to higher ATP synthesis. Instead, it contributed primarily to an essential,dihydroorotate dehydrogenase catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotespyrimidine biosynthesis by p70S6 kinase–mediated phosphorylation of CAD (carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydroorotase; Ser1859) and favors S-phase cell-cycle progression. Wefound increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacologicalinhibition of de novo pyrimidine biosynthesis using N-phosphonacetyl-L-aspartate and leflunomide, RNAi-mediated knockdown of p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic toFluR, but not FluS, cells. These results reveal a novel link between mTORC1-mediated metabolicreprogramming and Flu resistance identifying mitochondrial respiration and de novo pyrimidine biosynthesisas potential therapeutic targets.

Implications: This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidinebiosynthesis in a relevant disease setting. Mol Cancer Res; 12(9); 1205–15. �2014 AACR.

IntroductionFludarabine (Flu; also known as F-ara-A) is a purine

analogue that is indicated for the treatment of hematologicmalignancies, including chronic lymphocytic leukemia(CLL; ref. 1) and indolent non-Hodgkin lymphomas (2).Although Flu-based regimens have been successful in

improving the outcome in patients, primary or acquiredresistance limits the effectiveness of this therapy (1).Recent research in B-cell malignancies, including CLL

and non-Hodgkin lymphomas, suggests that constitutiveactivation of B-cell receptor–associated cellular signalingpathways and cues from the microenvironment are the keyregulators for survival and maintenance of these cancers, aswell as their response to chemotherapy (3). A criticaldownstream component of the B-cell receptor signalingpathway is the mTOR kinase that is regulated by thePI3K/Akt pathway (4, 5). The mTOR kinase occurs in2 distinct complexes: mTOR complex 1 (mTORC1) andmTOR complex 2 (mTORC2; ref. 6). Akt activatesmTORC1, which in turn phophorylates p70S6 kinase(p70S6K) and the eukaryotic-initiation-factor 4E-bindingprotein-1 [4EBP1, also known as neuroguidin (NGDN)],whereas mTORC2 phosphorylates and activates Akt(5, 6).Aberrant activation of mTORC1 occurs in the most

common human cancers, suggesting that mTORC1

1Department of Cancer Biology, Lerner Research Institute, ClevelandClinic, Cleveland, Ohio. 2Department of Biological, Geological and Envi-ronmental Sciences, Cleveland State University, Cleveland, Ohio. 3Depart-ment of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleve-land, Ohio. 4Department of Hematologic Oncology and Blood Disorders,Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Alexandru Almasan, Department of Cancer Biol-ogy, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195.Phone: 216-444-9970; Fax: 216-445-6269; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0124

�2014 American Association for Cancer Research.

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signaling confers survival and proliferative advantages tocancer cells (7). Therefore, allosteric inhibitors ofmTORC1,rapamycin, and its analogues (rapalogs), represent an attrac-tive therapy for various tumors, including hematologicmalignancies (8, 9). However, these drugs failed to inducesignificant apoptosis of either cycling or quiescent cells andshowed modest clinical responses that were also associatedwith toxicity (9). The mechanism of resistance to rapalogs isattributed to their ability to inhibit only one of severaldownstream targets of PI3K, leaving Akt unaffected. More-over, they also disrupt a feedback mechanism that dam-pens PI3K activity, leading to a compensatory upregula-tion of Akt activity, causing counterproductive prosurvivaleffects. On the contrary, the ATP-competitive dual PI3K/mTORC1/2 and mTORC1/2 inhibitors display potentanticancer properties both in vitro and in vivo in a widerange of malignancies, including leukemia (9, 10). Severalof these compounds are being tested in preclinical modelsand they show a consistently robust effect against tumorsdriven by PI3K/Akt signaling, whereas they are ineffectiveagainst tumors driven by mutations of RAS, which cansignal through multiple pathways, such as those for MEKand ERK (11).An alternative approach for inhibiting mTORC1 is to

target its downstream effectors. A previous study, usingunbiased genomic and metabolomic approaches, reportedthat gene sets related to specific metabolic pathways, includ-ing the pentose phosphate pathway, fatty acid biosynthesis,glycolysis, and cholesterol biosynthesis, comprised the top20 mTORC1-induced genes (12). mTORC1 stimulatesprotein synthesis by regulating mRNA translation andribosome biogenesis (13). Additional recent reports suggestregulation of glutamine (14) and pyrimidine metabolism bymTORC1 (15–17). Consistently, targeting the enzymescomprising metabolic pathways has been evaluated invarious mTORC1-dependent cancer settings (18, 19).Targeting downstream metabolic pathways is unlikely toelicit the same unwanted feedback signaling events thatseem to limit the usefulness of rapamycin and its analoguesin the clinic. In addition, it is possible that such metabolicinhibitors would elicit selective cytotoxic responses in thetumor, rather than the cytostatic effects routinely seenwith rapamycin.As mTORC1 is associated with poor treatment out-

comes in B-cell malignancies (20), we examined thesignificance of mTORC1 pathway activation in Flu-resis-tant (FluR) cells that were generated by chronic exposureto Flu (21). Moreover, we investigated the metabolicconsequences of mTORC1 activation in FluR cells, aim-ing to identify their selective vulnerability to interferencewith specific metabolic pathways. Our study revealsmTORC1-dependent increase in glycolysis and mito-chondrial respiration in FluR cells. In addition, there wasan increase in de novo pyrimidine biosynthesis, whichcontributed to addiction to mitochondrial respiration inFluR cells. We propose targeting de novo pyrimidinebiosynthesis and mitochondrial respiration as potentialstrategies to overcome Flu resistance.

Materials and MethodsReagentsFludarabine (9-b-D-arabinofuranosyl-2-fluoroadenine 50-

phosphate) was purchased from Sigma Aldrich, everolimusfrom Selleck, and rapamycin from Calbiochem. N-phos-phonacetyl-L-aspartate (PALA, NSC224131) was acquiredfrom theNCI/DTPOpenChemical Repository (http://dtp.cancer.gov) for a study in Dr. Christine McDonald's labo-ratory (ClevelandClinic). Cells were treated with 10 mmol/LFlu and 200 nmol/L everolimus, unless otherwise stated.

Cell lines and patient samplesHuman pre-B acute lymphocytic leukemic Nalm-6, Reh,

multiple myeloma RPMI-8226, histiocytic lymphomaU937, and acute T lymphocytic leukemic Molt-4 cell lineswere obtained from the ATCC. FluR cells were generatedby initially culturing cells with a lower concentration (1mmol/L) of Flu for short periods of time followed by 48hours of recovery time. The drug concentration wasincreased gradually until the desired resistance of twice theIC50 value was achieved. The resistant cells were intermit-tently treated with verapamil (Sigma Aldrich) to eliminatethe possibility of acquired resistance because of increasedexpression of efflux pumps. In addition to the derivativeFluR cells, we usedMec-1 andMec-2 cells (a gift fromDr. Y.Saunthararajah, Cleveland Clinic), which are CLL-derivedcell lines known to be inherently resistant to Flu (22, 23).Cells were maintained in RPMI-1640 medium supple-mented with 10% fetal bovine serum (Atlanta Biologi-cals), L-glutamine (Gibco BRL), and antibiotic-antimyco-tic (Invitrogen). Cell lines were verified periodically formorphological characteristics, growth rates, and responseto stimuli using Annexin V/propidium iodide (PI) stain-ing or Trypan blue exclusion. Passage number was notallowed to exceed 15 to 20, and cell lines were routinelytested to be mycoplasma free.Peripheral blood samples were obtained from patients

with CLL after patients gave informed consent according toprotocols approved by the Cleveland Clinic InstitutionalReview Board, according to the Declaration of Helsinki.Briefly, lymphocytes from blood samples were purified byFicoll-Paque PLUS (Amersham Biosciences) gradient cen-trifugation. Primary cells were cultured and cell death wasassayed as previously described (21, 24).

ImmunoblottingCell lysates for immunoblotting and immunoprecipita-

tion were prepared, as described previously (21). The pri-mary antibodies were used against p-p70S6K (T389),p70S6K, pCAD (S1859), cytochrome c, cleaved caspase-3(Cell Signaling Technology), and b-actin (Sigma). Second-ary antibodies were anti-mouse horseradish peroxidase(HRP; Millipore) and anti-rabbit HRP (Fisher Scientific).

Cell viability and apoptosis assaysApoptosis was measured using Annexin V–fluorescein

isothiocyanate and PI staining (BD Biosciences), as

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Mol Cancer Res; 12(9) September 2014 Molecular Cancer Research1206




described previously (21). Cell death data were acquired on aBD FACSCalibur flow cytometer (BD Biosciences) andanalyzed using CellQuest software. 3-(4,5-Dimethylthia-zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium inner salt (MTS) assay (ref. 25; Promega)was used to assess cell proliferation. Data are expressed as %reduction in metabolic activity, that is, 100 � {[(O.D. 490nm untreated) � (O.D. 490 nm treated)]/(O.D. 490 nmuntreated)� 100} versus the indicated concentrations of thedrug.For clonogenic assays, 20 cells/mL were seeded using

30% FBS containing RPMI media in poly-lysine–coatedplates, and treated as indicated. After 8 to 12 days, cellswere stained with crystal violet and colonies were scoredby an alpha image analyzer (Alpha Innotech Corp.). Thepercentage of surviving fraction was then calculatedaccording to the equation ¼ (number of counts in treatedsample/number of counts in NT sample) � 100 (26). Theinteraction between PALA and Flu in clonogenic assayswas determined using the isobolographic method of Chouand Talalay (27). The combination index was calculatedusing the Compusyn software (www.combosyn.com),combination index < 1 indicates synergism, fractionaffected ¼ (100 � % surviving fraction)/100.

Extracellular flux analysisA Seahorse Bioscience XF-24 Flux Analyzer (Seahorse

Bioscience) was used to measure the oxygen consumptionrate (OCR) and extracellular acidification rate (ECAR).Cell density titrations were first performed to define theoptimal seeding density for Nalm-6 and Nalm-6-FluR cells.Suspension cells were seeded in Seahorse Cell-Tak-coatedextracellular flux (XF) 24-well cell culture microplates in150 mL Seahorse assay medium [unbuffered DMEM(Sigma D5030), supplemented with 2 mmol/L glutamine,1 mmol/L pyruvate, and 11 mmol/L glucose] prewarmed to37�C. In the subsequent experiments, Nalm-6 andNalm-6-FluR cells were seeded in growthmedia in plates, as describedabove, with 95,000 Nalm-6 cells or 50,000 Nalm-6-FluRcells per well to ensure about 90% surface coverage at thetime of the experiment. The cells were incubated for 30minutes at 37�C to allow media temperature and pH toreach equilibrium. During this time, selective metabolicinhibitors were preloaded into injection ports of the cartridgeto achieve final concentrations of 2-DG (100 mmol/L),FCCP (1.5 mmol/L), oligomycin (1.5 mmol/L), rotenone(0.75 mmol/L), and antimycin A (0.75 mmol/L). Oligomy-cin and FCCP titrations were performed for each cell line.Before the first rate measurement, total volume was adjustedto 500 mL for mito-stress using the Seahorse media andincubated for an additional 15 minutes. At the end ofincubation, the plate was placed in the Seahorse XF24analyzer. During the assay, baseline rates were measured 3times. OCR was reported in nmol/min and ECAR in milli-pH (mpH)/min and further normalized for each cell type.Substrates and selective inhibitors were injected during themeasurements and mixed for 3 to 5 minutes. OCR andECAR were then measured 3 times each.

Cytochrome c releaseCells were washed in 1� PBS and resuspended in the lysis

buffer (20 mmol/L Hepes, 10 mmol/L KCl, 1.5 mmol/LMgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/Ldithiothreitol, and 250 mmol/L sucrose). To ensure com-plete cell lysis, cells were drawn into a 281/2- or 30 1/2-gaugeneedle using a syringe and then expelled a minimum of 20times. Unbroken cells were removed by spinning at 5,000rpm for 5minutes. The supernatant was again centrifuged at14,000 rpm for 30 minutes at 4�C to separate the mito-chondrial fraction (pellet) from the cytoplasm (supernatant).The protein was quantified using the Bradford method, 5�SDS sample buffer was added to the supernatants, andanalyzed on 15% SDS-PAGE gels to probe for cytochromec release.

ATP quantificationThe quantity of ATP was measured using the Mitochon-

drial ToxGlo luminescent cell viability assay (Promega),according to the manufacturer's protocol. Briefly, cells wereseeded in white 96-well microplates at 1.0 � 104 cells perwell in 100 mL growth media, and treated as indicatedin Fig. 3 for 2 hours at 37�C, and 5% CO2. Then, 100 mLluminogenic ATP detection reagent was added and lumi-nescence intensity from each well was measured using amultilabel plate reader (Wallac Victor 1420; Perkin Elmer).

siRNA transfectionTransfections were performed with control-GFP or S6K1

siRNAQiagen (Valencia) using the AmaxaNucleofector KitV (Lonza), according to the manufacturer's protocol. Inbrief, 3.0 � 106 cells were transfected with 500 nmol/LsiRNA using program D023.

Statistical analysisStatistical comparisons between 2 groups were conducted

by using the Student t test and between multiple groupsusing 2-way ANOVA with the Prism software (version5.01). Error bars indicate standard deviation, which wascalculated from three independent experiments performedin triplicates.

ResultsFludarabine resistance is associated with hypermTORC1 activationDeregulated mTORC1 activity is frequently associated

with a variety of human cancers (7), including leukemia(20, 28), and negatively influences the response to chemo-therapy (20). To determine how mTORC1 regulates Fluresistance, we derived FluR cells from initially sensitiveNalm-6 and Reh cells (21). Examination of phospho-p70S6K at Thr-389 (p-p70S6K T389) using immunoblot-ting as an assay ofmTORC1 activation status revealed highermTORC1 activation in FluR-Nalm-6, -Reh, and CLLderived Mec-2 cell lines compared with parental Flu-sensi-tive (FluS) Nalm-6 and Reh cells (Fig. 1A). Extending ourfindings to a panel of malignant B-cell lineage lines by

Targeting mTORC1 to Overcome Fludarabine Resistance

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comparing Flu sensitivity (Fig. 1C), determined by thedose-dependent effect of Flu on MTS reduction, andmTORC1 activation (Fig. 1B), we found a remarkablystrong correspondence between hyperphosphorylation ofp70S6K and Flu resistance. In addition, we identified asimilar relationship between p-p70S6K T389 (Fig. 1D)and Flu resistance (Fig. 1E) in primary CLL cells. Thus,Flu resistance is associated with hyper-mTORC1 activa-

tion in B-cell leukemia and lymphoma cell lines andprimary cells.

mTORC1 activation is critical for survival of FluR cellsNext, we studied the effect ofmTORC1 inhibition on cell

death using 2 different rapalogs, rapamycin and everolimus,alone or in combination with Flu, in FluS versus FluR cells.InNalm-6, a FluS cell line, 100 nmol/L rapamycin alone did

Figure 1. Fludarabine resistance is associated with mTORC1 activation. A and B, protein expression analysis of p-p70S6K T389, as a marker ofmTORC1 activation in the indicated cell lines by immunoblot. Total p70S6Kwas used as a loading control. IC50, as determined in C, is indicated at the bottom.C, dose–response for the effect of 24-hour fludarabine (Fd, Flu) treatment on cell growth in the indicated cell lines, as determined by the MTS assay. Dataare expressed as mean � SD (n ¼ 3). D, protein expression analysis of p-p70S6K T389 and p70S6K in the indicated primary CLL samples. Numbersindicate CLL patient numbers. FluR, fludarabine resistant; FluS, fludarabine sensitive. E, effect of 48-hour fludarabine (Flu) treatment on apoptosis in theindicated primary CLL samples, as determined by Annexin V/PI staining and flow cytometry. % cell death following Flu treatment was normalized to% cell death in control cells using the formula: (Livecontrol � LiveFlu/Livecontrol) � 100.

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not induce apoptosis and, in fact, may have led to reducedcleaved caspase-3 in the presence of Flu (Fig. 2A). Incontrast, in Nalm-6-FluR cells, mTORC1 inhibition alonedid induce cleaved caspase-3 (Fig. 2A). However, rapamycin(Fig. 2A) did not sensitize FluR cells to Flu. Rapamycin (100nmol/L) inhibited mTORC1 as measured by decreasedphosphorylation of p70S6K in FluR cells (Fig. 2B). AnnexinV/PI staining further confirmed that rapamycin inducedapoptosis in Nalm-6-FluR, but not in parental FluS Nalm-6cells (Fig. 2C). Similar data were obtainedwith everolimus inNalm-6 and Nalm-6-FluR cells (Fig. 2D). Importantly, wefound similar results in primary CLL cells cultured ex vivo,indicating that rapamycin (Fig. 2E) or everolimus (Fig. 2F)alone induce significant cell death (P < 0.05), but do notenhance sensitivity to Flu, as measured by Annexin V/PIstaining. These findings suggest that even though constitu-tive mTORC1 activation is critical for survival of FluR cells,mTORC1 inhibition does not overcome Flu resistance.

High basal mTORC1 activation leads to higher aerobicglycolysis and oxygen consumption rates in FluR cellsThe efficacy of mTORC1 inhibition is limited by com-

pensatory activation of oncogenic pathways because of loss ofnegative feedback on the upstream PI3K/Akt pathway andby regulation of mTORC1 by other signaling pathways(9, 11, 29). Therefore, we intended to investigate whethertargeting downstream functions of mTORC1 activation was

an effective alternative to overcome Flu resistance. As recentstudies suggest that activation of oncogenic pathways,including mTORC1, must induce metabolic reprogram-ming in order to provide ATP and substrates for biosynthesisto support tumor growth (30), we next investigated whetherFluR cells had different metabolic requirements than FluScells.We measured two metabolic parameters: the ECAR and

OCR using a label-free system with the Seahorse XF-24Metabolic Flux Analyzer. ECAR correlates with the rate ofglycolysis because lactic acid is produced from pyruvategenerated through glycolysis, to replenish theNADþ neededfor glycolysis. OCR represents mainly the mitochondrialrespiration rate. We found that Nalm-6-FluR cells had asignificantly higher basal rates of glycolysis (P < 0.001; Fig.3A) and mitochondrial respiration (P < 0.0001; Fig. 3B)compared with Nalm-6 cells. Everolimus treatment signif-icantly inhibited both ECAR (P < 0.02; Fig. 3A) and OCR(P < 0.001; Fig. 3B) in FluR cells, suggesting that mTORC1regulates both glycolysis as well as mitochondrial respirationin FluR cells. As 2-deoxyglucose (2-DG) is an inhibitor ofhexokinase, the first enzyme required for glycolysis, it alsoinhibits glycolysis and thus, glucose utilization. Addition of2-DG blocked ECAR in both untreated as well as ever-olimus-treatedNalm-6-FluR cells (Fig. 3A), confirming thatECAR was a specific measure of glycolysis. Moreover, ever-olimus treatment for 16 hours had no effect, whereas

Figure 2. mTORC1 inhibitioncauses moderate cell death in FluRcells and does not enhance thecytotoxic efficacy of Flu. A,Western blot analysis for cleavedcaspase-3 and b-actin, as aloading control, in Nalm-6 andNalm-6-Flu-resistant (Nalm-6-FluR) cells following inhibition ofmTORC1 using rapamycin (Rap) incombination with Flu. B, Nalm-6-FluR cells were treated with theindicated concentrations ofrapamycin and cell lysatesanalyzed by Western blotting forp-p70S6K T389 and p70S6K. C,Nalm-6 and Nalm-6-FluR cellswere treated with the indicatedconcentrations of rapamycin for 48hours and cell death wasdetermined by Annexin V/PIstaining. D, Nalm-6 and FluR cellswere treated with everolimus (Ev)and analyzed by Western blottingfor the levels of indicated proteins.b-Actin was used as a loadingcontrol. Primary CLL cells weretreated with Flu � rapamycin orrapamycin alone (E) and Flu �everolimus or everolimus alone (F)for 48 hours and cell death wasdetermined by Annexin V/PIstaining. Data, mean � SD (n ¼ 7);�, P < 0.05.






bendamustine (Bd), which is known to induce apoptosis inFluR cells (21), led to cytochrome c release in FluR cells (Fig.3C), indicating that the decrease in OCR following ever-olimus treatment was not an outcome of mitochondrialmembrane permeabilization (31).We next defined the metabolic profile of Nalm-6 and

Nalm-6-FluR cells using a series of mitochondrial chemicalprobes (32). Oligomycin blocks ATP synthesis (and degra-dation) by the F0/F1 F-type ATPase, therefore, reducing theOCR in cells in which oxygen consumption is coupled toATP synthesis. A decrease in basal OCR on addition ofoligomycin thus provides an estimation of mitochondrialATP synthesis. Trifluorocarbonylcyanide phenylhydrazone(FCCP) disrupts the proton gradient across the inner mito-chondrial membrane and therefore uncouples the electron

transport chain from oxidative phosphorylation. As a result,the electrons continue to pass through the chain and reduceoxygen to water, but with no ATP synthesis taking place. Asa consequence, mitochondrial oxygen consumption abrupt-ly increases when FCCP is added to coupled cells. Moreover,the response to the combination of rotenone and antimycinA, which blocks the respiratory chain at complexes 1 and 3,respectively, provides a measure of non-mitochondrial oxy-gen consumption. The nearly complete inhibition of OCRwith rotenone and antimycin A confirmed that OCR is,indeed, a measure of mitochondrial oxygen consumption(Fig. 3B). Oligomycin treatment reduced the OCR, whichthen abruptly rose when FCCP was added (Fig. 3B). Thesedata indicate that mitochondrial function is not compro-mised in either Nalm-6 or Nalm-6-FluR cells. Nevertheless,

Figure 3. Constitutive mTORC1activation leads to metabolic re-programming in FluR cells.Untreated Nalm-6 parental (95,000cells/well), untreated or 16-houreverolimus (Ev)-treated Nalm-6-FluR (50,000 cells/well) cells wereseeded in V7 Seahorse tissueculture plates. A, the basal ECARwas calculated for each well for 45minutes. In the case of Nalm-6-FluR-untreated and everolimus-treated cells, ECAR wassubsequently measured for another45minutes following 100mmol/L 2-deoxyglucose (2-DG) injection as acontrol to validate ECAR as aspecific measure of glycolysis. B, aseries of basalOCRweremeasuredfor untreated or everolimus-treatedNalm-6 parental and derivativeNalm-6-FluR cells for the first 45minutes and then followingsequential injection of 1.5 mmol/Loligomycin, 1.5 mmol/L FCCP, and0.75 mmol/L rotenone þ antimycinA.C,Nalm-6FluRcellswere treatedas indicated for 16 hours.Mitochondria-free cytosol was thenprepared and cytochrome c releasewas analyzed by Western blotting.D, Nalm-6 and Nalm-6-FluR cellswere plated at 10,000 cells/well in96-well plates and treated asindicated for 2 hours and ATP wasassayed using the mitochondrialToxGlo assay from Promega. Data,counts per second (cps) ofluminescence intensity per 10,000cells. Nalm-6, Nalm-6-FluR, andMec-2 cells were treated with 200mmol/L 2-DG or cultured inglucose-free media for 72 hours (E),or treated with 0.75 mmol/Lrotenone and antimycin A for theindicated times and cell death wasdetermined by Annexin V/PIstaining (F). Data, mean � SD(n ¼ 3); �, P < 0.05; ��, P < 0.01;��, P < 0.001; ��, P < 0.0001.

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there was only a low decrease in basal OCR on addition ofoligomycin (

of B-cell receptor–associated signaling pathways, includ-ing mTORC1, is a potential treatment target in B-cellmalignancies, including CLL (28). p-p70S6K T389 acti-vation status per se has not been previously studied in thecontext of CLL or Flu responsiveness. We show thatmTORC1 activation correlates with Flu resistance in apanel of leukemic cell lines and in primary CLL cells.Despite high mTORC1 activity in FluR cells, mTORC1inhibition by rapalogs had limited effect on cell death,likely because of the previously identified feedback acti-vation of other oncogenic pathways (37).To address these limitations, we evaluated an alternative

approach by targeting downstream metabolic reprogram-ming associated with mTORC1 activation in FluR cells(12). Consistent with the well-established role of mTORC1in regulation of cellular metabolism, our study highlights 3important aspects of metabolic reprogramming in FluRcompared with parental Nalm-6 cells. FluR cells exhibited:(i) accelerated rates of glycolysis and mitochondrial respira-tion, (ii) higher de novo pyrimidine biosynthesis, as suggestedby hyper-phosphorylation of CAD, and (iii) cell death in

response to inhibition of mitochondrial respiration and denovo pyrimidine biosynthesis.An increased rate of glycolysis in the presence of sustained

OCR has been previously reported in leukemic cells, usingelectrons from non-glucose carbon sources (38). Glutamine-dependent, glucose-independent Krebs cycle activity hasbeen also reported in glioblastoma and melanoma cells(38). Our data suggest that both FluS and FluR cells utilizeglycolysis for ATP synthesis; therefore, cell death occurs inboth cases in response to 2-DG. However, FluR cells aresensitive, to a greater extent, to 2-DG, which indicates thatFluR cells are more dependent on glycolysis for ATPsynthesis and overall survival, which, in turn, can beexplained by an overall increase in biosynthetic pathways,such as pyrimidine biosynthesis.Nevertheless, the higher celldeath in Nalm-6 cells than FluR cells in response to glucosestarvation seems contradictory. However, it suggests that theresistant cells have adapted to survive without exogenousglucose. Thus, Flu-sensitive cells require exogenous glucose,and hence they die in response to glucose starvation. Incontrast, FluR cells make their own glucose by activation of

Figure 4. Constitutive mTORC1activates CAD in FluR cells. A,Western blot analysis of Ser1859-CAD and p-P70S6K proteinexpression in the indicated celllines. B, Nalm-6 and Nalm-6-FluRcells were treated with everolimusfor 24 hours, and cell lysates wereanalyzed byWestern blotting for p-p70S6K (T389) and pCAD (S1859).C, combination index fractionaffected plot of the effect ofcombination of fludarabine (Flu)and PALA on clonogenic cellsurvival in Nalm-6-FluR cells.Combination index

endogenous glucose-deprivation response pathways, such asautophagy (39) which, therefore, do not die in response tolack of glucose in the cell culture media. High glycolysis andintracellular utilization of glucose coexisting with lowerdependence on exogenous glucose because of increasedexpression of the glucose deprivation response network,including unfolded protein response, autophagy, glucagonsignaling, and gluconeogenesis, genes, has been describedbefore in the context of acquired resistance to lapatinib inbreast cancer cell lines (40). Selective targeting of thesepathways associated with glucose-deprivation could over-come resistance (40). Similarly, we recently reported thatFluR cells could be selectively targeted by inhibition ofautophagy (21). Thus, glucose deprivation response path-ways could potentially be targeted to overcome Fluresistance.Treatment with mitochondrial toxins induced robust cell

death in FluR cells. Although we observed higher OCR inFluR than in parental Nalm-6 cells, with carefully titratedconcentrations of FCCP both cell lines demonstrated basalOCR close to their maximal capacities. Yet, coupling effi-ciency was low in both cell lines. Moreover, the 2 cell linesshowed no significant difference in ATP levels.Overall, theseresults suggest that the higher OCR in FluR cells was notcoupled to higher ATP synthesis. Mitochondrial respirationin hematopoietic and various other cell types is known to beaffected by de novo pyrimidine synthesis in a Krebs cycle- andglucose-independent manner (38). Moreover, mTORC1activation was recently shown to enhance glutamine flux

through pyrimidine biosynthesis (1, 16) and leflunomidewas reported to overcome Flu resistance in CLL (41).Consistent with those data, we found higher pCAD(S1859) levels in FluR cells. Moreover, inhibition of pyrim-idine biosynthesis using 2 different inhibitors, PALA andleflunomide, reduced clonogenic survival of FluR cells.Importantly, PALA acted synergistically with Flu in induc-ing cell death in FluR cells. These findings conclusivelyestablish that constitutive mTORC1 activation promotes denovo pyrimidine synthesis in FluR cells, to which they areaddicted.Notably, p70S6K knockdown induced remarkable cell

death in FluR cells compared with FluS cells. This furthersupports the importance of the mTORC1/p70S6K/CADaxis in regulating pyrimidine biosynthesis and, therefore,survival in FluR cells. The fact that rapalog treatment,despite reducing active p70S6K levels more effectivelythan S6K knockdown, was less effective in inducing celldeath seems intriguing. However, rapalog treatment willalso affect other targets of mTORC1 which, in turn, maybe associated with prosurvival pathways (29). For exam-ple, mTORC1 inhibition activates the ULK-1 (Unc-51-like kinase, or ATG1 (autophagy-related gene 1) complexwhich, in turn, will activate autophagy, which is indeed awell-established prosurvival pathway (42). Consistently,we have previously reported that FluR cells depend onautophagy for their survival (21). Therefore, these findingsfurther underscore the importance of targeting down-stream pathways in mTORC1-dependent cancers.

Figure 5. Model for targetingmetabolic vulnerability of FluR cells. 1. HyperactivemTORC1 is associatedwith Flu resistance. 2.mTORC1 causes higher rates ofglycolysis, as measured by ECAR and mitochondrial respiration, as measured by OCR, both of which are essential for FluR cell survival. Inhibition ofmitochondrial respiration using rotenone and antimycin induces a more dramatic cell death than inhibition of glycolysis using 2-deoxyglucose, However, theincrease inOCR is not related to ATP synthesis. 3. In fact, constitutivemTORC1activation causesCADS1859phosphorylation in FluR cells, which leads todenovo pyrimidine biosynthesis and promotes survival in these cells. As such, FluR cells are also highly susceptible to inhibition of de novo pyrimidinebiosynthesis using PALA and leflunomide, 4. DHODH, an essential enzyme in de novo pyrimidine biosynthesis, requires mitochondrial respiratory chainelectron acceptors to oxidize DHO to orotate, Thus, high mitochondrial respiration contributes to increase in de novo pyrimidine biosynthesis, in addition toother functions in FluR cells.






We recognize that OxPhos inhibition may cause celldeath because of multiple reasons, for example, inhibitionof recycling of NADþ (43), inhibition of de novo pyrim-idine biosynthesis (44), reactive oxygen species (RCS) (45)and, disruption of mitochondrial membrane potential(MMP) leading to Bax/Bak oligomerization (38). Indeed,uridine supplementation could only modestly rescue thecell death caused by rotenone and antimycin treatment inthese cells (data not shown). Nevertheless, our data sug-gest that one of the reasons should be de novo pyrimidinebiosynthesis given high proportion of S-phase cells andhigh pCAD S1859. The DHODH enzyme, a criticalcomponent of this pathway, is located in the innermitochondrial membrane and must use mitochondrialelectron transfer chain (ETC) components, that is, ubiqi-none as the proximal acceptor and coenzyme q as theultimate electron acceptor, in order to carry out oxidationof DHO to orotate.In summary, we established mTORC1 activation, as

measured by p-p70S6K T389, and downstream pCADS1859 as potential biomarkers of Flu resistance in leukemiccells (Fig. 5). FluR cells depend on mTORC1-dependent denovo pyrimidine biosynthesis and mitochondrial respirationfor survival. Thus, directly targeting de novo pyrimidinebiosynthesis pathway enzymes using PALA and leflunomide,or targeting mitochondrial respiration, represent effectivestrategies to overcome Flu resistance.

Disclosure of Potential Conflicts of InterestB.T. Hill reports receiving commercial research support from Millennium Phar-

maceuticals. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A. Sharma, S.C. Erzurum, A. AlmasanDevelopment of methodology: A. Sharma, A.J. Janocha, S.C. Erzurum, A. AlmasanAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A. Sharma, A.J. Janocha, M.R. SmithAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): A. Sharma, A.J. Janocha, B.T. Hill, M.R. Smith, S.C. Erzurum,A. AlmasanWriting, review, and/or revision of the manuscript: A. Sharma, A.J. Janocha,B.T. Hill, M.R. Smith, S.C. Erzurum, A. AlmasanAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S.C. ErzurumStudy supervision: A. Almasan

AcknowledgmentsThe authors thank Drs. C. McDonald, Y. Sountharajah, and N. Gupta (Cleveland

Clinic), R. Dalla Favera (Columbia University Medical Center) for critical reagents.Dr. C. Talerico (Cleveland Clinic) provided substantive editing and comments.

Grant SupportThis work was supported by a research grant from NIH CA127264 (awarded

to A. Almasan), HL103453 and HL60917 (awarded to S.C. Erzurum), anda fellowship from Cleveland State University (Cellular and Molecular MedicineProgram to A. Sharma).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received March 4, 2014; revised May 27, 2014; accepted July 7, 2014;published OnlineFirst July 24, 2014.

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2014;12:1205-1215. Published OnlineFirst July 24, 2014.Mol Cancer Res Arishya Sharma, Allison J. Janocha, Brian T. Hill, et al. Fludarabine Resistance in Malignant B Cells

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