inhibition of akt potentiates 2-dg induced apoptosis via ... · signaling and regulation inhibition...

Signaling and Regulation

Inhibition of Akt Potentiates 2-DG–Induced Apoptosis viaDownregulation of UPR in Acute Lymphoblastic Leukemia

Joanna DeSalvo1, Jeffim N. Kuznetsov2, Jianfeng Du1, Gilles M. Leclerc1, Guy J. Leclerc1,Theodore J. Lampidis3,4, and Julio C. Barredo1,2,4

AbstractThe ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor

genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanismof cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis andN-linked glycosylation, in acute lymphoblastic leukemia (ALL).We found that, unlike most other cancer phenotypesin which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis.Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant,revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential forN-linkedglycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and inductionof ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity inALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers(IRE1a, GRP78, P-eIF2a, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we findthat pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cellsto remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance ofT-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling.Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, andAKT andAMPK, traditionallythought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine celldeath or survival. Mol Cancer Res; 10(7); 969–78. �2012 AACR.

IntroductionAcute lymphoblastic leukemia (ALL) is themost common

malignancy in children and adolescents and is a leading causeof cancer-related deaths in these patients (1). Current clinicalpractices have had only minimal impact on cure rates forpatients with resistant phenotypes or after relapse (2, 3). Anumber of ALL phenotypes exhibit mutations that lead toinactivation or constitutive activation of oncogenic pathwayssuch as LKB1, PTEN, PI3K/Akt and RAS, which have beenlinked to the regulation of energy metabolism in general andglucose metabolism in particular (4–6). T-ALL is known tohave a high rate of PTENmutations that lead to constitutiveactivation of Akt (7). Our laboratory has previously shownthat the master energy regulator AMPK has significant

feedforward and feedback cross-talk with these pathwaysand that AMPK is a suitable target for ALL therapy(8, 9).Consequently, although targeting glucosemetabolismin ALL has not been explored, the above-described biologyof ALL lymphoblasts suggests a significant therapeuticpotential.The increased glycolytic rate exhibited by cancer cells

compared with their nonmalignant counterparts, even in thepresence of sufficient oxygen, was first described by Otto H.Warburg (10). This unique alteration in glucose metabolismprovides a biochemical basis for targeting glycolysis as aselective anticancer treatment strategy. Indeed, the glucoseanalog 2-deoxy-D-glucose (2-DG) has shown activity againstsolid tumors in preclinical models and early phase humanclinical trials (11), but its role as an antileukemic agent hasnot been explored. 2-DG can induce cell growth arrest andcell death by inhibiting 2 key glycolytic enzymes, hexokinaseand phosphoglucose isomerase (PGI; ref. 12). Followingfacilitated diffusion into cells through glucose transporters,2-DG is converted to 2-DG-6-phosphate by hexokinase, butunlike glucose-6-phosphate, 2-DG-6-phosphate cannot befurther metabolized by PGI, and its accumulation inhibitsthe glycolytic pathway (13). Inhibition of glycolysis has beenshown to be a main mechanism of 2-DG's cytotoxicity inhypoxic cells of solid tumors (14). However, because of itsstructural similarity with mannose, 2-DG has also been

Authors' Affiliations: Departments of 1Pediatrics Hematology and Oncol-ogy, 2Biochemistry andMolecular Biology, 3Cell Biology andAnatomy, and4Sylvester Comprehensive Cancer Center, University of Miami MillerSchool of Medicine, Miami, Florida

Note: J. DeSalvo and J.N. Kuznetsov contributed equally to this work.

Corresponding Author: Julio C. Barredo, Department of Pediatric Hema-tology-Oncology, University of Miami Miller School of Medicine, P.O. BOX016960, Miami, FL 33101. Phone: 1-305-243-2448; Fax: 1-305-243-2691;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0125

�2012 American Association for Cancer Research.

MolecularCancer

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on March 23, 2020. © 2012 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst June 12, 2012; DOI: 10.1158/1541-7786.MCR-12-0125

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shown to be a potent inhibitor of N-linked glycosylation(12, 15). Indeed, 2-DG can be incorporated in place ofmannose into lipid-linked oligosaccharide (LLO) chains(15), but unlike mannose, 2-DG cannot accommodate therequired complex branching structure of these molecules,leading to premature termination of LLO synthesis, andaccumulation ofmisfolded, nascent ER-synthesized proteins(16).The 2-DG–induced accumulation of misfolded glycopro-

teins in the ER lumen leads to ER stress and induction of theunfolded protein response (UPR), a protective physiologicresponse aimed at reducing the presence of unfolded proteinsand consequently ER stress (11, 12). The UPR is mediatedvia 3 ER transmembrane receptors: protein kinase dsRNA-like ER kinase (PERK), activating transcription factor 6(ATF6), and inositol-requiring enzyme 1a (IRE1a), whichare activated upon dissociation from the main ER chaperoneprotein GRP78 to fully engage the UPR (17). In addition toits role in the removal of excess misfolded proteins from theER lumen for proteosomal degradation, GRP78 also func-tions to suppress proapoptotic pathways triggered by ERstress (18). However, the protective capacity of the UPR islimited, and prolonged ER stress will eventually triggerirreversible proapoptotic signals such as increased expressionof CHOP (CCAAT/enhancer binding protein homologoustranscriptional factor; ref. 19). In most malignant cell types,CHOP induction is considered sufficient to induce ERstress/UPR-mediated cell death, whereas in B-lineage lym-phocytes, apoptotic death is determined by the homeostaticbalance betweenmultipleUPRproteins, rather than changesin any single UPR protein (20).In this study, because of the dual nature of 2-DG as an

inhibitor of glycolysis and an inducer of ER stress, weevaluated its effects on these pathways in B-precursor andT-cell ALL cell models and primary cells. These studiesuncovered that unlike most cancer phenotypes, which donot undergo cell death when treated with relatively lowdoses (<8 mmol/L) of 2-DG under normoxic as opposedto hypoxic conditions, ALL cells undergo apoptosis pri-marily through activation of ER stress. Moreover, wefound differential sensitivity to inhibition of N-linkedglycosylation versus aerobic glycolysis within ALL sub-types, in which Bp-ALL cells show significantly greatersensitivity to 2-DG than T-ALL cells. We showed that thedecreased sensitivity of T-ALL is conferred by Akt andconsequently cotargeting Akt signaling enhances cell deathin 2-DG–treated ALL cells. Finally, we establish that Aktand AMPK differentially modulate the ability of ALL cellsto effectively activate UPR, which buffers the proapoptoticeffects of 2-DG–induced glycoprotein misfolding and ERstress.

Material and MethodsCell culture and reagentsThe ALL cell lines CCRF-CEM (T-ALL), Jurkat

(T-ALL), and NALM6 (B precursor-ALL) were maintainedin RPMI-1640 medium (Cellgro) supplemented with

10% heat-inactivated FBS (Sigma-Aldrich) and antibiotics(penicillin, 100 IU/mL; streptomycin, 100mg/mL;Cellgro).SupB15 cells (Bp-ALL, BCR-ABL [t(9;22)]) were main-tained in Iscove's Dulbecco's modified Eagle's medium plusheat-inactivated FBS and antibiotics. Primary ALL cells wereobtained from patients with ALL at the University of Miamifollowing Institutional Review Board–approved informedconsent. Primary cells were cocultured using a human bonemarrow stromal cell feeder layer (2.5 � 106 cells/75 cm2)immortalized by human telomerase reverse transcriptase(hTERT) transfection (provided by Dr. D. Campana,SJCRH, Memphis, TN). 2-DG, 2-fluorodeoxy-D-glucose,and D-mannose were from Sigma-Aldrich, and Akt InhibitorX was from Calbiochem.

Cell viability and apoptosis assaysCell viability was assayed by trypan blue exclusion using

the Vi-CELL XR analyzer (Beckman Coulter). Cell deathwas determined either using the Vi-CELL XR or by flowcytometry analysis of Annexin V–fluorescein isothiocyanate(FITC)/propidium iodide (PI) staining using BD Pharmi-gen FITC Annexin V apoptosis detection kit. Labeled cellswere then analyzed by BDTM LSR II flow cytometer in theappropriate channel and FACSDiVa software version 4.1.2(BD Biosciences). Events positive for Annexin V and PIstaining were gated and used to define the population ofdead/dying cells. The level of cell death is expressed as thepercentage of death measured in untreated cells. Apoptosiswas expressed as a percentage (%) of cells stained withAnnexin V–FITC/PI in the population (mean � SEM, n¼ 3). Synergy analysis was determined using Chou's com-bination index (CI) analyses using the following equation:CI ¼ [(D1-combination/D1-single) þ (D2-combination/D2-single)].Assessment of synergy was carried out by quantitating druginteraction with the Calcusyn computer program (Biosoft).CI values of <1, 1, and >1 indicate synergy, additivity, andantagonism, respectively (21). Statistical differences in cellproliferation or cell death were assessed by one-way ANOVAfollowed by theNewman–Keuls multiple comparison test orby unpaired Student t test using GraphPad PRISM.

Construction of stable shRNA-expressing cell linesTo downregulate the expression of Akt1 and GRP78, we

used lentiviral particles generated from cotransfection ofp8.2vpdeltaR, pVSVG-pseudotyped lentiviral and pLKO.1plasmids encoding specific hairpin RNA sequences in HEK-293 cells (Clontech). The hairpin oligonucleotides for shorthairpin RNA (shRNA)-Akt1 (22) and GRP78 (23) werehybridized and the duplex ligated into pLKO.1puro(Addgene plasmid #10878). Negative control lentiviralshRNAs (sc-108080) were obtained from Santa Cruz Bio-technology. To knockdown AMPKa1, lentiviral particlescontaining up to 5 different constructs encoding target-specific 19 to 25 nt shRNA were used (sc-296730V; SantaCruz Biotechnology). Stable ALL cell lines expressing eachone of these shRNAs were developed by transducing CCRF-CEM andNALM6 cells by spinoculation and selection with1mg/mL puromycin as described elsewhere (24). Each stable

DeSalvo et al.

Mol Cancer Res; 10(7) July 2012 Molecular Cancer Research970




shRNA-expressing cell line was validated for knockdown byimmunoblotting.

Protein extracts and immunoblotsCells were harvested at the specified time points, washed

with 1� PBS, and sonicated in 50 mmol/L Tris-HCl (pH7.4) containing protease inhibitors (Thermo). Proteins (50mg per lane) were resolved by SDS-PAGE electrophoresisand transferred onto polyvinylidene difluoride membranes.b-Actin was used as a loading control. The immunoblotspresented are representative of 3 independent experiments.

Results2-DG induces growth inhibition and cell death in ALLcells under normoxiaTo evaluate the clinical relevance of glycolytic inhibitors in

childhood ALL, we first determined the effects of 2-DG and2-FDG on ALL proliferation and cell death under normoxic(pO2 ¼ 21%) and hypoxic (pO2 ¼ 0.5%) conditions. Forthis, ALL cell models for T-ALL (CCRF-CEM and Jurkat,both PTEN mutants), Bp-ALL (NALM6 and SupB15, achemotherapy-resistant BCR-ABL[t(9;22)]-expressing cellline), and representative primary ALL cells (PATIENT)were treated with a range of doses of 2-DG or 2-FDG forup to 72 hours. As shown in Fig. 1, 2-DG (0.5–4 mmol/L)

induced growth inhibition (Fig. 1A) and cell death (Fig. 1B)under normoxic conditions in all ALL cell models andprimary ALL cells tested. Significantly lower cytotoxicitywas observed when cells were treated under hypoxic condi-tions (not shown). Although growth inhibition was com-parable across T- and B-lineage phenotypes, T-ALL cells(CCRF-CEM and Jurkat) exhibited relative resistance to 2-DG–induced cell death compared with B-lineage ALL cells(Fig. 1B). Primary Bp-ALL cells tested exhibited sensitivityto 2-DG under normoxic conditions in the same range asobserved in the representative cell models. In contrast,treatment with equimolar concentrations of the more selec-tive glycolytic inhibitor 2-FDG had negligible effects onCCRF-CEM cells and induced less cell death than 2-DG inNALM6 and SupB15 cells under normoxia (Fig. 1C).It has been reported that in cancer phenotypes in which

low-dose treatment with 2-DG (<4 mmol/L) induces celldeath under normoxia, 2-DG acts through inhibition of N-linked glycosylation (12, 14). D-Mannose has been shown toreverse both the growth inhibitory and cytotoxic effectsassociated with inhibition of N-linked glycosylation butcannot reverse the cytotoxic effects triggered by inhibitionof glycolysis (25, 26). In addition, 2-FDG is known topreferentially inhibit glycolysis with minimal effects on N-linked glycosylation. On the basis of the profile of 2-DG and2-FDG–induced cytotoxicity in ALL cells, we postulated

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Figure 1. Cytotoxicity of 2-DG in ALL cells under normoxia. Growth inhibition (A) and cell death (B) in T-ALL (CCRF-CEM and Jurkat) and Bp-ALL (NALM6,SupB15, and primary patient Bp-ALL) cells treatedwith 2-DG (4mmol/L) for 72 hours under normoxia (pO2¼ 21%). Cell death (C) in CCRF-CEM, NALM6, andSupB15 cells treated with 2-FDG (4 and 8 mmol/L) for 72 hours under normoxia. Cell death (D) in CCRF-CEM, NALM6, and SupB15 cells treated with2-DG (4 and 8mmol/L) and eitherwith (MAN, 2 and4mmol/L) orwithout (CTRL) D-mannose for 72 hours under normoxia. Growth inhibition and cell deathwereexpressed relative to control values (mean �SEM, n ¼ 3), or as a percentage (%) of cells in the population (mean �SEM, n ¼ 3), respectively. � and #denoteP < 0.0001 andP < 0.01, respectively, for glycolytic inhibitor-treated cells versus CTRL (panels A, B, and C), or for 2-DG alone versus 2-DGþMAN (D).

2-DG Induces UPR-Mediated Cell Death in ALL

www.aacrjournals.org Mol Cancer Res; 10(7) July 2012 971




that inhibition of N-linked glycosylation by 2-DG leads tocell death in T-ALL, and although it seems to be thepredominant mechanism leading to cytotoxicity in Bp-ALL,inhibition of glycolysis also contributes to cell death in thelatter phenotype. Consequently, we cotreated CCRF-CEM,NALM6, and SupB15 cells with 2-DG andD-mannose andevaluated the effects on cell death. Our results indicated thatmannose completely reverses the cytotoxic effects of 2-DGin CCRF-CEM at 4 and 8 mmol/L doses of 2-DG, but onlyat 4 mmol/L 2-DG dose in NALM6 (Fig. 1D). Partialreversal was seen in NALM6 at higher dose of 2-DG (8mmol/L) and in SupB15 cells at both doses (Fig. 1D).Therefore, inhibition of N-linked glycosylation is the pre-dominant mechanism by which low-dose 2-DG induces celldeath in ALL cells. However, phenotypic differences exist,and it is possible that in Bp-ALL, inhibition of aerobicglycolysis also contributes to 2-DG–induced cytotoxicity.

2-DG induces changes in the expression andphosphorylation/activation of Akt, AMPK, and ERstress/UPR-associated signaling proteins in ALL celllinesTo further understand the molecular mechanisms by

which 2-DG induces cell death in ALL, we examined theexpression of critical signaling proteins associated withenergy metabolism in general, glucose metabolism in par-ticular, and cell proliferation. As shown in Fig. 2A, 2-DGinduced significant activation of P-AMPK (T172) in bothcell lines. Consistent with increased P-AMPK expression,

P-mTOR (S2448) and its downstream target P-p70S6K(T389) were downregulated by 2-DG through the inhibi-tory effect of AMPK on mTOR activity (27), and thedownregulation of mTOR signaling was greater in NALM6cells as compared with CCRF-CEM cells (Fig. 2A). ThePTEN-mutant CCRF-CEM cells exhibited both high basalexpression and sustained increased expression of P-Akt(S473) (6 vs. 24 hours), whereas in NALM6 cells, the basalexpression of P-Akt (S473) was lower, and the strongupregulation of P-Akt (S473) observed at 6 hours subsidedby 24 hours. Our data show a strong correlation between thehigh basal and sustained expression of P-Akt followingtreatment with 2-DG in CCRF-CEM cells and their relativeresistance to this agent (Fig. 1B). We postulated that PTENinactivation in CCRF-CEM cells and subsequent hyperac-tivation of the Akt pathway leads to relative resistance of T-ALL cells to 2-DG. Because of the high prevalence ofinactivating mutations in patients with T-ALL (7), thismechanism of resistance is of clinical relevance.Inhibition of N-linked glycosylation by 2-DG has been

shown to induce accumulation of misfolded nascent glyco-proteins in the ER lumen, leading to ER stress and UPRactivation (25, 28).We analyzed the expression of ER stress/UPR markers in CCRF-CEM and NALM6 cells treatedwith 2-DG. In both cell lines, 2-DG (4 mmol/L) increasedthe expression of GRP78, GRP94, IRE1a, P-eIF2a (S51),and CHOP, confirming induction of ER stress/UPR, andtheir increased expression correlated with PARP cleavage, amarker for apoptosis (Fig. 2B). These data suggested that

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Figure 2. 2-DG activates AMPK,Akt, and UPR signaling proteins inALL. A, immunoblotting of AMPKand Akt/mTOR signaling factors inCCRF-CEM and NALM6 cellstreated with 2-DG (4 and 8mmol/L)for 6 and 24 hours under normoxia.B, immunoblotting of UPRsignaling factors in CCRF-CEMandNALM6cells treatedwith 2-DG(4mmol/L) for 24 hours. C,Westernblots of Akt/mTOR and UPRsignaling proteins in primarypatient Bp-ALL cells treatedwith 2-DG (4 mmol/L) for 24 hours. D,immunoblotting of UPR factors inCCRF-CEM and NALM6 cellstreated with 2-DG and either with(þ) orwithout (�) D-mannose (MAN,10 mmol/L) for 24 hours.

DeSalvo et al.





2-DG induces ALL cell death through ER stress/UPR-mediated mechanisms. The changes in Akt/mTOR andUPR signaling observed in these cell lines were replicatedusing representative primary ALL cells, validating theirclinical relevance (Fig. 2C; representative Bp-ALL sample).To confirm that the putative inhibition of N-linked glyco-sylation by 2-DG led to induction of ER stress/UPR, wetreated the cell line models with 2-DG in the presence orabsence of mannose. Western blots revealed that the addi-tion of mannose (10 mmol/L) resulted in downregulation ofthe UPR markers GRP78, P-eIF2a (S51), and CHOP inboth CCRF-CEM and NALM6 cells (Fig. 2D), suggestingthatmannose relieves ER stress–induced cytotoxicity in ALLcells by reversing 2-DG–induced inhibition of N-linkedglycosylation.

Inhibition of Akt sensitizes ALL cells to 2-DG–inducedcell death via a UPR-mediated mechanismOn the basis of upregulation of P-Akt (S473) in response

to 2-DG and the relative resistance of cells, harboringinactivating PTEN mutations and constitutive activation ofAkt, we hypothesized that Akt plays a central prosurvival rolein ALL cells treated with 2-DG. To test this, we treatedCCRF-CEMcells with a pharmacologic inhibitor of Akt and

examined its effects on 2-DG–mediated cell death. Figure3A shows that inhibition of Akt signaling using the Aktinhibitor X (AIX, 10 mmol/L) sensitized CCRF-CEM cellsto even a very low dose of 2-DG (0.5 mmol/L). Thiscombination led to more than 85% apoptotic death ascompared with very modest effects with each agent alone(Fig. 3A; 2-DG–8.1%; AIX–6.9%, P < 0.001). In addition,we found that this combination exhibited synergism (CI ¼0.36), as determined using the equation by Chou (21).Similar effects, albeit of a lesser magnitude, were observed inNALM6 (CI¼ 0.60; data not shown).Western blot analysisof CCRF-CEM cells treated with both agents (2-DG–0.5and 10mmol/L; AIX–10mmol/L) showed significant down-regulation of P-Akt (S473) and the UPR markers GRP78,IRE1a, P-eIF2a (S51), and CHOP, whereas the level of P-AMPK (T172) was significantly increased (Fig. 3B).To confirm these results, we used a genetic approach based

on the lentiviral delivery of shRNAs to specifically knock-down Akt1 expression in ALL cells. We found that shAkt1-expressing ALL cells treated with 2-DG (0.5, 1.5, and5.0 mmol/L) exhibited significant increase in apoptoticdeath compared with shRNA scramble controls (shSCR;P < 0.001 for CCRF-CEM; P < 0.01 for NALM6; Fig. 3C).As expected, Western blotting showed downregulation of

Figure 3. Inhibition of Aktsynergistically sensitizes ALL cells to2-DG and downregulates expressionof UPR factors. A, level of apoptosisin CCRF-CEM cells treated with2-DG (0.5 mmol/L) and the Aktinhibitor X (AIX, 10 mmol/L) eitheralone or in combination for 24 to72 hours under normoxia. B,immunoblotting of P-Akt (S473),P-AMPK (T172), and UPR markers inCCRF-CEM cells treated with 2-DG(0.5 and 10 mmol/L) in presence orabsence of AIX (10 mmol/L) for24 hours. C, effects of Akt1downregulation on 2-DG–inducedapoptosis in stably transducedCCRF-CEM and NALM6 cellsexpressing either shRNAs againstAkt1 (shAkt1) or control/scrambledshRNAs (shSCR) and treated with2-DG (0.5–5.0 mmol/L) for 72 hoursunder normoxia. D, immunoblottingof Akt1, P-AMPK (T172), and UPRsignaling proteins in the CCRF-CEMand NALM6 cells treated in (C) for24 hours. � and # denote P < 0.001and P < 0.01, respectively, for singledrug versus combination (A), orshSCR versus shAkt1 (C).

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Akt1 in shAkt1-transduced cells compared with shSCRcontrol cells. Moreover, as was the case in the AIX-treatedcells, genetic downregulation of Akt lead to increasedexpression of P-AMPK (T172) and decreased expression ofUPR markers IRE1a, GRP78, P-eIF2a (S51), and CHOP,compared with cells treated with 2-DG alone (Fig. 3D).Taken together, our results showed that Akt signaling iscritical for ALL cell proliferation and survival in response to2-DG treatment and supports the role of Akt as a positiveregulator of the prosurvival arm of the UPR (29).

Inhibition of AMPK restores UPR function and rescuesALL cells from ER stress–mediated cell death followingtreatment with 2-DG alone or in combination with AktdownregulationDuring the course of our investigations, we uncovered that

2-DG induced P-AMPK (T172) in ALL cells (Fig. 2A) andthat inhibition of Akt further enhanced AMPK activation(Fig. 3). To determine the role of AMPK in 2-DG–treatedALL cells, we used shRNAs to downregulate AMPKa1 inCCRF-CEM and NALM6 cells. Cells were treated with 2-DG (0.5 and 2.5 mmol/L) � AIX (10 mmol/L), and theexpression of AMPK, Akt, UPRmarkers, and cell death wereexamined.We found that downregulation of AMPK rescuedCCRF-CEM and NALM6 cells from cell death induced bythe combination of 2-DGþAIX (Fig. 4A and B; P < 0.0001and P < 0.001 for shAMPK vs. shSCR cell death in CCRF-CEM and NALM6, respectively). Importantly, these effects

were accompanied by increased induction of UPR markersin both AMPK knockdown cell lines treated with 2-DG þAIX (Fig. 4C and D), indicating that AMPK knockdownrestored the ability of ALL cells to effectively engage theUPR.

Inhibition of GRP78 sensitizes ALL cells to 2-DG–induced cell deathTo definitively establish that the ability of ALL cells to

effectively engage the UPR is central to the ability of ALLcells to buffer 2-DG–induced cytotoxicity, we used a geneticapproach to inhibit the main effector of UPR signaling—GRP78 (17, 18) and evaluated apoptotic death in 2-DG–treated ALL cells. Downregulation ofGRP78 sensitizedALLcell models to equimolar concentrations of 2-DG, leading toa significant increase in cell death compared with shSCR-expressing cells (Fig. 5B, P < 0.001 for CCRF-CEM/shSCRvs. CCRF-CEM/shGRP78; Fig. 5C, P < 0.01 for NALM6/shSCR vs. NALM6/shGRP78). These data confirmed thatthe ability of ALL cells to effectively engage the UPR isessential to buffer the proapoptotic effects of 2-DG.

DiscussionReprogramming tumor metabolism has recently been

proposed as an emerging hallmark of cancer (30). Thisincludes reprogramming of glucose metabolism in generaland the glycolytic pathway in particular, to allow cancer cells

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2-DG (mmol/L):AIX (10 μmol/L):

P-Akt (Ser473)

P-mTOR (Ser2448)

P-eIF2α (Ser51)

AMPKα

β-Actin

IRE1α

GRP78

A

C D

B

Figure 4. Inhibition of AMPK rescues ALL cells from 2-DG–induced cell death. Apoptosis in CCRF-CEM (A) and NALM6 (B) cells expressing either scrambleshRNAs (shSCR) or shRNAsagainstAMPK (shAMPK) and treatedwith 2-DG (0.5–2.5mmol/L) andwith orwithout AIX (10mmol/L) for 72hours under normoxia.� and # denote P < 0.0001 and P < 0.001, respectively, for shSCR versus shAMPK. Western blot analysis of AMPK, Akt/mTOR, and UPR signaling pathwayproteins in the shSCR- and shAMPK-expressing CCRF-CEM (C) and NALM6 (D) cells treated as described in panels A and B for 24 hours.

DeSalvo et al.





to generate ATP through aerobic glycolysis, whereas thisswitch is limited in normal cells (31). Although there issubstantial evidence to support increased glycolysis in tumorversus normal tissue, recently there have been reports indi-cating that glycolysis is closely linked with the cell cycle andthat increased glucose metabolism is a consequence ofgrowth signaling within a cell (normal or tumor) and notnecessarily as a result of "rewiring" of a cancer cell (32).Nonetheless, increased glucose metabolism seems to be ahallmark of cancer, which is likely due to their uncontrolledand constant movement through the cell cycle as well asother factors associated with oncogenic transformation(30, 33).Recently it has been shown that ALL lymphoblasts exhibit

increased glycolytic rate even in the presence of sufficientoxygen (34). On this basis, we examined the effects oftargeting glucose metabolism and found that unlike in mostcarcinomas, 2-DG induced significant apoptosis in ALL cellmodels and primary ALL cells under normoxic conditions.Nevertheless, phenotypic differences in sensitivity were seenbetween Bp-ALL and T-ALL, with the latter exhibitingrelative resistance due to the hyperactivation of Akt signalingin this PTEN-mutant prevalent phenotype. Most cancerphenotypes that exhibit resistance to 2-DG under normoxiabuffer the downstream proapoptotic signals induced by thisglycolytic inhibitor by alternatively using other energysources, fueling oxidative phosphorylation via the tricarbox-ylic acid cycle (TCA) to generate ATP (35, 36). Therefore,our data suggest that ALL cells may be either unable tocompensate via oxidative phosphorylation or that 2-DGinterferes with pathways critical for cell survival independentof glycolysis. Using a sugar essential for N-linked glycosyl-ation (D-mannose), we showed that 2-DG's known ability toinhibit the N-linked glycosylation pathway contributessignificantly to ALL cell death under normoxia and that inT-ALL cells, inhibition of this pathway seems to be thepreferential event leading to cell death. In contrast, in Bp-ALL cells, D-mannose only partially reversed the 2-DG

cytotoxicity, suggesting that 2-DG induces cell death inBp-ALL cells via inhibition of both N-linked glycosylationand glycolysis. Conversely, the more selective glycolyticinhibitor 2-FDG induced cell death in Bp-ALL, but not inT-ALL, consistent with increased dependence on glycolysisby Bp-ALL as compared to T-ALL (37). Our data supportthe notion that phenotypic differences in the regulation ofglucose metabolism lead to the differential sensitivity ofT- versus Bp-ALL cells to 2-DG and that further elucidationof these phenotypic differences may lead to lineage-specifictherapeutic strategies.Because of its structural similarity to mannose, a sugar

central to N-linked glycosylation of proteins in the ERlumen, 2-DG can enter the glycosylation pathway anddisrupt the elongation of polysaccharide chains linked tonascent proteins, resulting in accumulation of misfoldedproteins in the ER lumen (12, 25) and induction of UPR(38). Although the role of UPR is to alleviate sustained ERstress by inhibiting protein translation, activating chaper-ones, and enhancing proteosomal degradation of misfoldedproteins (17), the persistent ER stress will turn on theproapoptotic arm of UPR regulated by CHOP (19). Ourdata indicate that 2-DG induces ALL cell death via amechanism mediated by ER stress/UPR as evidenced byincreased expression of several UPR markers, includingCHOP, which correlates with PARP cleavage and evidenceof cell death shown by Annexin V/PI staining. Moreover, weshow that the addition of D-mannose not only rescues 2-DG–treated cells but also correlates with the downregulationof UPR markers. Therefore, our data are consistent withpublished reports indicating that inhibition of glycolysismodulates prednisolone resistance in ALL and that veryhigh doses of 2-DG (40 mmol/L) induce cytotoxicity inlymphoma cell lines (39, 40).Mammalian cells exhibit redundancy in the regulatory

pathways that coordinate essential physiologic processesaimed at buffering proapoptotic signals. This redundancyis significant in pathways that pair cellular energetics

Figure 5. Downregulation of GRP78sensitizes 2-DG–treated ALL cells. Western blotanalysis of GRP78 (A) in stably transducedCCRF-CEM and NALM6 cells expressingshRNAs against GRP78 (shGRP78) andtreated with 2-DG (0.5–2.5 mmol/L) for24 hours under normoxia. Level of apoptosisin stably transduced CCRF-CEM (B) andNALM6 (C) cells expressing either scrambleshRNAs (shSCR) or shRNAs against GRP78(shGRP78) and treated with 2-DG(0.5, 1.0, and 2.5 mmol/L) for 72 hoursunder normoxia. � and # denote P < 0.001and P < 0.01, respectively, for shSCR versusshGRP78.

45

40

35

30

25

20

15

10

5

0

shSCR

shSCR

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CCRF-CEM

NALM6

NALM6

02-DG (mmol/L):

GRP78

β-Actin

0.5 2.5 0 00.5 0.5 0 1.01.0 0.52.5

shSCRshGRP78

shGRP78

shSCR*

*

#

#

shGRP78

shGRP78

80

60

40

20

02-DG (mmol/L): 2-DG (mmol/L):0 00.5 2.5 0.5 1

Ap

op

tosis

(%

)

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op

tosis

(%

)

B C

A






(AMPK/mTOR) with oncogenic (Akt) pathways related togrowth/survival of ALL cells and glucose metabolism (41,42). Indeed, the high frequency of PTEN inactivation in T-ALL results in constitutive activation of Akt (43), andresistance to TKI–induced apoptosis in BCR/ABL-positiveleukemia has been associated with PTEN downregulation(44). Phosphorylation/activation of Akt results in upregula-tion of the glucose transporter GLUT1 and promotes thetranslocation of hexokinase into the mitochondria (45). Ithas also been reported that in addition to UPR, ER stressactivates parallel endogenous cell survival mechanisms thatdirectly buffer apoptotic signals through the Akt and ERKpathways (29) and via the antiapoptotic proteins cIAP-2 andXIAP (46). We show that the inhibition of Akt in 2-DG–treated ALL cells results in synergistic cell death. Moreover,both the pharmacologic and genetic inhibition of Akt led tosignificant upregulation of AMPK activity and concomitantdownregulation of UPR factors in 2-DG–treated cells. It hasbeen shown that Akt can negatively regulate AMPK activityin various cell models (47–49) and that AMPK can act as asuppressor of UPR activity (9).

We propose that it is the inhibition of the prosurvivalUPRresponse byAMPK that significantly potentiates apoptosis inresponse to 2-DG treatment, rather than CHOP-mediatedapoptosis, induced in response to prolonged UPR andextensive ER stress. Indeed, shRNA-mediated inhibition ofAMPK rescued ALL cells from 2-DG–induced cytotoxicityand correlated with restoration of the essential UPR-signal-ing factors. Therefore, restoration of effective UPR is neededfor ALL cells to buffer and reduce the proapoptotic signalsmediated by sustained 2-DG–induced ER stress. The pro-survival role of the UPR in determining the cellular responseto 2-DG in ALL was further confirmed using shRNAinhibition of GRP78, which also sensitized ALL cells to2-DG.On the basis of these data, we propose amodel for themechanism of 2-DG–induced cell death via ER stress/UPR-mediated apoptosis in which AMPK and Akt modulate theUPR (Fig. 6).In summary, we show for the first time that 2-DG (at low

dose) induces cell death in ALL cells under normoxia bytriggering ER stress/UPR-mediated apoptosis. The sensitiv-ity of ALL cells to 2-DGunder normoxia places them amongthe few cancer phenotypes that behave this way, which isimportant when considering 2-DG as a potential antileu-kemic agent as leukemia cells circulate freely through nor-moxic and hypoxic environments. The resistance to glyco-lytic inhibitors observed in PTEN-mutant T-ALL cellsunderscores the role of Akt signaling as a prosurvival pathwaythat buffers ER stress/UPR-mediated cell death. The cross-talk between the AMPK and Akt pathways and glucosemetabolism represents synthetically lethal interactions andsupports further consideration of 2-DG, alone or in com-bination with selected targeted agents, as an antileukemicagent for future translation into clinical trials for resistant/refractory or relapsed ALL.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. DeSalvo, J.N. Kuznetsov, J. Du, G.M. Leclerc, G.J.Leclerc, T.J. Lampidis, J.C. BarredoDevelopment ofmethodology: J.DeSalvo, J.N.Kuznetsov, J.Du,G.M. Leclerc, G.J.Leclerc, T.J. Lampidis, J.C. BarredoAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. DeSalvo, J.N. Kuznetsov, J. Du, G.M. Leclerc, G.J. Leclerc, T.J.LampidisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): J. DeSalvo, J.N. Kuznetsov, J. Du, G.M. Leclerc, G.J. Leclerc, T.J.LampidisWriting, review, and/or revision of the manuscript: J. DeSalvo, J.N. Kuznetsov, G.M. Leclerc, G.J. Leclerc, T.J. Lampidis, J.C. BarredoAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. DeSalvo, J. Du, G.M. Leclerc, G.J. Leclerc, T.J. LampidisStudy supervision: G.M. Leclerc, G.J. Leclerc, T.J. Lampidis, J.C. Barredo

Grant SupportThis work was supported by STOP Children's Cancer Fund and the Women's

Cancer Association of the University of Miami, FL.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received February 29, 2012; revised May 9, 2012; accepted May 9, 2012;published OnlineFirst June 12, 2012.

2-DG

P-AMPK

GRP78

P-AKT

CHOP

Apoptosis Cytosol

ER Lumen

ATPN-linkedglycosylationinhibition

ER stress(aberrant, faultyglycoproteins)

Blocked UPR(Akt inhibition andincreased AMPK)

IRE1α PERKATF6

ProlongedUPR

CHOP-mediatedapoptotic signaling

Glycolysisinhibition

Figure 6. Proposed mechanism of action for 2-DG in ALL cells undernormoxia. 2-DG interferes with N-linked glycosylation, leading toaccumulation of misfolded glycoproteins, which triggers ER stress andinduction of UPR. Binding of the misfolded glycoproteins to the centralUPR regulator GRP78will result in its dissociation from theUPReffectors(IRE1a, ATF6, and PERK), allowing them to fully engage UPR. GRP78also acts a major chaperone during protein folding, removes excessmisfolded proteins from the ER lumen for proteosomal degradation, andfunctions to suppress proapoptotic pathways triggered by CHOP.Separately, 2-DG inhibits glycolysis, decreases ATP levels, and leads toactivation of AMPK. Our data show that AMPK activation blocksexpression of UPR signaling and significantly potentiates apoptosis in 2-DG–treated ALL cells. Akt promotes UPR signaling and cell survival viadownregulation of AMPK. Therefore, we hypothesize that it is theinhibition by AMPK of the prosurvival GRP78-mediated UPR signalingthat significantly potentiates apoptosis in response to 2-DG plus AIXtreatment, rather than the classical CHOP-mediated apoptotic signalinginduced by extensive ER stress and prolonged UPR.

DeSalvo et al.





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2012;10:969-978. Published OnlineFirst June 12, 2012.Mol Cancer Res Joanna DeSalvo, Jeffim N. Kuznetsov, Jianfeng Du, et al. Downregulation of UPR in Acute Lymphoblastic Leukemia

Induced Apoptosis via−Inhibition of Akt Potentiates 2-DG

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