Mitochondrial BAX Determines the Predisposition to ... · translocation of BAX and BAK to the mitochondria establishes an equilibrium between cytosolic and mitochondrial protein pools

Cancer Therapy: Preclinical

Mitochondrial BAX Determines the Predispositionto Apoptosis in Human AMLFrank Reichenbach1,2,3, Cornelius Wiedenmann1, Enrico Schalk4, Diana Becker5,Kathrin Funk1,3, Peter Scholz-Kreisel6, Franziska Todt1, Denise Wolleschak4,Konstanze D€ohner7, Jens U. Marquardt5, Florian Heidel8,9, and Frank Edlich1,10

Abstract

Purpose: Cell-to-cell variability in apoptosis signaling contri-butes to heterogenic responses to cytotoxic stress in clinicallyheterogeneous neoplasia, such as acutemyeloid leukemia (AML).The BCL-2 proteins BAX andBAK can commitmammalian cells toapoptosis and are inhibited by retrotranslocation from the mito-chondria into the cytosol. The subcellular localization of BAX andBAK could determine the cellular predisposition to apoptoticdeath.

ExperimentalDesign: The relative localization of BAXandBAKwas determined by fractionation of AML cell lines and patientsamples of a test cohort and a validation cohort.

Results: This study shows that relative BAX localization deter-mines the predisposition of different AML cell lines to apoptosis.Human AML displays a surprising variety of relative BAX localiza-

tions. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITDstatus, and leukocytosis. Analysis of a validation cohort of 80elderly patients treated with myelosuppressive chemotherapyconfirmed that relative BAX localization correlates with proba-bility of disease progression, FLT3-ITD status, and leukocytosis.Relative BAX localization could therefore be helpful to identifyelderly or frail patients who may benefit from cytotoxic therapy.

Conclusions: In this retrospective analysis of two indepen-dent AML cohorts, our data suggest that Bax localizationmay predict prognosis of patients with AML and cellularpredisposition to apoptosis, combining the actual contribu-tion of known and unknown factors to a final "common path."Clin Cancer Res; 23(16); 4805–16. �2017 AACR.

IntroductionAcute myeloid leukemia (AML) is a disease characterized by

clinical and biological heterogeneity. Recently, genomics havefacilitated insights into the genetic landscape of AML and iden-tified novelmutations with functional implications (1–3). Genet-ic alterations have been described to be of major prognosticrelevance (4–6). Genetic diversity, however, is not the sole deter-

minant of relapse, drug resistance, and aggressiveness of leukemiabiology (7). Large-scale genetic analysis approaches naturally lackinsights on posttranscriptional protein expression, protein regu-lation, and localization, that is, the actual level of activation. Cell-to-cell variability in protein levels and in some cases subcellularlocalization induce large differences in the responsiveness touniform stimuli between individual cells and descendants fromdifferent clones (8–10). Within a clonal cell population, somecells rapidly induce programmed cell death in response to deathreceptor–induced apoptosis, while other cells appear more resis-tant and survive (11). This "intratumoral" heterogeneity and theunderlying differences in cell signaling critically influence cellularpredisposition to apoptosis upon cytotoxic stress (e.g., induced bychemotherapy). Differential sensitivity of malignant cells to che-motoxic stress has become a central challenge in oncology.Therefore, the identification of prognostic factors and the analysisof individual cellular predisposition to apoptosis is a major focusof translational cancer research. Investigation of the apoptoticmachinery may offer novel strategies to sensitize resistant cells tocytotoxic agents.

Analysis of signaling between prosurvival BCL-2 proteins, theproapoptotic BCL-2 proteins BAX and BAK, and BH3-only pro-teins (sharing only the BH3 domain with BCL-2) on the outermitochondrial membrane has been proposed to predict cellularpredisposition to chemotoxic cell death. Cellular cytotoxicresponse is suggested to depend on the presence of prosurvivalBCL-2 proteins and the accumulated BH3-only proteins on themitochondria, termed "mitochondrial priming," resulting per-haps from prior sublethal cell stresses (12–15). Prosurvival BCL-2proteins inhibit BAX and BAK via direct interactions or by

1Institute for Biochemistry and Molecular Biology, University of Freiburg, Frei-burg, Germany. 2Spemann Graduate School of Biology and Medicine, SGBM,Freiburg, Germany. 3Faculty of Biology, University of Freiburg, Freiburg, Ger-many. 4Department of Hematology and Oncology, Medical Center, Otto-von-Guericke University Magdeburg, Magdeburg, Germany. 5Department of Medi-cine I, University of Mainz, Mainz, Germany. 6Institute for Medical Biostatistics,Epidemiology and Informatics, University Mainz, Mainz, Germany. 7Departmentof Internal Medicine III, University Hospital of Ulm, Ulm, Germany. 8InternalMedicine II, Hematology and Oncology, Jena University Hospital and MedicalFaculty, Friedrich-Schiller-University, Jena, Germany. 9Leibniz Institute onAging, Fritz-Lipmann-Institute, Jena, Germany. 10BIOSS, Centre for BiologicalSignaling Studies, University of Freiburg, Freiburg, Germany.

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

Corresponding Authors: Frank Edlich, University of Freiburg, Stefan-Meier-Str.17, Freiburg 79104, Germany. Phone: 4976-1203-97482; Fax: 761-203-5253;E-mail: [email protected]; Florian Heidel, Fritz-Lipmann-Institute, Beutenbergstrasse 11, Jena 07745, Germany. E-mail:[email protected]; and Jens U. Marquardt, University of Mainz,Langenbeckstr. 1, Mainz 55131, Germany. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-1941

�2017 American Association for Cancer Research.

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sequestering "activator" BH3-only proteins, thereby preventingtheir interactionwith BAX and BAK (16–21). In response to stress,BH3-only protein signaling is thought to activate BAX and BAK,which in turn permeabilize the outer mitochondrial membraneand release of cytochrome c (cyt c). Cytosolic cyt c results inmitochondrial dysfunction and initiates the caspase cascade thatefficiently dismantles the cell (22, 23). The activation of BAX orBAK commits the cell to apoptosis necessitating a tight control ofthese proapoptotic BCL-2 proteins (24). In healthy cells, BAX andBAK are controlled by constant retrotranslocation of mitochon-dria-associated protein into the cytosol (25, 26). Permanenttranslocation of BAX and BAK to the mitochondria establishesan equilibrium between cytosolic and mitochondrial proteinpools (25, 27). These processes are dependent on the porinVDAC2, which serves as a platform for BAX retrotranslocation(28). Differential rates of BAX and BAK shuttling determinelocalizations of BAX (predominantly in the cytosol) and BAK(largely on the mitochondria; ref. 26). Retrotranslocation of BAXand BAKdepends on interactionswith prosurvival BCL-2 proteins(25, 26). Importantly, BAX retrotranslocation rates determine thesize of the mitochondrial BAX pool and cellular response toapoptosis stimulation (29). Moreover, BH3-only proteins reducethe rate of BAX retrotranslocation (25), suggesting a reflection ofthe BCL-2 protein signaling on the mitochondria (and "mito-chondrial priming") and perhaps other factors in the equilibriumbetween cytosolic and mitochondrial BAX.

Here, we provide first evidence for the correlation of thesubcellular BAX (and BAK) localization with susceptibility tocytotoxic therapy in human AML cell lines and primary AMLblasts. While BAX shift toward the mitochondria correlates withincreased cellular sensitivity to chemotoxic stress and beneficialprognostic markers, BAX shift toward the cytoplasm rather corre-lates with cellular resistance to chemotherapeutic stress andadverse prognostic markers. Thus, relative BAX localization hasthe potential to combine all relevant factors to assess the cellularsusceptibility to apoptosis induction as one of the "final effectors"of cell death.

Materials and MethodsCell culture

HeLa cells were cultured in DMEM medium supplementedwith 10mmol/LHEPES and 10%heat-inactivated FBS in 5%CO2at 37�C. All AML cell lines and primary AML cells were cultured in

RPMI1640 medium supplemented with 10 mmol/L Hepes, 10%heat-inactivated FBS, 4.5 g/L glucose, and 1 mmol/L sodiumpyruvate in 5% CO2 at 37�C. Cells were regularly (in 4-weekintervals) tested for potential mycoplasma infection using theVenor GeM kit (Biochrom).

Whole-cell lysis and subcellular fractionationFor the standard curve whole-cell lysates, HeLa cell lysate was

used to establish a standardized mix of all analyzed proteins,ensuring similar standardization of all patient samples. HeLa cellswere harvested, washed with PBS, and subsequently resuspendedin cell lysis buffer (20mmol/L Tris, 100 mmol/L NaCl, 1 mmol/LEDTA, 0.5% Triton X-100, pH 7.5) supplemented with proteaseinhibitors. Upon incubation on ice for 15 minutes, the sampleswere centrifuged at 15,000 � g for 10 minutes at 4�C. Thesupernatants were subjected to acetone precipitation, followedby resuspension in SDS sample buffer and storage at �80�C. Toobtain mitochondrial and cytosolic fractions of patient bonemarrow samples,mononuclear cellswere isolated by Ficoll-Paquedensity gradient centrifugation. Cells were washed with PBS,resuspended in RPMI supplemented with 10% DMSO and20% FCS, and stored in liquid nitrogen. Aspirates were thenthawed and centrifuged for 5 minutes at 1,500 � g at 4�C. Themean percentage of blasts in the bone marrow aspirates (n ¼ 44)was 64% (95% CI: 54%–74%). Upon washing with PBS, the cellpellet was resuspended in SEM buffer (10 mmol/L HEPES,250 mmol/L sucrose, pH 7.2) supplemented with protease inhi-bitors and homogenized using the MINILYS (PeqLab) system.Subsequently, sampleswere centrifuged at 1,500� g for 5minutesat 4�C. The supernatant was transferred to a new tube andcentrifuged for 30minutes at 13,000� g at 4�C.While sedimentedmitochondria were washed two times, the supernatant of thisstep, the cytosolic fraction, was ultracentrifuged at 150,000� g for1 hour at 4�C. Finally, the samples were separated by SDS-PAGEand subjected to Western blot analysis.

Quantification of Western blot dataBand intensities were captured using the Fujifilm LAS-4000

imager and quantified using ImageJ. On the basis of a whole-cell lysate standard curve within each individual blot, BAX(E63, Abcam) and BAK (Millipore) were then quantified usingSigmaPlot. The amounts of mitochondrial and cytosolic pro-tein were then determined as relative to COX IV (Invitrogen)and b-actin (Millipore), respectively. Finally, ratios were builtfrom the mitochondrial and cytosolic values to obtain therelative protein localization. Accumulation of the cleaved formof the caspase substrate PARP was analyzed by Western blot(pAB, Cell Signaling Technology) to determine apoptosisprogression.

PatientsForty-eight bone marrow samples of the test cohort were

obtained from the Hematology Tumor Bank Magdeburg (HTM)at theDepartment ofHematology andOncology,Medical Center,Otto-von-Guericke University (Magdeburg, Germany). Thesesamples were obtained after written informed consent of patientsand in accordancewith theDeclarationofHelsinki. This studywasapproved by the institutional review board of the UniversityHospital Magdeburg and the local ethics committee (file#15/2008). The majority of patients were diagnosed with AML(n¼45; 93.8%).Othermyeloid diagnoses includedblast phase of

Translational Relevance

Differential sensitivity of malignant cells to chemotoxicstress based on cell-to-cell variability in cell death signalinghas become a central challenge in oncology. The proapoptoticBCL-2 proteins BAX and BAK can commit human cells toapoptosis and are regulated by dynamic shuttling betweencytosol and mitochondria. The analysis of the relative BAX/BAK localization in human cells and pretreatment patientsamples from two cohorts of patientswithAMLpredict cellulardisposition to apoptosis and treatment response. Our datasuggest relative BAX/BAK localization as potential tumor-based biomarker to predict therapeutic response to cytotoxicstimuli in AML and perhaps other tumor entities.

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chronic myeloid leukemia (n¼ 1), blastic plasmacytoid dendriticcell neoplasm (BPDCN; n ¼ 1), and one patient was diagnosedwith acute biphenotypic leukemia. Three of all samples (6.3%)were from relapsed patients. Analyzing the AML samples (n¼ 45)only, three were taken in relapse (6.7%), and 74.4% (32/43)

were de novo AML. Of these AML samples, 20.9% (9/43) wereFLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplica-tion) positive, and 18.6% (8/43) were at good, 51.2% (22/43)were at intermediate, and 30.2% (13/43) were at poor riskaccording ELN criteria (6), respectively.

Figure 1.

Relative BAX localization predicts therapeutic response in 48 patients with AML. A, Western blot analysis of the mitochondrial and cytosolic pools of BAX andBAK in bone marrow samples of patients with AML. Separation of cytosol and heavy membrane fraction (mitochondria) was controlled using b-actin and COX IV,respectively. The relative BAX and BAK levels were normalized using loading controls and HeLa whole-cell lysate (protein standard). B, BAX (and BAK) constantlytranslocates and retrotranslocates forming an equilibrium between cytosolic and mitochondrial pools dependent on the combined signaling of all involvedprosurvival BCL-2 proteins (red, e.g., BCL-2, Bcl-xL,Mcl-1), proapoptoticBH3-only proteins (green, e.g., Bim, tBid) andproteins outside theBCL-2 family in response tosubtoxic cell stress. This equilibrium was assessed by determining the relative cytosolic protein pool [rel. cytosolic BAX, combining cytosolic BAX (light blue)with the cytosol marker b-Actin (dark gray)] and assigning relative mitochondrial protein [rel. mitochondrial BAX, mitochondrial BAX (dark blue) per mitochondrialmarker COX IV (light gray)]. Relative protein localization results from the quotient of relative mitochondrial protein and relative cytosolic protein. Thus, ashift of the protein to the mitochondria results in a high relative protein localization value, whereas low relative protein values indicate a protein shifttoward the cytosolic pool. C, Relative protein localization (log10 scale) of BAX and BAK in 48 AML patient bone marrow samples. P value according to the t test isdisplayed. D, Relative cytosolic BAX (log10 scale) versus relative mitochondrial BAX (log10 scale) is displayed for 48 patient bone marrow samples. Lack ofcorrelation according to Pearson method is shown by the displayed r and P values. E, Kaplan–Meier survival curves of patients with high (red), intermediate high(gray), intermediate low (black), and low (blue) relative BAX localization values (n ¼ 12 in each group). P value according to odds test is shown.

BAX Determines the Predisposition to Apoptosis

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Figure 2.

Relative BAX localization correlates with clinical markers in 48 patients with AML. A, Relative BAK localization versus relative BAX localization (double log10 scale) isdisplayed for 48 patient bonemarrow samples. r and P values according to Pearson correlation are displayed.B,Distribution ofFLT3-ITD–positive samples in the quartileof the patient collective with low relative BAX localization values (low) or high mitochondrial BAX (25% highest relative BAX localization values, high) or intermediatelow (medL) or intermediate high (medH) relative BAX localization values is displayed (n ¼ 12 in each group). (Continued on the following page.)

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SNV analysesDNA from 40 available samples (low n¼ 8;medL n¼ 9;medH

n ¼ 11; high n ¼ 12) were isolated and genotyped using theIllumina OmniExpressExome 1.2 Chip. The data were analyzedusing Illumina Genome Studio 2011.1 Genotyping Module toobtain normalized genotype data. CNV analysis was carried outusing the CNVpartition 3.2.0 algorithm within Genome Studiousing default parameters. Somatic copy-number alterationswere determined using GISTIC 2.0. Identification of differentialsingle nucleotide variants (SNV) was performed using PLINK(P < 0.001).

Statistical analysisFor the main statistical analysis, Sigma Plot was used. The

logistic regression and ROC calculation was done using SASSoftware 9.4. A significance level of a ¼ 5% was used for testing;however, all P values have explorative character only. For theROS, curve area under curve and 95% confidence intervals werecalculated using the Mann–Whitney method.

Caspase-3/7 assayFor caspase assays, cells were washed with ice-cold 1� PBS and

resuspended in ice-coldCell Lysis Buffer (20mmol/L TRIS pH7.4,100mmol/L NaCl, 1mmol/L EDTA, including protease inhibitorcocktail and 0.5% Triton X-100).Whole-cell lysate was incubatedwith Caspase-3/7 substrate (BD Pharmingen) for 60 minutes at37�C, and protein concentration was determined by a BradfordAssay (Roth). Substrate cleavage was measured for 50 cycles with10-seconddelay (excitation at 380nm, emission at 430–460nm).Kinetics were determined and calculated to the amount of proteinper sample.

Lactate dehydrogenase assayCells were seeded in 96-well plate format with mock-treated

samples and lysis control on the same plate. Cells were treatedwith 1 or 20 mmol/L daunorubicin, 1 mmol/L actinomycin D,100 mmol/L etoposide, 5 mmol/L doxorubicin, 10 mmol/L ABT-737, 10 mmol/L UMI-77, or 1 mmol/L staurosporin for 24 hours.Then, 50 mL supernatant was transferred to fresh a 96-well plateand mixed with substrate (Promega). Lactate dehydrogenase(LDH) activity was monitored over 25 cycles with a 25-seconddelay, and kinetics were calculated.

ResultsHigh mitochondrial BAX levels correlate with improved AMLpatient survival

The regulation of BAX and BAK by steady translocation to themitochondria and retrotranslocation back into the cytosol ofnonapoptotic cells raises the question whether BAX/BAK locali-

zation could predict cellular predisposition to apoptosis. There-fore, the relative distribution of BAX and BAK between cytosolicand mitochondrial pools in primary bone marrow samples ofhuman AML was analyzed in a test cohort by fractionating 48patient samples into cytosol andmitochondria (Fig. 1A). To allowcomparison of samples not analyzed on the same Western blot,relative mitochondrial and relative cytosolic protein was deter-mined using the fractionation loading controls COX IV andb-actin, respectively. All detected proteins were normalized usinga titration of the same HeLa cell extract and thus the sameamounts of all detected proteins for the evaluation of everyanalyzed sample. Both relative protein pools were then combinedto calculate the relative protein localization (relative BAX local-ization ¼ mitochondrial BAX/cytosolic BAX), with high relativeprotein localization values pointing to increased mitochondrialprotein and low values to predominantly cytosolic protein inde-pendent of cellular protein levels (Fig. 1B). Surprisingly, theanalysis of patient bone marrow samples revealed a highinterindividual variability of relative BAX and BAK localizations(Fig. 1C; Supplementary Table S1; Supplementary Fig. S1).Nonetheless, relative BAX localizations show a general trendtowards cytosolic localizations, whereas BAK is present in largermitochondrial pools (Supplementary Fig. S2A). Comparison ofcytosolic and mitochondrial pools of BAX or BAK shows noapparent correlation (Fig. 1D; Supplementary Fig. S2B). Theabsence of correlations between the levels of mitochondrialand cytosolic BAX suggests that BAX localization does notdepend on cellular BAX levels. Also the maintenance of aconstant ratio between mitochondrial and cytosolic BCL-2protein pools is not apparent.

The analysis of test cohort quartiles [low,mostly cytosolic; high,largely mitochondrial, medium high (medH) and medium(medL)] of relative BAX localization reveals a strikingly bettersurvival probability among patients with high relative BAX local-ization (Fig. 1E). Low relative BAX localization values increase theodds of death during treatment by 11.5 times. The samedifferenceis apparent analyzing only patients from the test cohort with denovo AML (Supplementary Fig. S3). On the basis of the similarresponse of patients with intermediate or low relative BAX local-ization, the use of relative BAX localization as continuous value isnot appropriate to distinguish responder and nonresponder(Supplementary Fig. S4). However, categorizing patients withAML according to their relative BAX localization could predicttreatment response.

Cytosolic BAX localization is associated with decreasedpredisposition to apoptosis and FLT3-ITD

Among test cohort patients, relative BAX localization showsa strong positive correlation with relative BAK localization(Fig. 2A). A strong correlation between cytosolic BAX and relative

(Continued.) P value is provided for the relative risk of FLT3-ITD positivity in the different quartiles. C, Relative BAX localization (log10 scale) is displayed forFLT3-ITD–positive (n ¼ 9) and negative (n ¼ 35) patient samples. P value according to one-way ANOVA using Holm–Sidak method is displayed. D, Graphicalrepresentation of the genetic alterations in the respective BAX localizations. Amplifications are depicted in red and losses are depicted in blue. Lower part representsrecurrent genetic alterations determined by GISTIC analyses. E, Pretreatment blast count is displayed for patient samples with low, intermediate low (medL),intermediate high (medH), and high relative BAX localization and thus mitochondrial BAX (n ¼ 12 in each group). P values according to one-way ANOVA usingHolm–Sidak method are shown. F, Pretreatment blast count versus relative BAX localization (double log10 scale) is displayed for 48 patient bone marrowsamples. Pearson correlation is shown by r and P values.G, Comparison of apoptotic response of primary AML samples from seven patients after 24-hour treatmentwith doxorubicin (Doxo, 5 mmol/L), daunorubicin (Dauno, 1 or 20 mmol/L), actinomycin D (ActD, 1 mmol/L), etoposide (Eto, 100 mmol/L), or staurosporine(STS, 1 mmol/L). H, Cell death measured by the release of LDH of seven primary AML patient samples in response to doxorubicin treatment for 24 hours (5 mmol/L,as in G) versus relative BAX localization (log10 scale). Pearson correlation is shown by r and P values.






BAX localization suggests high variability of cytosolic BAX poolscompared with similar mitochondrial levels detected among thepatients with AML (Supplementary Fig. S5A and S5B). Theseresults indicate that cells tolerate a larger variety of BAX concen-trations in the cytosol, probably explained by the fact that cyto-solic BAX is monomeric and lacks apparent protein interactions(30). Of note, the strong positive correlation between cytosolicBAX and BAK suggest that cells also tolerate a large variety ofcytosolic BAK despite a greater tendency of this BCL-2 protein tolocalize on the mitochondria (Supplementary Fig. S5C). Thesubcellular distribution of BAX and BAK is clearly regulated bythe same retrotranslocation process in pretreatment bonemarrowsamples of patients with AML. Therefore, the localization analy-sis of BAX is sufficient to assess the cellular BAX/BAK regulation.

The potential of relative BAX localization as a diagnostic toolwas analyzed in the test cohort of 48 patients with AML (Sup-plementary Table S2.) and correlated with clinical data. Exem-plarily, FMS-like tyrosine kinase 3 internal tandem duplications(FLT3-ITD), a bona fide mutational marker of negative prog-nostic impact in AML (31, 32), were investigated. Strikingly,FLT3-ITD–positive patients show low relative BAX localizationvalues, suggesting increased cytosolic BAX and increased protec-tion against apoptosis (Fig. 2B). Accordingly, FLT3-ITD is asso-ciated with a nonfavorable prognosis (Supplementary Fig. S6A).FLT3-ITD has been associated with activation of both prosurvivalBCL-2proteins andBH3-only proteins and could therefore inducechanges in the relative BAX localization (33–36). Analysis ofpatient collective quartiles [low, mostly cytosolic; high, largelymitochondrial, medium high (medH) and medium (medL)] ofrelative BAX localization confirms predominant occurrence ofFLT3-ITD in samples with low relative BAX localization values(Fig. 2C). The relative risk of FLT3-ITD in this group is at least 6.25times of that in the groups with medium and high relative BAXlocalization values. The same coherence is apparent based on theanalysis of the relative BAK localization, again suggesting a BAXlocalization–based analysis is sufficient for the regulatory status ofboth prosurvival BCL-2 proteins (Supplementary Fig. S6B). Onthe other hand, the analysis based on BAX levels instead of BAXlocalization shows FLT3-ITD occurrence in patients with highcellular BAX levels (Supplementary Fig. S7). Remarkably, patientswith intermediate BAX localization values show inferior survivalcompared with high relative BAX localization patients despite asimilar chance of FLT3-ITD (Figs. 1E and 2B). Of note, one FLT3-ITD–positive individual with high relative BAX localization valueshows prolonged survival following chemotherapy. FLT3-ITDthus may influence relative BAX localization, but many otherfactors (e.g., genetic or epigenetic) also determine BAX localiza-tion and the predisposition to apoptosis.

Consistently, single-nucleotide variants (SNVs) occurring inpatient samples of high or other relative BAX localization wereanalyzed. Overall, distribution and frequency of SNVswas similaracross the different BAX localizations reflecting a quite homog-enous pattern of genetic alterations and very little genomicdiversity among the different patient groups (Fig. 2D). Notably,patient samples with high relative BAX localization displayed asignificantly lower number of genetic losses compared with allother samples. GISTIC analyses further confirmed a high concor-dance of driving copy-number alterations across the differentgroups and very few specific alterations (Supplementary TableS3). Notably, while no recurrent losses were detected in high BAXlocalization, both intermediate and low relative BAX localization

quartiles showed losses on 5q31.3. Nevertheless, comparison ofthe differences between SNVs in BAX high versus the othersubgroups showed little overlap among the different subgroups,indicating the different BAX localizationsmight be determined bydiverse molecular mechanisms and cell stress rather than com-mon genetic alterations (Supplementary Fig. S8).

In an attempt to identify further factors that are reflected by therelative BAX localization, we analyzed potential and knownpredictors of response to therapy and survival in AML. In the testcohort, patient age seems to have no influence on relative BAXlocalization (Supplementary Fig. S9A). Surprisingly, the estab-lished overall risk rating shows no striking congruence withrelative BAX localization (Supplementary Fig. S9B). However,patients with therapy-associated AML present with elevatedmito-chondrial BAX levels (Supplementary Fig. S10). This association isconceivable, as prior nontoxic cellular stress could be reflected inthe relative BAX localization (29). Strikingly, the initial whiteblood count is significantly increased in patients with low relativeBAX localization comparedwith patients with BAX shifted to theirmitochondria (Fig. 2E). Relative BAX localization shows a neg-ative correlation with initial leukocytosis (Fig. 2F). A shift of theBAX population towards the mitochondria and high predisposi-tion to commitment to mitochondrial apoptosis thus is associ-ated with lower initial leukocyte counts.

Treatment of primary AML samples of seven patients with sixdifferent apoptotic stimuli shows reduced apoptotic response byAML cells with low relative BAX localization (Fig. 2G). Patientsamples with high relative Bax localization show increased apo-ptotic response, resulting in a correlation of relative BAX locali-zation and apoptotic response, particularly for doxorubicin- (anddaunorubicin-) treated cells (Fig. 2H). Relative Bax localizationcan identify cellular predisposition to apoptosis.

Relative BAX localization determines predisposition of AMLcell lines to apoptosis

The link between BAX localization and predisposition to apo-ptosis in AML was further tested by determining the relativeBAX localization in eight different AML cell lines (Fig. 3A; Sup-plementary Fig. S11). Consistent with the AML patient data, theanalysis shows increased pools of mitochondrial BAK comparedwith BAX (Supplementary Fig. S12A). However, in AML celllines, this difference is much more pronounced, while relativecytosolic BAX and relative mitochondrial BAX also lack an appar-ent correlation (Supplementary Fig. S12B).

The effects of relative BAX localization in AML cell lines on theinduction of cell death were investigated in response to cytotoxicstress induced by daunorubicin. Application of 1 mmol/L dauno-rubicin for 24 hours resulted 2%–16% cell death depending onthe cell line. The signal-to-noise ratio was increased using20 mmol/L daunorubicin (37), resulting in robust cell deathinduction in seven of the eight tested AML cell lines (Fig. 3B).However, HEL cells did not induce cell death even at 20 mmol/Ldaunorubicin, revealing remarkably reduced predisposition tocell death compared with other cell lines. Noteworthy, HEL cellsshow a particular low relative BAX localization (Fig. 3C). Treat-ment with cytarabine (ara-C) for 24 hours did not induce signif-icant apoptosis in HEL or OCI-AML3 cells (SupplementaryFig. S13). Comparing the apoptotic response of HEL cells andOCI-AML3 cells with increased mitochondrial BAX pools inresponse to daunorubicin treatment demonstrates a robust acti-vation of caspases-3/7 inOCI-AML3 cells, whereasHEL cells show

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Figure 3.

Relative BAX localization determines predisposition to apoptosis in AML cell lines. A, Western blot analysis of the mitochondrial and cytosolic BAX in four AMLcell lines (OCI-AML3, HEL, Kasumi-1, THP-1). Separation ofmitochondria and cytosol was controlled using COX IV and b-actin, respectively. The relative BAX andBAKlevels were normalized using loading controls and protein standard (HeLa whole-cell lysate, used as a protein standard to normalize all analyzed proteins),as in Fig. 1A. B, Cell death of AML cell lines in response to daunorubicin (Dauno; 20 mmol/L) measured by the release of LDH relative to lysed cells (% of control). HELcells (light blue) show high tolerance of this extreme chemotoxic stress, whereas other cells, for example, OCI-AML3 cells (dark blue), profoundly induceapoptosis. C, Relative BAX localization (log10 scale) versus cell death measured by the release of LDH of eight AML cell lines shown in C. D, Caspase-3/7 activitymeasured in OCI-AML3 and HEL cells in the presence and in the absence of daunorubicin. Caspase activity is displayed in relative fluorescence units (RFU).Data represent averages� SEM; n� 3. P value according to one-wayANOVA is displayed. E,Western blot analysis of PARP cleavage in OCI-AML3 and HEL cells withand without daunorubicin (D). Similar sample loading is controlled by b-actin. F, Comparison of apoptotic response of AML cell lines after 24-hour treatmentwith ABT-737 (10 mmol/L), UMI-77 (10 mmol/L), actinomycin D (ActD, 1 mmol/L), etoposide (Eto, 100 mmol/L), or staurosporine (STS, 1 mmol/L). G, Effectof FLT3 inhibitor (Calbiochem, 0.5 mmol/L) treatment for 1 and 6 hours on relative Bax localization in MV4-11 cells (FLT3-ITD–positive) and THP-1 cells(FLT3-ITD–negative) normalized to mock treatment. Data represent averages � SEM; n ¼ 3. P value according to one-way ANOVA is displayed.






low levels of active caspase after 24hours (Fig. 3D). This differenceis corroborated by accumulation of the cleaved form of thecaspase substrate PARP in OCI-AML3 cells but not HEL cells (Fig.3E). These results corroborate the correlation between BAX local-ization and apoptotic response, resulting from altered BAX retro-translocation rates, as previously shown inHCT116 andMEF cells(29). In parallel, HEL cells show increased resistance againstdifferent apoptotic stimuli and the BH3 mimetic UMI-77 (Fig.3F; Supplementary Fig. S14). Among the 8AML cell lines,MV4-11cells contain the FLT3-ITD. FLT3 inhibition results in a shift ofBAX towards the mitochondria suggesting an increase of apopto-sis predisposition of MV4-11 cells but not THP-1 cells (Fig. 3G).However, MV4-11 cells are not particularly protected againstapoptosis induction, suggesting an influence on relative BAXlocalization by FLT3 activity among other factors. The responsesof AML cell lines to apoptotic stimuli suggest that relative BAXlocalization could predict predisposition to apoptosis.

Relative BAX localization is associatedwith FLT3-ITD in AML ofthe elderly

Next, we focused on the patients with an age of 60 years andolder who are frequently not eligible for intensive chemother-apy or hematopoietic stem cell transplantation. Association ofthe susceptibility to cytotoxic therapy with relative BAX local-ization could facilitate treatment decisions toward intensivetreatment versus nonintensive approaches such as targeted- orimmune-therapies and best supportive care. Among elderlypatients (n ¼ 25), a pronounced difference in relative BAXlocalization became evident when comparing FLT3-ITD–posi-tive to FLT3-ITD–negative patients (Fig. 4A). While 60% ofelderly patients with low relative BAX localization levels harborFLT3-ITD mutations, no elderly patient with high relative BAXlocalization values is FLT3-ITD–positive (Fig. 4B). These obser-vations are supported by the analysis based on relative BAKlocalization (Supplementary Fig. S15). In addition, differentblast counts between samples with low and high relative BAXlocalization values were detected (Fig. 4C). Therefore, it istempting to speculate on a potential use of relative BAXlocalization to guide clinical decisions as diagnostic or prog-nostic tool for elderly patients with AML.

BAX and BAK localization is highly variable and correlates withclinical markers in human AML

To evaluate our previous insights into the localization of BAXand BAK in human AML and correlation of BAX localizationwith clinical markers, we analyzed a validation cohort of 80elderly patients with AML treated with myelosuppressive che-motherapy within the AMLSG. Consistently with the testcohort, correlation between mitochondrial and cytosolic pro-tein levels is not apparent (Fig. 5A; Supplementary Table S4;Supplementary Fig. S16). As observed in the test cohort, a widevariety of BAX/BAK localizations were detected with a generaltrend toward cytosolic BAX localization and mitochondrialBAK (Fig. 5B). A strong correlation also exists between therelative localizations of BAX and BAK in the validation cohort(Fig. 5C), demonstrating similar regulation of both proapop-totic BCL-2 proteins in human AML.

Different relative BAX/BAK localizations were detectable in thevalidation cohort depending on the mutational status of FLT3,although the difference was less pronounced compared with thetest cohort (Fig. 5D). The same effect could be observed for thesubgroup of intermediate risk patients according to ELN riskcriteria (Supplementary Fig. S17). When applying the test cohortcriteria and categorizing patients according to low (mostly cyto-solic), medium low (medL) medium high (medH), and high(largely mitochondrial) BAX localization, decreased risk ofpatients with high mitochondrial BAX for FLT3-ITD positivitycan be confirmed (Fig. 5E). Relative BAX localization also corre-lates with leukocytosis in the validation cohort (Fig. 5F; Supple-mentary Fig. S18), suggesting a similar correlation between rel-ative BAX localization and predisposition to apoptosis in bothpatient cohorts.

Relative BAX localization correlates with therapeutic responsein AML of the elderly

Next, we analyzed the potential link between relative BAXlocalization and therapeutic response. Of note, 16 patients fromthe validation cohort that underwent allogeneic stem cell trans-plantation were excluded, as their therapeutic response is ratherrelated to immunogenic control than to susceptibility to cytotoxictherapy. The analysis shows a tendency of mitochondrial BAX

Figure 4.

Relative BAX localization is associated with clinical AML markers in elderly patients with AML. A, Relative BAX localization (log10 scale) in 60þ years oldFLT3-ITD–positive (n ¼ 7) and -negative (n ¼ 16) patients. P value according to one-way ANOVA using Holm–Sidak method is displayed. B, Distribution ofFLT3-ITD–positive samples among60þ years old patientswith low (n¼8), intermediate low (medL, n¼ 7), intermediate high (medH, n¼6), and high (n¼ 5) relativeBAX localization values is displayed. P values are shown for the relative risk of FLT3-ITD positivity in the different quartiles. C, Pretreatment blast count for60þ years old patients with low (n ¼ 8), intermediate low (medL, n ¼ 7), intermediate high (medH, n ¼ 6), and high (n ¼ 5) relative BAX localization isdisplayed. P values according to one-way ANOVA using Holm–Sidak method are shown.

Reichenbach et al.





localization towards positive response to chemotherapy (Supple-mentary Fig. S19). Moreover, patients with high relative BAXlocalization are less likely refractory to chemotherapy (Fig. 6A).This group of patients also shows a trend toward improvedsurvival (Fig. 6B). However, elderly patients from the test cohortwith high relative BAX localization values show strikinglyimproved survival (Fig. 6C). The effects observed in the inten-sively treated validation cohort (Fig. 6B) are less pronouncedcompared with the test cohort, where patients received nonmye-losuppressive regimens (cytoreductive therapies or best support-ive care; Figs. 1E and 6C). Again, the analysis of relative BAXlocalization as a continuous value provides no significant differ-ence in receiver operating curve for validation cohort patients(Supplementary Fig. S20). The analysis basing on categorizedBAXlevels is showing a clear trend in both patient cohorts but resultsare still suffering from the small number of participants. Logisticregression on survival as a function of relative BAX localization,sex, FLT3-ITD, ELN-risk, and total-risk showed a significant influ-ence of increased mitochondrial BAX levels OR ¼ 1.61 (95%confidence interval, 1.1–2.4), while all other showed trends onsurvival influence but failed to reach significance (SupplementaryTable S5).

DiscussionBAX and BAK share common regulation of their localization in

primary AML patient samples and AML cell lines. In healthy cells,both proapoptotic BCL-2 proteins are retrotranslocated from themitochondria into the cytosol by the same prosurvival BCL-2protein-dependentmechanism(25, 26). Shuttling at similar rates,BAX and BAK share similar apoptotic activity and subcellularlocalization (26). Therefore, the analysis of the relative BAXlocalization is sufficient to determine the status of BAX/BAKsignaling. The total cellular protein level is not linked to BAXregulation. Increased BAX level could even be associated withmore apoptosis-resistant scenarios (Supplementary Fig. S6). Thisargues against previous attempts to predict cellular survival orchemotherapy response solely by analyzing cellular BAX levels(38, 39). Also, the determination of the BAX/BCL-2 ratio, aspreviously suggested (40, 41), considers a potentially importantinteraction for BAX retrotranslocation, but neglects BAK-depen-dent apoptosis and potential cell protection from BAX/BAKactivity by other prosurvival proteins, such as Bcl-xL and Mcl-1(25). The analysis of relative BAX/BAK localization includes thecombined activities of prosurvival BCL-2 proteins and potential

Figure 5.

Localizations of BAX and BAK and clinical markers correlate in a validation cohort of 80 patients with AML. A, Relative cytosolic BAX (log10 scale) versus relativemitochondrial BAX (log10 scale) is displayed for 80 bone marrow samples from an evaluation cohort of elderly patients. Lack of correlation according toPearson's method is shown by the displayed r and P values. B, Relative protein localization (log10 scale) of BAX and BAK in 80 AML elderly patient bone marrowsamples. P value according to t test is displayed. C, Relative BAK localization versus relative BAX localization (double log10 scale) is displayed for 80 elderlypatient bonemarrow samples. r andP values according Pearson correlation are displayed.D,Relative BAX localization (log10 scale) inFLT3-ITD–positive (n¼ 27) and-negative (n ¼ 52) elderly patients. E, Distribution of FLT3-ITD–positive samples among elderly patients with low (n ¼ 50), intermediate low (medL, n ¼ 15),intermediate high (medH, n ¼ 5), and high (n ¼ 9) relative BAX localization values is displayed. P values are shown for the relative risk of FLT3-ITD positivityin the different quartiles. F, Pretreatment blast count versus relative BAX localization (double log10 scale) is displayed for 80 elderly patient bone marrowsamples. Pearson correlation is shown by r and P values.






influence of BH3-proteins, like Bim, on BAX/BAK retrotransloca-tion (25, 26, 29). The equilibrium between cytosolic and mito-chondrial BAXpools displays a surprising diversity in AMLpatientsamples that reflects the heterogeneous response to chemotoxicstress. Significant differences mainly in the cytosolic BAX poolhold the promise of discriminating between clinically differentAML.

Cellular survival requires fast BAX retrotranslocation from themitochondria (26, 27). Consequently, a shift of the equilibriumbetween cytosolic and mitochondrial BAX toward the mitochon-dria results in increased apoptosis induction in response toapoptotic stimuli (29). In parallel to other cell cultures, reducedpredisposition to apoptosis is observed in HEL cells with cytosol-shifted BAX compared with other AML cell lines. The other testedcell lines show a similar response to apoptotic stimuli, corrobo-rating previous results from BH3 profiling (42). While BH3profiling has the potential to dissect contributions of differentkey proteins, cell permeabilization, required for this type ofanalysis, removes cytosolic protein pools and triggers reequili-bration potentially interfering with the determination of cel-lular predisposition to apoptosis. Relative BAX localizationreveals the status of cellular BAX/BAK regulation, includingcontributions of known and unknown factors but cannotdissect their contributions.

Relative BAX localization itself is influenced by a plethora offactors, probably including genetic aberrations, for example,FLT3-ITD. However, genetic determinants of BAX regulation areheterogeneous in the analyzed patients' samples. The use ofgenomic data for risk stratification is probably prone to under-score the effect of mutational events on transcriptional, transla-tional, and posttranslational consequences. They may be furtherinfluenced by the microenvironment or epigenetic alterations.Presence of complex aberrant cytogenetic changes has beendescribed to correlate with resistance to low-dose cytarabinetreatment before (43). However, even patients without any cyto-genetic aberrations do not uniformly respond to chemotherapy.Most recently, an integrative genomic and clinical analysis of1,540AMLpatients enrolled in three prospective clinical trials andtreated with myelosuppressive chemotherapy identified a set of5,234 driver mutations with more than one driver mutationoccurring in the vast majority of patients (86%; ref. 44). More-

over, cooccurring mutations influenced outcome and survivalalso beyond the previously described categories (6), highlightingthe complexity of risk-assessment in AML. Here, determination ofcellular susceptibility toward induction of apoptosis could poten-tially serve as a rapid and suitable assessment to predict thera-peutic response and to stratify patients towardsmyelosuppressivechemotherapy versus rather nonintensive therapy such as targetedagents, epigenetic treatment, or immunotherapy.

Clinically, the predictive value of certain prognostic markershas a distinct impact, depending on the patient's fitness andcomorbidities. AML of the elderly or frail patient requires detailedassessment of physical status and past medical history beforepatients are stratified toward intensive treatment versus nonin-tensive therapies or best supportive care. The majority of patientsdiagnosed with AML is above the age of 60 years and many ofthem do not have any curative options due to comorbidities orlack of response. Achievement of remission leads to prolongedsurvival and improved quality of life of the patient; however,which patients will respond is difficult to predict. When focusingon these patient-oriented aspects, prediction of response by usingone major regulator of cellular resistance could facilitate stratifi-cation of elderly and frail patients to chemotherapeuticapproaches versus targeted therapies or best supportive care.

In principle, BAX localization can be analyzed within a shortperiod of time (less than 72 hours) and requires low amounts ofcells (

clone to cytotoxic therapies. BAX localization shift toward thecytosol, on the other hand, leads to a resistance phenotype and isassociated with a high tumor burden and FLT3-ITD, described tocoincide with poor prognostic outcome. These correlations areapparent, although not homogeneous, in both cohorts of patientswith AML analyzed in this article. Of note, small sample size andheterogeneity of consolidation (e.g., allogeneic stem cell trans-plantation) or second-line therapies (demethylating agents, tar-geted therapies) may significantly influence analysis of survival asreadout for response. Still, BAX localizationmay predict responseand outcome and its predictive value is even better detectable inanalysis of rather small but more homogeneous subgroups (Fig.6B and C). Prospective analysis of response rates to first-linetreatment especially in a higher number of older patients withAML is necessary to validate its predictive value in the future.

Our findings indicate the potential use of BAX localization toassess for response to cytotoxic therapy in AML but also in thetreatment of other tumor entities, as the underlying BAX regula-tion is a general mechanism that prevents BAX activity in humancells. Nevertheless, this analysis identifies also the limits of ouranalysis, as we assess biologic response of the dominant AMLclone at diagnosis. Subclones may be selected or emerge duringchemotherapy. Overall, we provide first evidence that BAX local-ization is associated with response to chemotherapy.

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

Authors' ContributionsConception and design: F. Reichenbach, C. Wiedenmann, F. EdlichDevelopment of methodology: F. Reichenbach, C. Wiedenmann, F. Edlich

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Reichenbach, C. Wiedenmann, E. Schalk,D. Wolleschak, K. D€ohner, J.U. Marquardt, F. HeidelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Reichenbach, C. Wiedenmann, K. Funk, P. Scholz-Kreisel, F. Todt, K. D€ohner, J.U. Marquardt, F. Heidel, F. Edlich, D. BeckerWriting, review, and/or revision of the manuscript: F. Reichenbach,C. Wiedenmann, E. Schalk, P. Scholz-Kreisel, K. D€ohner, J.U. Marquardt,F. Heidel, F. EdlichAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Reichenbach, F. HeidelStudy supervision: F. Edlich

Grant SupportThis work was supported by the DFG Emmy Noether program, the Collab-

orative Research Cluster (CRC) 746, the Else Kr€oner-Fresenius-Stiftung, theWilhelmSander-Stiftung, the SpemannGraduate School of Biology andMedicine(SGBM,GSC-4)and theCentre forBiological SignallingStudies (BIOSS,EXC-294)funded by the Excellence Initiative of the German Federal and State Governments(F. Reichenbach,C.Wiedenmann, F. Todt, F. Edlich). F.Heidelwas supportedby agrant of the German Research Foundation, Collaborative Research Cluster (CRC)854 (Project A20), the ProExcellence Research Initiative "RegenerAging" (State ofThuringia), the Thuringian country programme ProExzellenz (RegenerAging -FSU-I-03/14) of the Thuringian Ministry for Research (TMWWDG), and theGerman Jose-Carreras Leukemia Society (DJCLS SP12/08). K. D€ohner was sup-ported by a grant of the German Research Foundation, Collaborative ResearchCluster (CRC) 1074 (Project B3) and the AMLSG study group. J.U. Marquardt issupported by the German Cancer Aid (DKH 110989), the Wilhelm Sander-Stiftung, and the Volkswagen Foundation (Lichtenberg program).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 5, 2016; revised September 5, 2016; accepted April 11, 2017;published OnlineFirst April 18, 2017.

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Mitochondrial BAX Determines the Predisposition to ... · translocation of BAX and BAK to the mitochondria establishes an equilibrium between cytosolic and mitochondrial protein pools