targetingglutaminemetabolismandredoxstate for …...2019/04/25 · 1department of biochemistry and...

Translational Cancer Mechanisms and Therapy

Targeting Glutamine Metabolism and Redox Statefor Leukemia TherapyMarkA.Gregory1,TravisNemkov1, HaeJ. Park1,VadymZaberezhnyy1, SarahGehrke1,Biniam Adane2, Craig T. Jordan2, Kirk C. Hansen1, Angelo D'Alessandro1, andJames DeGregori1

Abstract

Purpose: Acute myeloid leukemia (AML) is a hematologicmalignancy characterized by the accumulation of immaturemyeloid precursor cells. AML is poorly responsive toconventional chemotherapy and a diagnosis of AML isusually fatal. More effective and less toxic forms of therapyare desperately needed. AML cells are known to be highlydependent on the amino acid glutamine for their survival.These studies were directed at determining the effectsof glutaminase inhibition on metabolism in AML andidentifying general weaknesses that can be exploitedtherapeutically.

Experimental design: AML cancer cell lines, primary AMLcells, and mouse models of AML and acute lymphoblasticleukemia (ALL) were utilized.

Results: We show that blocking glutamine metabolismthrough the use of a glutaminase inhibitor (CB-839) signifi-

cantly impairs antioxidant glutathione production inmultipletypes of AML, resulting in accretion of mitochondrial reactiveoxygen species (mitoROS) and apoptotic cell death.Moreover,glutaminase inhibition makes AML cells susceptible to adju-vant drugs that further perturbmitochondrial redox state, suchas arsenic trioxide (ATO) and homoharringtonine (HHT).Indeed, the combination of ATO or HHT with CB-839 exacer-bates mitoROS and apoptosis, and leads to more completecell death in AML cell lines, primary AML patient samples, andin vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicatingALL cells in vitro and in vivo.

Conclusions: Targeting glutamine metabolism in combi-nation with drugs that perturb mitochondrial redox staterepresents an effective and potentially widely applicable ther-apeutic strategy for treating multiple types of leukemia.

IntroductionAcute myeloid leukemia (AML) is a genetically heteroge-

neous disease, which is characterized by the aberrant growthof myeloid progenitor cells with a block in cellular differenti-ation. AML is the most common adult acute leukemia andcomprises 20% of childhood leukemias (1, 2). AML is anaggressive malignancy and long-term (5 year) survival isless than 50% even for younger patients that can withstandintensive cytotoxic chemotherapy. Outcomes are far worse inelderly and less fit younger patients who can only receive lessintensive chemotherapy (median survival < 1 year; ref. 3).Significant improvement of AML patient outcomes will requirethe development of new therapeutic strategies that are both

more effective and less toxic than currently used regimens.Although in recent years there have been significant advancesin our understanding of AML biology and the various geneticalterations that drive the disease, this knowledge has not, as ofyet, led to development of targeted therapies that are capableof achieving long-term remissions. Moreover, for targeted ther-apies that have shown some modicum of clinical efficacy, suchas those targeting FLT3 and IDH2, their utility is limited tosubsets of AML that exhibit mutations in these genes (2, 4).

It has long been known that tumor cells rewire their metabo-lism to enable and sustain cell growth and proliferation andreprogrammed cellular metabolism is now recognized as a hall-mark of cancer (5). It is feasible that the unique metabolicdependencies of tumor cellsmaybe targeted for therapeutic effect.Many types of tumor cells, including AML, are highly dependenton glutamine for glutaminolysis, a mitochondrial pathway thatinvolves the initial deamidation of glutamine by the enzymeglutaminase (GLS andGLS2), yielding glutamate, which can thenbe converted into a-ketoglutarate to fuel the tricarboxylic (TCA)cycle or used as a precursor for synthesis of glutathione (GSH), animportant antioxidant factor (6–9). Our previous work hasshown that inhibition of FLT3 in FLT3-mutated AML shuts downglutamine metabolism, leading to depletion of GSH and celldeath by apoptosis due to overwhelmingmitochondrial oxidativestress (10). In addition, we and others have shown that inhibitionof glutaminase in FLT3-mutated AML has similar effects as FLT3inhibition on GSH levels and redox state. (11, 12).

In this study, we sought to determine if glutaminase inhibitioncould universally compromise GSH metabolism and redox

1Department of Biochemistry and Molecular Genetics, School of Medicine,University of Colorado Anschutz Medical Campus, Aurora, Colorado. 2Divisionof Hematology, University of Colorado Anschutz Medical Campus, Aurora,Colorado.

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

Corresponding Authors: James DeGregori, University of Colorado AnschutzMedical Campus,Mail Stop8101; P.O. Box6511,. 12801 East 17thAveue,Aurora, CO80045. Phone: 303-724-3230; Fax: 303-724-3215; E-mail:[email protected]; and Mark A. Gregory,[email protected]

doi: 10.1158/1078-0432.CCR-18-3223

�2019 American Association for Cancer Research.

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state in multiple types of AML and thus demonstrate broadtherapeutic potential. Indeed, we found that a glutaminase inhib-itor (CB-839) inhibits GSH production, induces mitochondrialreactive oxygen species (mitoROS) and causes apoptotic celldeath in many types of AML, as well as in acute lymphoblasticleukemia (ALL). Moreover, we demonstrate that combining aglutaminase inhibitor with a drug that further induces mitoROSand apoptosis is an effective therapeutic strategy for eliminatingleukemia cells both in vitro and in vivo.

Materials and MethodsCell culture

The AML cell lines Molm13 and MV4-11 (FLT3-ITDþ), EOL-1(FLT3WT but FLT3-dependent; ref. 13), HL-60, Kasumi-1,OCI-AML3, THP-1, and U937 were obtained from D. Graham(Emory University SOM). MonoMac6 (FLT3V592A) was obtainedfrom K. Bernt (University of Colorado SOM). KG1a was obtainedfromGail Eckhardt (University of Texas at Austin Dell SOM). TheCML cell lines K562 and KU812 and ALL cell line SUP-B15 werepurchased from ATCC. Z-119 Phþ ALL cells were obtained fromZ. Estrov (MDAnderson). All cell lineswere cultured inRPMI1640medium w/10% FBS, except for HL-60 [Iscove's ModifiedDulbecco's Medium (IMDM)/20% FBS], K562 (IMDM/10%FBS)and SUP-B15 and Z-119 (RPMI1640/20% FBS with 5 mmol/Lb-mercaptoethanol). Cell lines were authenticated by short tan-dem repeat examination and tested negative for mycoplasmausing the e-Myco plus PCR Detection Kit (iNtRON) in January2017. Murine Bcr-Ablþ ARF�/� ALL cells (14) were cultured inRPMI1640 medium/10% FBS and 2� L-glutamine and murineMLL-ENL/FLT3-ITDþ AML cells (15) in 50% IMDM/RPMI1640with 20% FBS prior to inoculation into mice.

Metabolic tracing experimentsCells were seeded at 3 � 105/mL (replicates of 3) and treated

with vehicle (DMSO) or CB-839 at 500 nmol/L for 8 hours,followed by incubation in glutamine-free RPMI1640 supplemen-ted with 13C5,

15N2-labeled L-glutamine at 300 mg/L (CambridgeIsotope Laboratories) for up to 12hours, in the presence of vehicle

or drug. Flash frozen cell pellets (�1 � 106 cells) or supernatants(50 mL) were extracted and subjected to analysis by ultra-highpressure liquid chromatography and mass spectrometry(UHPLC/MS) as was previously described (16). Metaboliteassignments, isotopologue distributions, and correction forexpected natural abundances of 13C and 15N isotopes wereperformed using MAVEN (Princeton University, Princeton, NJ)and manually validated.

ROS and GSH measurementsCells (1� 105) were treated with drug as indicated for 20 to 24

hours, washed in PBS, resuspended in 150 mL of 10 mmol/LMitoPY1 (Tocris Bioscience) or 5 mmol/L CM-H2DCFDA(Invitrogen) in serum-free media, and incubated at 37�C for 60to 90minutes (forMitoPY1) or 15minutes (for H2DCFDA). Cellswerewashed in cold FACSbuffer, resuspended in FACSbuffer andimmediately analyzed by flow cytometry. To measure reducedGSH levels, the GSH-Glo Assay (Promega) was utilized accordingto the manufacturer's instructions. GSH data were normalizedto cell number.

Cell viability assaysCells were seeded at 0.5 to 1.0 � 105/mL in triplicate wells of

48-well tissue culture plates. Where indicated, the cells weretreated with drug for a period of 48 to 72 hours. After treatment,a sample of cells from each well was stained with propidiumiodide (PI; 10 mg/mL) and viable cells (PI�) were counted with aflow cytometer (Millipore Guava easyCyte 8HT). Alternatively,cells were stained using 7-aminoactinomycin D (7-AAD)/anti-Annexin V (Nexin reagent, Millipore Sigma) to detect apoptoticcells. Adenosine triphosphate (ATP) levels were measured usingthe Cell Titer-Glo Assay (Promega). ATP data were normalized tocell number.

Mouse experimentsNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)were obtained fromThe

Jackson Laboratory and bred in house. The patient sample forxenograft (obtained from D. Pollyea, University of ColoradoSOM) came from a 54-year-old female with AML expressingFLT3-ITD andNPM1mutations. The leukemia was engrafted intofemale NSG mice as previously described (10). Treatment wasstarted when peripheral blast count was 3% to 10% (mean of 6%for all groups). For monitoring leukemic burden in mice, bloodwas stained with anti-human CD45-FITC and HLA-ABC-PE-Cy7(BD Biosciences) and analyzed by flow cytometry. For theMLL-ENL/FLT3-ITD AML and Bcr-Ablþ ALL models, 6- to10-week-old female C57BL/6 mice (National Cancer Institute)were utilized. C57BL/6 mice were inoculated intravenouslywith 1 � 106 MLL-ENL/FLT3-ITD AML luciferase cells or5 � 105 ARF�/� p185 Bcr-Abl/GFP B-ALL cells. After 3 to 5 days,treatment was started. Treatment at the indicated doses of drugswas 5 days on/2 days off. In the ALLmodel, leukemic burden wasmonitored by quantifying GFP/B220þ and Mac1� blasts in theperipheral blood (PB) as previously described (17). In theAMLmodel, leukemic burden was monitored by bioluminescentimaging using a Xenogen IVIS200 imaging device and standardprotocols. For in vivo delivery, CB-839 was solubilized in 25%HP-b/10 mmol/L citrate pH 2.0 (vehicle), and ATO and HHT insterile saline. The Institutional AnimalCare andUseCommittee atUniversity of Colorado approved all mouse experiments underprotocol No. 00170.

Translational Relevance

AML is a difficult to treat leukemia for which more effectiveand better-tolerated therapies are required. Here, we demon-strate that glutaminase inhibition using CB-839 generallyimpairs redox metabolism in AML cells by limiting synthesisof antioxidant glutathione, resulting inmitochondrial reactiveoxygen species (ROS) accumulation and apoptosis. Moreover,we demonstrate that CB-839 makes AML cells more sensitiveto anti-leukemic drugs that induce mitochondrial oxidativestress, for example ATO andHHT. The combination of ATO orHHT with CB-839 exacerbates mitochondrial ROS and apo-ptosis, and leads tomore complete leukemic cell eradication inmodels of both AML and another type of leukemia, ALL. Thisnovel strategy of targeting glutamine metabolism in combi-nation with drugs that perturb mitochondrial redox statecould represent a very effective and broadly applicable ther-apeutic strategy for treating multiple types of leukemia andpotentially other glutamine addicted cancers.

Gregory et al.

Clin Cancer Res; 2019 Clinical Cancer ResearchOF2




Statistical analysisData are expressed as the mean � SEM. Comparisons between

2 values were performed by Student t test (unpaired 2-tailed) orby Mann–Whitney U test (for analysis of in vivo experiments),unless otherwise indicated. Significance was defined at �P� 0.05,��P � 0.01, ��P � 0.001. Combination indices were calculatedusing the median-effect principle and the Combination Index-Isobologram Theorem (CompuSyn software).

ResultsOur previous studies have shown that impeding glutamine

uptake or metabolism using either a FLT3 inhibitor or glutamin-ase inhibitor, respectively, impairs antioxidant defenses by lim-iting GSH synthesis and leads to the accumulation of mitoROS inFLT3-mutated AML cells (10, 11). FLT3, however, is mutated inonly a subset of AMLs. Thus, we wanted to determine if gluta-minase inhibition could similarly impair glutamine/GSHmetab-olism in AML cells of other genotypes, indicating the potentialfor broader therapeutic utility. To this end, we usedmetabolic fluxanalysis using 13C,15N-labeled glutamine (Supplementary Fig.S1A) in 2 FLT3 wild-type (WT) AML cell lines, OCI-AML3 andEOL-1. These cells were treated with vehicle or the glutaminaseinhibitor CB-839 for 8 hours followed by incubationwith labeledglutamine (up to 12 hours). As shown in Figure 1A, the cellstreated with CB-839 displayed severe impairment of glutamineflux into glutamate and its downstream metabolites aspartate,alanine and a-ketoglutarate. Because a-ketoglutarate (a-KG)feeds into the TCA cycle, flux into TCA cycle intermediates(succinate, malate, and citrate) was severely reduced. In addition,we observed decreased flux into purine nucleotide synthesisintermediates (glycine and aspartate) and the adenylate pool(AMP, ADP, and ATP; Supplementary Fig. S1B). As previouslyobserved in FLT3 mutated AML (11), CB-839 also severelyimpaired flux into antioxidant GSH, leading to lower overall GSHlevels (Supplementary Fig. S1C).

The observed depletion of a-KG and GSH could both contrib-ute to CB-839-induced apoptosis, through energy (ATP) deple-tion due to TCA cycle/adenosine deficiency and/or through oxi-dative stress due to lack of antioxidant capacity. In order to parsethe importance of the TCA cycle for cell survival, we addeddimethyl-glutarate to EOL-1 cells upon treatment with CB-839.Dimethyl-glutarate is a membrane-permeable dimethyl-ester ofa-KG, which is metabolized to a-KG by cellular esterases. Asshown in Figure 1B, a-KG was capable of reversing ATP depletioncaused byCB-839 and also substantially suppressed the inductionof apoptosis. Unexpectedly, however, a-KG also substantiallysuppressed the induction of mitoROS caused by CB-839, indi-cating that exogenous a-KG may be used for generation andmaintenance of GSH. Indeed, a-KGwas able to prevent depletionof GSH in EOL-1 cells upon treatment with CB-839 (Supplemen-tary Fig. S1D). Toparse the importance ofGSH for cell survival, weadded cell-permeable GSH (GSH-MEE) to EOL-1 cells upontreatment with CB-839. GSH-MEE was completely incapable ofrescuing ATP levels (Fig. 1C) and whereas treatment with exog-enous GSH-MEEwas only capable of partial maintenance of GSHlevels (Supplementary Fig. S1D), this was sufficient to significant-ly suppress induction of mitoROS and subsequent apoptosis(Fig. 1C). These results suggest a prominent role for GSH deple-tion and the induction of mitoROS in the causation of apoptosisand AML cell death upon glutaminase inhibition.

To determine if GSH depletion is a universal consequence ofglutaminase inhibition in AML, we treated a panel of AML celllines (EOL-1, Molm13, MonoMac6, MV4-11, HL-60, Kasumi-1,OCI-AML3, and THP-1), along with U937 (lymphoma), andK562 and KU812 (chronic myeloid leukemia, CML) cell lineswith CB-839 for 24 hours. As shown in Fig. 2A, CB-839 caused areduction in GSH levels (normalized to untreated cells; unnor-malized data shown in Supplementary Fig. S2A) in all AML celllines and in the blast crisis CML cell line, KU812, whereas this wasnot observed in U937 or K562 cells. Moreover, the degree of GSHdepletion closely mirrored the amount of mitoROS (Fig. 2B) andapoptosis (Fig. 2C) induction caused by CB-839 treatment. Onthe contrary, therewas no correlationwith overall cellular levels ofreactive oxygen species (Supplementary Fig. S2B). These resultsagain demonstrate thatmaintenance ofmitochondrial redox stateis critical for AML cell survival. Obviously, some AML cell linesdisplayed greater sensitivity to glutaminase inhibition thanothers. In attempts to find a biomarker of glutaminase inhibitorsensitivity, we examined expression of glutaminase (GLS), c-MYC,a major regulator of glutamine metabolism (18), and glutamate-cysteine ligase (GCLC), the rate-limiting enzyme of GSH synthe-sis; however, we found no correlation between expression of thesegenes and CB-839 sensitivity (Supplementary Fig. S2C). None-theless, all of the AML cell lines showed at least a significantreduction in GSH levels upon CB-839 treatment, revealing aweakness that could potentially be exploited by a novel combi-nation therapeutic strategy (Fig. 2D): glutaminase inhibition,while able to induce some degree of mitoROS and apoptosis byitself through inhibition of GSH production, may sensitize AMLcells to a secondoxidative stressor, leading tooverwhelming levelsof mitoROS andmore complete killing of leukemia cells. Indeed,we found that CB-839 sensitizes Molm13 AML cells to the killingeffect of H2O2 (Supplementary Fig. S2D).

To further explore the therapeutic strategy described above,we tested the efficacy of arsenic trioxide (ATO) combined withCB-839onAMLcell lines in vitro. ATO is a chemotherapy drug thatpromotes generation of ROS andmitochondrial apoptosis, and ithas longbeen known tohave antileukemic activity (19). As shownin Fig. 3A, ATO by itself induces mitoROS in a dose-dependentmanner; however, ATO induces higher levels of mitoROS whenused in combination with CB-839. Moreover, ATO and CB-839synergized in killing EOL-1 cells (Fig. 3B). Combination index(CI) values were synergistic (<0.6) at all combination doses testedand were strongly synergistic (<0.3) with most combinations (allCI values listed in Supplementary Table S1). Effects on viabilitycorrelated with increased apoptosis (Fig. 3C). Comparable resultswere obtained using Molm13 cells; whereas ATO did not induceappreciable mitoROS in these cells by itself, it strongly inducedmitoROS when used in combination with CB-839 (Fig. 3D),which correlated with increased cell killing (Fig. 3E) and apopto-sis (Fig. 3F). As shown in Supplementary Fig. S3A and S3B, thelipophilic antioxidant vitamin E markedly suppressed bothmitoROS accumulation and subsequent apoptosis, suggestingthat mitoROS induced by CB-839/ATO treatment is causative inapoptotic cell death. This drug combination had similar effects oncell viability and apoptosis in several additional AML cell lines,including OCI-AML3, MV4-11, KG1a, HL-60, and MonoMac6(Figs. S4A-F); however, this combination did not have consider-able synergistic effects on the CB-839-refractory cell line K562(Supplementary Fig. S4G). Additionally, CB-839 and ATO hadsynergistic effects on cell viability using 2 human primary AML

Glutaminase/Redox-Targeted Therapy for Leukemia

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Figure 1.

The glutaminase inhibitor CB-839 impairs energy and redox metabolism in FLT3WT AML cells. A, Glutamine flux analysis of OCI-AML3 and EOL-1 AML cellstreated with vehicle (DMSO) or CB-839 (500 nmol/L). Cells were pretreated for 8 hours followed by 13C,15N-glutamine labeling for 0, 3, 6, and 12 hours, asindicated, followed by UHPLC/MS analysis. Levels of 13C and 15N containing isotopologues (based on signal intensity) are shown. MþXþY¼molecular ion peakþNo. of labeled 13Cþ No. of labeled 15N. B and C, EOL-1 cells were treated with CB-839 (0, 0.5, or 1 mmol/L, as indicated) with simultaneous addition of cell-permeable a-KG (4mmol/L a-KG; B, or cell-permeable GSH (4mmol/L GSH; C, for 24 hours and ATP levels and % of cells positive for MitoROS (detected usingthe dye MitoPY1) were measured. Apoptosis was measured after 48 hours. For B and C, asterisks indicate statistical significance for comparisons of a-KG or GSHtreated cells with control cells (0 a-KG or GSH) under the same CB-839 treatment conditions.

Gregory et al.





samples (Supplementary Figs. S4H and S4J; SupplementaryTable S1), which corresponded with the induction of mitoROS(Supplementary Figs. S4I and S4K). The clinical characteristics ofall primary patient samples used in this study are summarized inSupplementary Table S2.

We next wanted to test the therapeutic efficacy of CB-839combinedwithATO in vivo. For this purpose, we used a geneticallyengineered mouse model of MLL-ENL/FLT3-ITD-initiatedAML (15). Briefly, fetal liver cells isolated from C57Bl/6 (B6)mice were transduced with retroviruses encoding MLL-ENL andFLT3-ITD together with luciferase. Transplantation of these cellsgenerates a myeloid leukemia similar to human AML. Theseprimary mouse AML cells, which can be cultured in vitro, are thencapable of transferring disease to unconditionedB6 recipientmicewith high penetrance. Three days after transferring the primaryAML cells to recipient mice, therapy was initiated using vehicle,ATO, CB-839, or CB-839 and ATO in combination (n¼ 5 for eachgroup). Leukemic burden was quantified using bioluminescentimaging on days 4 and 11 after the start of therapy (Fig. 3G).By day 11, leukemic burden was significantly lower in the com-

bination treated mice compared with the groups treated witheither ATO or CB-839 alone (Fig. 3G and H), indicating superiortherapeutic efficacy.

To determine if CB-839 will effectively combine with anotherdrug that induces mitochondrial ROS, we next explored homo-harringtonine (HHT). HHT is a protein translation inhibitorindicated for the treatment of tyrosine kinase inhibitor-resistantCML (20), which was recently found to effectively combine withFLT3 inhibitors for the treatment ofAML (21).Wehavepreviouslyshown that drugs that combine well with FLT3 inhibitors arefrequently potent inducers of mitoROS (10). Indeed, we foundthat HHT strongly induced mitoROS in Molm13 AML cells andmitoROS was exacerbated when HHT was used in combinationwith CB-839 (Fig. 4A). The CB-839/HHT combination alsocaused a significant increase in levels of total cellular ROS (Sup-plementary Fig. S5A). Moreover, HHT strongly synergized withCB-839 in killing Molm13 cells (Fig. 4B; Supplementary TableS2), which correlated with induction of apoptosis (Fig. 4C). Asshown in Supplementary Fig. S3C and S3D, vitamin E signifi-cantly suppressed both mitoROS accumulation and apoptosis,

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The glutaminase inhibitor CB-839 decreases GSH levels, and induces mitoROS and apoptosis in AML cells. A–C, The AML cell lines EOL-1, Molm13, MonoMac6,MV4-11, HL-60, Kasumi-1, OCI-AML3, and THP-1, and the lymphoma-derived cell line U937 and the CML cell lines K562 and KU812 were treated with CB-839 (0,0.5, or 1 mmol/L, as indicated) and GSH (A) andmitoROS (B) were measured after 24 hours using MitoPY1. Apoptosis was measured after 48 hours (C). D,Modelshowing mechanism of redox-directed combination therapy for treating leukemia. Use of a glutaminase inhibitor (drug 1, e.g., CB-839) blocks glutaminemetabolism and production of antioxidant GSH andmakes leukemia cells vulnerable to a pro-oxidant agent (drug 2, e.g., ATO or HHT), leading to superinductionof mitoROS and subsequent leukemia cell death via apoptosis. For A, B, and C, asterisks indicate statistical significance for comparisons of CB-839 treated cellsto untreated cells (0 CB).






suggesting thatmitoROS induced uponCB-839/HHT treatment iscausative in apoptotic cell death. Similar effects on cell viabilityusing this drug combination were observed in the EOL-1 cells(Fig. 4D). To determine if there is something distinct about theAML cells that survive CB-839/HHT or ATO therapy, Molm13cells were treated with these drugs for 72 hours (effects oncell viability are shown in Supplementary Fig. S5B) and then

removed from drug and allowed to grow out for 6 days (Note thatfor cells treated with CB-839 combined with 0.4 mmol/L ATO or10 nmol/L HHT, no surviving cells could be recovered.). Supple-mentary Figs. S5C to S5E shows that cells surviving drug therapyhad similar levels of ATP, GSH, and mitoROS. Moreover, whenthese cellswere retreatedwith the samedrugs (Supplementary Fig.S5F), the effects on viabilitywere very similar to the effects seen on

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CB-839 cooperates with the pro-oxidant drug ATO in inducing mitoROS, apoptosis, and AML cell elimination in vitro and in vivo. EOL-1 (A–C) or Molm13 (D–F)were treated with CB-839 and ATO alone or together as indicated (increasing concentrations of ATO of 0, 0.2, 0.4, and 0.8 mmol/L indicated by triangles) andmitoROS were measured using MitoPY1 (A and D). After 72 hours, the number of viable cells (based on PI exclusion) was counted by flow cytometry (B and E)and apoptosis was measured after 48 hours (C and F).G and H,MLL-ENL/FLT3-ITDþ luciferase expressing mouse leukemia cells were transplanted into Bl6 miceand after 3 days treatment was initiated with vehicle (n¼ 5), CB-839 (n¼ 5; 200mg/kg twice daily p.o.), ATO (n¼ 5; 8 mg/kg once daily i.p.), or CB-839 andATO in combination (n¼ 5) for up to 11 days (4 days on/2 days off/5 days on therapy). Leukemic burden was measured by bioluminescence using an in vivoimaging system (IVIS) on the indicated days (G). H, IVIS images from day 11. For A, C, D, and F, asterisks indicate statistical significance for comparisons ofCB-839 treated cells with control cells (0 CB) under the same ATO treatment conditions.

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HHT induces mitoROS and cooperates with CB-839 in eliminating AML cells in vitro and in vivo. A–C, Molm13 cells were treated with CB-839 and HHT alone ortogether as indicated for 20 hours (triangles: 0, 5, 10, and 20 nmol/L HHT) andmitoROS were measured using MitoPY1 (A). After 48 hours, the number of viablecells (based on PI exclusion) was counted by flow cytometry (B) and apoptosis was measured after 24 hours (C). D, EOL-1 cells were treated with CB-839 andHHT alone or together as indicated for 72 hours (triangles: 0, 5, 10, and 20 nmol/L HHT) and the number of viable cells (based on PI exclusion) was counted byflow cytometry. E,MLL-ENL/FLT3-ITDþ luciferase expressing mouse leukemia cells were transplanted into Bl6 mice and after 4 days, treatment was initiatedwith vehicle (n¼ 5), CB-839 (n¼ 5; 200mg/kg twice daily p.o.), HHT (n¼ 5; 1 mg/kg once daily i.p.), or CB-839 and HHT in combination (n¼ 5) for up to 15 days(5 days on/2 days off therapy). Leukemic burden was measured by bioluminescence using an in vivo imaging system (IVIS) on the indicated days. F, IVIS imagesfrom day 8. For A and C, asterisks indicate statistical significance for comparisons of CB-839 treated cells with control cells (0 CB) under the same HHT treatmentconditions.






drug naive cells (Supplementary Fig. S5B). Thus, it would seemthat there is nothing unique about the cells that survive therapyand they are not inherently therapy-resistant.

To test the efficacy of the CB-839/HHT combination in vivo, weutilized the MLL-ENL/FLT3-ITD AML mouse model describedabove. Five days after leukemia transfer, therapy was initiatedusing vehicle, CB-839, HHT, or CB-839 andHHT in combination(n ¼ 5 per group). As shown in Fig. 4E, by day 8 of therapy,leukemic burden was significantly lower in the combinationgroup comparedwith the CB-839 andHHT groups, and remainedsignificantly lower on day 15 (Fig. 4E and F). These resultsdemonstrate the therapeutic efficacy of the CB-839 plus HHTcombination in vivo, at least in thismodel of AML. To determine ifCB-839/HHT or CB-839/ATO therapy has any toxic effects inmice, B6 mice were treated for 2 weeks with these therapies andblood tests were performed. Complete blood count (CBC) anal-ysis showed that CB-839/HHT and CB-839/ATO did cause some

degree of hematologic suppression, including modest, yet statis-tically significant, reductions in hemoglobin, red blood cells,platelets, white blood cells, and lymphocytes (SupplementaryFig. S6A); however, all of these counts remained within thenormal range (Charles River Mouse Biochemistry Reference).Markers of kidney and liver toxicity were not elevated (Supple-mentary Fig. S6B) and there was no significant weight loss in themice over the course of therapy (Supplementary Fig. S6C). Fur-thermore, these drug combinations did not show any consider-able effects on the viability of normal human CD34þ hemato-poietic progenitor cells (Supplementary Fig. S6D).

To determine if CB-839 plus HHT shows antileukemic activityin human primary AML cells, we tested the effects of this com-bination on 4 AML patient samples. As shown in Fig. 5A–D andSupplementary Table S1,HHT synergizedwithCB-839 in primaryAMLcell killing. Additionally,we show that thedrugs cooperate ininducing mitoROS (Supplementary Fig. S7A and S7C) and

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CB-839 cooperates with HHT in eliminating primary human AML cells in vitro and in vivo. A–D, Primary AML cells from 4 different patients (626, 111, 115, 041) wereseeded at 5� 105/well and treated with CB-839 and HHT alone or together as indicated for 72 hours (triangles: 0, 5, 10, and 20 nmol/L HHT; 0, 5, and 10 nmol/Lfor 041) and the number of viable cells (based on PI exclusion) was counted by flow cytometry. E, Primary AML cells (AML626) were treated with CB-839 andHHT alone or together as inA for 24 hours and then added to humanmethylcellulose complete media. After 16 days, the number of colony-forming units wascounted. Nonoverlapping confidence intervals indicate statistical significance. F,NSGmice were engrafted with primary human AML cells and groups of micewere treated with vehicle (n¼ 5), CB-839 (n¼ 5; 200mg/kg twice daily p.o.), HHT (n¼ 5; 1 mg/kg once daily i.p.), or CB-839 and HHT in combination (n¼ 10) for25 days. Leukemic burden was monitored by PB draws and quantitation of leukemic cells (human CD45þ, HLA-ABCþ cells) by flow cytometry on the indicateddays is shown.

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CB-839 cooperates with HHT in eliminating ALL cells in vitro and in vivo. A–C, Z-119 B-ALL cells were treated with CB-839 and HHT alone or together as indicatedfor 36 hours (triangles: 0, 5, 10, and 20 nmol/L HHT) andmitoROS were measured using MitoPY1 (A). After 72 hours, the number of viable cells (based on PIexclusion) was counted by flow cytometry (B) and apoptosis was measured after 48 hours (C). D,ARF�/� p185 Bcr-Abl/GFPmouse B-ALL cells weretransplanted into Bl6 mice and after 5 days, treatment was initiated with vehicle (n¼ 5), CB-839 (n¼ 5; 200mg/kg twice daily p.o.), HHT (n¼ 5; 1 mg/kg oncedaily i.p.), or CB-839 and HHT in combination (n¼ 5) for up to 15 days (5 days on/2 days off therapy). After 7 and 10 days of therapy, PB from all mice wasimmunostained for B220 and Mac-1 and analyzed by flow cytometry. The percentage of GFPþ cells in the B-lineage (B220þ, Mac-1�) populationwas determinedand plotted. E, Kaplan–Meier curve showing survival of mice receiving the indicated therapy. Mice were sacrificed when moribund, and all showed clear evidenceof leukemia in blood, bone marrow, and spleen. For A and C, asterisks indicate statistical significance for comparisons of CB-839–treated cells with control cells(0 CB) under the same HHT treatment conditions. For E, statistical significance determined using the log-rank (Mantel–Cox) test.






apoptosis (Supplementary Fig. S7B). To determine if this drugcombination would affect cells capable of forming leukemic cellcolonies, we performed methylcellulose colony-forming unit(CFU) assays. As shown in Fig. 5E, HHT in combination withCB-839 severely suppressed colony formation, indicating anability to eliminate leukemia stem/progenitor cells. To determineif CB-839 and HHT cooperate in eliminating primary AML sam-ples in vivo, we utilized a patient-derived xenograft mouse modelas previously described (10). Briefly, primary AML cells wereengrafted into immunodeficient NSG mice, and after �4 weeks,leukemic burden in the PBwas determined by flow cytometry, themice were divided into groups with approximately equal meanleukemic burden (�6%), and therapy was initiated using vehicle(n ¼ 5), CB-839 (n ¼ 5), HHT (n ¼ 5), or CB-839 and HHT incombination (n ¼ 6). Fig.5F shows that after 11 days of therapy,leukemic burden in PBwas significantly lower in the combinationgroup compared with the individual treatment groups andremained lower out to day 25, again demonstrating the superi-ority of the CB-839/HHT combination in treating AML. Finally,wewanted todetermine ifCB-839plusHHT combination therapycan show efficacy in treating a different type of leukemia, ALL.Thus, we tested the effects of the drug combination using theZ-119 human ALL cell line. Similar to what was observed in AMLcells, HHT induced a greater amount of mitoROS when used incombination with CB-839 (Fig. 6A), which correlated withdecreased cell viability (Fig. 6B) and increased apoptosis(Fig. 6C). Comparable effects on cell viability (SupplementaryFig. S7D) and apoptosis (Supplementary Fig. S7E) were observedusing a second ALL cell line, SUP-B15. To determine if CB-839plus HHT shows efficacy in treating ALL in vivo, we utilized amousemodel of Bcr-Ablþ B-cell ALL as described previously (17).Bl6 mice were transplanted with mouse ARF�/� p185 Bcr-Ablþ

B-ALL cells and after 3 days to allow for engraftment, mice begantherapy with vehicle, HHT, CB-839, or HHT and CB-839 incombination (n¼ 5 per group). After 7 days of therapy, leukemiccells in PB were quantified by flow cytometry (GFPþ/B220þ).(Note that 3 mice in both the vehicle and CB-839 alonegroups had already succumbed to leukemia at this point).Fig. 6D shows that although CB-839 by itself had no significanteffect, HHT in combination with CB-839 was far more effectivethan HHT alone in controlling leukemic burden (as shown ondays 7 and 10), which resulted in significant extension ofsurvival (Fig. 6E). Taken all together, these results strongly arguethat the strategy of combining a glutaminase inhibitor, such asCB-839, together with a pro-oxidant drug, such as HHT or ATO,has substantial therapeutic potential for treating multiple typesof leukemia.

DiscussionIt has long been recognized that tumor cells exhibit meta-

bolic characteristics that distinguish them from normaldifferentiated nonproliferative cells (5, 22, 23). The alteredglucose metabolism that is frequently observed in tumors hasbeen extensively studied and is already being exploited forcancer therapy (24). Altered glutamine metabolism in cancer,however, has only recently begun to be investigated. Glutamineis the most abundant amino acid in plasma and most tumorsutilize glutamine at a much higher rate compared with normalcells. Glutamine metabolism contributes to energy production,macromolecular synthesis, and redox homeostasis, and is

essential for survival of some cancer cells that have becomeaddicted to glutamine (6, 25, 26).

Several recent studies have shown that targeting glutamineuptakeormetabolismcanhaveantileukemic activity inAML(7–9,11, 12). However, most of these studies have focused on the roleof glutamine in supporting energy metabolism rather on its rolein maintaining redox homeostasis. Our recent studies ofFLT3-mutated AML have shown that inhibition of FLT3 severelyimpairs glutamine metabolism, which leads to a deficiency inGSH levels and accumulation of mitoROS that is causativein apoptotic cell death (10). Similar effects were observed inFLT3-mutated AML cells upon direct inhibition of glutamin-ase (11). In this report, we show that a glutaminase inhibitorcauses decreased GSH levels and increased mitoROS andapoptosis in an array of different AML cell lines, including thosethat are FLT3 WT. Furthermore, we show that addition of exog-enousGSH,whichhas no effect on energy production, is sufficientto suppress mitoROS levels and apoptosis upon glutaminaseinhibition, strongly arguing that GSH-dependent redox homeo-stasis is critical for maintaining cell survival in AML. Importantly,this dependence on glutamine/redox metabolism is independentof FLT3 status, and thus glutaminase inhibitors may have broadtherapeutic utility in the treatment of various genotypes of AML.

Numerous redox-directed therapeutic agents have been inves-tigated in recent years for the treatment of cancer, so far withlimited success, at least as monotherapy (27–30). Given thatglutaminase inhibition leads to a compromised redox state, wesurmised that this would sensitize leukemia cells to adjuvant pro-oxidant drugs, leading tomore apoptosis and far greater leukemiacell elimination. Indeed, we show that 2 different FDA-approvedleukemia drugs that are potent inducers of mitoROS, ATO andHHT, have enhanced antileukemic activity in AML when com-bined with the glutaminase inhibitor CB-839, both in vitro and invivo. Additionally, we show that CB-839 in combination withHHT shows efficacy in treating a different form of leukemia, ALL.Moreover, the ability of this combination therapy to eliminateprimary AML colony-forming cells is indicative of the potential toeliminate leukemia stem cells (LSCs). LSCs have been shown torely heavily on upregulated mitochondrial metabolism for theirsurvival (31, 32), and thus these cellsmaybe especially vulnerableto therapies that compromise mitochondrial redox state. Certain-ly, venetoclax in combination with hypomethylating agents(HMA) has been shown to be effective at both eradicating AMLLSCs (33, 34) and augmentingmitoROS and apoptosis inductioncompared with HMA alone (35). There are other hematologicmalignancies, such as multiple myeloma, which have beenreported to be highly dependent on glutamine metabolism forsurvival (36, 37). Furthermore, subsets ofmany solid tumor types,including lung, breast, kidney, and prostate cancer, have beenshown to be susceptible to glutaminase inhibition (38–43).Therefore, the use of a glutaminase inhibitor in combinationwith a pro-oxidant drug could represent a strategy with broadtherapeutic utility in the treatment of various cancers.

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

Authors' ContributionsConception and design: M.A. Gregory, J. DeGregoriDevelopment of methodology: M.A. Gregory, T. Nemkov, V. Zaberezhnyy,B. Adane, K.C. Hansen, A. D'Alessandro

Gregory et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.A. Gregory, T. Nemkov, V. Zaberezhnyy,B. Adane, A. D'AlessandroAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.A. Gregory, T. Nemkov, H.J. Park, S. Gehrke,C.T. Jordan, A. D'Alessandro, J. DeGregoriWriting, review, and/or revision of the manuscript: M.A. Gregory, H.J. Park,S. Gehrke, K.C. Hansen, J. DeGregoriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. ZaberezhnyyStudy supervision: M.A. Gregory, H.J. Park, K.C. Hansen, J. DeGregori

AcknowledgmentsWe thank Dan Pollyea (University of Colorado SOM) for providing primary

AML samples. We thank Timothy Pardee (Wake Forest SOM) and RichardT.Williams (St. Jude Children's ResearchHospital) for theMLL-ENL/FLT3-ITDþ

AML and Bcr-Ablþ ALL mouse models, respectively. Grants from the NIH/NCI

(K22-CA133182) and the Cancer League of Colorado to M.A. Gregory, andthe V Foundation (T2016-012) to J. DeGregori, supported these studies.A. D'Alessandro was supported by funds from the Boettcher Foundation,Webb-Waring Early Career award 2017. Grants from the NIH/NCI(K22-CA133182) and the Cancer League of Colorado to M.A. Gregory,and the V Foundation (T2016-012) to J. DeGregori, supported these studies.A. D'Alessandrowas supported by funds from the Boettcher Foundation,Webb-Waring Early Career award 2017.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 1, 2018; revised March 2, 2019; accepted March 29, 2019;published first April 2, 2019.

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Gregory et al.




Published OnlineFirst April 2, 2019.Clin Cancer Res Mark A. Gregory, Travis Nemkov, Hae J. Park, et al. Leukemia TherapyTargeting Glutamine Metabolism and Redox State for

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