Combination of Imatinib with CXCR4 Antagonist BKT140 ...mct.aacrjournals.org/content/molcanther/13/5/1155.full.pdfin the bone marrow niche (12–14). Furthermore, stroma-derived CXCL12

Small Molecule Therapeutics

Combination of Imatinib with CXCR4 Antagonist BKT140Overcomes the Protective Effect of Stroma and Targets CMLIn Vitro and In Vivo

Katia Beider1, Merav Darash-Yahana2, Orly Blaier1, Maya Koren-Michowitz1, Michal Abraham3, Hanna Wald3,Ori Wald2, Eithan Galun2, Orly Eizenberg3, Amnon Peled2, and Arnon Nagler1

AbstractFunctional role of CXCR4 in chronic myelogenous leukemia (CML) progression was evaluated. Elevated

CXCR4 significantly increased the in vitro survival and proliferation in response to CXCL12. CXCR4

stimulation resulted in activation of extracellular signal-regulated kinase (Erk)-1/2, Akt, S6K, STAT3, and

STAT5 prosurvival signaling pathways. In accordance, we found that in vitro treatment with CXCR4

antagonist BKT140 directly inhibited the cell growth and induced cell death of CML cells. Combination of

BKT140 with suboptimal concentrations of imatinib significantly increased the anti-CML effect. BKT140

induced apoptotic cell death, decreasing the levels of HSP70 and HSP90 chaperones and antiapoptotic

proteins BCL-2 and BCL-XL, subsequently promoting the release of mitochondrial factors cytochrome c

and SMAC/Diablo. Bone marrow (BM) stromal cells (BMSC) markedly increased the proliferation of CML

cells and protected them from imatinib-induced apoptosis. Furthermore, BMSCs elevated proto-oncogene

BCL6 expression in the CML cells in response to imatinib treatment, suggesting the possible role of BCL6 in

stroma-mediated TKI resistance. BKT140 reversed the protective effect of the stroma, effectively promoted

apoptosis, and decreased BCL6 levels in CML cells cocultured with BMSCs. BKT140 administration in vivo

effectively reduced the growth of subcutaneous K562-produced xenografts. Moreover, the combination of

BKT140 with low-dose imatinib markedly inhibited tumor growth, achieving 95% suppression. Taken

together, our data indicate the importance of CXCR4/CXCL12 axis in CML growth and CML–BM stroma

interaction. CXCR4 inhibition with BKT140 antagonist efficiently cooperated with imatinib in vitro and in

vivo. These results provide the rational basis for CXCR4-targeted therapy in combination with TKI to

override drug resistance and suppress residual disease. Mol Cancer Ther; 13(5); 1155–69. �2014 AACR.

IntroductionChronic myelogenous leukemia (CML) is a myelopro-

liferative disease of hematopoietic stem cells (HSC) that isdriven by the constitutively active oncogenic tyrosinekinase BCR-ABL (1, 2). Inhibition of BCR-ABL activity bytyrosine kinase inhibitors (TKI), such as imatinib mesy-late, revolutionized the treatment of CML and remains amajor therapeutic strategy for patients with CML (3).However, existence of quiescent leukemic stemcells (LSC)that are resistant to TKIs andmay be responsible for CMLresistance and recurrence continues to pose challenges for

CML cure (4, 5). Thus, novel therapies that eradicate LSCsare in need.

The bone marrow microenvironment is believed tohave a role in protecting LSCs and CML cells from TKIs-induced apoptosis (6). CXCL12, also called stromal cell–derived factor-1 (SDF-1), a chemokine produced by bonemarrow stromal cells (BMSC), signals through the cog-nate chemokine receptor CXCR4, which is broadlyexpressed by normal and malignant cells of hematopoi-etic and nonhematopoietic origin (7). Data from knock-out mice indicate that the CXCR4 receptor plays animportant role in hematopoiesis, development, andorganization of the immune system (8–11). CXCR4 andits ligand CXCL12 have a key role in the traffickingand retention of normal hematopoietic as well as LSCsin the bone marrow niche (12–14). Furthermore, stroma-derived CXCL12 supports the proliferation of HSCs andprevents their terminal differentiation (15). In CML,leukemia–stroma interactions are impaired. CML adhe-sion to ECM components and bone marrow stromawas shown to mediate malignant phenotype and pro-tect CML cells from TKI treatment through variousmolecular mechanisms (16–18). Our previous work

Authors' Affiliations: 1Hematology Division and CBB, Sheba MedicalCenter, Tel-Hashomer; 2Goldyne Savad Institute of Gene Therapy, Hadas-sah Hebrew University Hospital, Jerusalem; and 3Biokine TherapeuticsLtd., Science Park, Ness Ziona, Israel

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

Corresponding Author: Arnon Nagler, Division of Hematology & CBB,Chaim Sheba Medical Center, Tel-Hashomer, Israel. Phone: 972-3-5305830; Fax: 972-3-5304792; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0410

�2014 American Association for Cancer Research.

MolecularCancer

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Published OnlineFirst February 6, 2014; DOI: 10.1158/1535-7163.MCT-13-0410

http://mct.aacrjournals.org/

showed that the CXCR4-dependent migration of imma-ture CD34þ CML cells is impaired and that the integrin-dependent migration and adhesion in response toCXCL12 are decreased (19). Others have found that theBCR-ABL oncoprotein interferes with CXCR4-mediatedsignaling, and suppresses the CXCL12-induced chemo-tactic response of CML cells (20, 21). Importantly, recentstudies demonstrated that CXCR4 can be elevated byTKI treatment andmay induce stroma-promoted chemo-resistance (22, 23).

Elucidating the role of the bone marrowmilieu and theCXCR4/CXCL12 axis in the response to TKIs and indisease progression may provide a rational basis for thedevelopment of novel therapies for CML eradication. Apotential strategy to overcome the stroma-provided drugresistance is to interrupt the CML-stroma cross-talk uti-lizing CXCR4 neutralization methods. We hypothesizedthat CXCR4 blockadewith specific antagonists can inhibitthe survival and spread of CML and LSC cells and mayrestore their sensitivity to TKIs in the bonemarrowmicro-environment context.

The present work demonstrates that CXCR4 over-expression in the CML cell line K562 does not increasethe migratory potential of CML cells, but rather sup-ports their survival and growth, therefore confirmingthe prosurvival role of CXCR4 in CML. Previously, wehave shown that the high affinity CXCR4 antagonistBKT140 demonstrates an effective antitumor effect bothin vitro and in vivo in various hematologic malignanciesand enhances the proapoptotic effect of antileukemiccompounds (24). In the current study, we evaluated theeffect of BKT140 on CML cell viability and sensitivity toimatinib in the presence of BMCSs. Our results indicatethat CML cells are protected by the bone marrow stromafrom imatinib-induced apoptosis, and significantlyupregulate expression of the antiapoptotic factor BCL6in response to imatinib treatment. BKT140 enhancesimatinib activity and disrupts the stroma-mediatedprotection. Moreover, BKT140 demonstrates a potentanti-CML activity in vivo in a xenograft model.

Materials and MethodsCell lines and CML patient samples

CML cell lines K562 (obtained from American TypeCulture Collection) and LAMA-84 (obtained fromDSMZ), and natural killer (NK) cell line YTS (kindlyprovided by Prof. Ofer Mandelboim, Hebrew University,Jerusalem, Israel)weremaintained at log growth inRPMI-1640 medium (Gibco Laboratories) containing 10% fetalcalf serum (FCS), sodium pyruvate, 1 mmol/L L-gluta-mine, 100 U/mL penicillin, and 0.01 mg/mL streptomy-cin (Biological Industries). K562 and LAMA-84 cells wereauthenticated by STR DNA fingerprinting using Amp-FISTR Identifier Kit (Applied Biosystems). YTS cells werenot authenticated.

Bonemarrow samples from informed-consented newlydiagnosed patients with CML were obtained in accor-

dance with institutional guidelines. Mononuclear cellswere collected after standard separation on Ficoll–Paque(Pharmacia Biotech).

InhibitorsBKT140, a CXCR4 antagonistic peptide, was provided

by Biokine Therapeutics and dissolved in sterile saline.Imatinib was purchased from Cayman and dissolved insterile dimethyl sulfoxide (DMSO). Final concentration ofDMSO in cultured cells was less than 0.001%.

Cell line transductionThe retroviral vector, pLNC/Luc, was prepared by

introducing the firefly luciferase gene (luc) downstreamof the cytomegalovirus (CMV) promoter in the plasmidvector pLNC carrying the Neomycin resistance gene(25). K562 cells were stably transduced with a retroviralvector, using transfer vector-pLNC/Luc and the pack-aging plasmid-pCL. K562L cells were then transducedagain with lentiviral vectors to produce K562 cellscoexpressing the luc and the GFP genes (K562LG) orthe luc and the bicistronic CXCR4-GFP genes (K562LG-CXCR4). This was done using a three-plasmid system:transfer vector-pHR’-CMV-GFP-WPRE or pHR’-CMV-CXCR4-IRES-GFP-WPRE; envelope coding plasmid-VSV-G and a packaging construct-CMVDR8.91. The NKcell line, YTS, was similarly transduced and YTSG andYTSG-CXCR4 cells were generated to be used as anegative (Ph-) control.

Analysis of positively transduced cellsCells were analyzed for GFP on FACSCalibur (Beckton

Dickinson) using CellQuest software. Luciferase activitywas evaluated after positive selection by luminometerusing the Luciferase assay system according to the man-ufacturer’s instructions (Promega Corp.).

Flow cytometry analysis of CXCR4 and CXCR7expression

Expression levels of the chemokine receptors CXCR4and CXCR7 were evaluated by immunostaining withphycoerythrin (PE)-conjugated anti-CXCR4 monoclonalantibody (12G5 clone; R&D Systems) and PE-conjugatedanti-CXCR7antibody (eBioscience). InprimaryCMLsam-ples, counterstaining with anti-CD34 fluorescein-conju-gated antibody was performed.

Cell migration assayMigration assay was performed in triplicates using

5-mm pore size Transwells (Costar). The lower compart-ment was filled with 600 mL of 1% FCS RPMI-1640medium containing CXCL12 (500 ng/mL; PeproTechEC), and 5 � 105 cells in 100 mL of 1% FCS RPMI-1640medium were applied to the upper compartment. Theamount of cells migrated within 4 hours to the lowercompartment was determined by fluorescence-activat-ed cell sorting (FACS) and expressed as a percentage ofthe input.

Beider et al.

Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics1156




RT-PCR analysisTotal RNA from cells was extracted using TRIzol

reagent (Invitrogen) according to the manufacturer’sinstructions. Subsequently, cDNA was generated from1 mg of total RNA using High-Capacity cDNA ReverseTranscriptionKit (AppliedBiosystems). CXCR4 andBCL-6 mRNA levels were evaluated using SYBR Green quan-titative PCR (qPCR). The qPCR reaction contained 100 ngof total RNA-derived cDNAs, forward and reverse pri-mers (300 nmol/L), and PerfeCta SYBR Green FastMix(Quanta Biosciences), and was performed using StepO-nePlus Real Time PCR system (Applied Biosystems).Changes in expression levels were normalized to controlb2-microglobulin using DDCt method of relative quanti-fication using StepOne Software v2.2. Primer sequencesare presented in Supplementary Table 1.

Survival assayK562 or bonemarrow-derived primary CML cells were

cultured2� 105 cells/mL, in triplicates, in 0.1%FCS, in thepresence or absence of CXCL12. Cultured cells werecollected after 48 hours incubation and the number ofcells was enumerated by FACS. Events were acquiredduring 30 seconds. Dead cells were eliminated by stainingwith propidium iodide (PI). Relative number of viablecells in each sample was determined. To confirm thenormalized flow rate and ensure accurate cell count, fixedcell concentration was counted before the experiment.

Apoptosis assayApoptosiswasdeterminedby stainingwithAnnexinV-

FITC and PI according tomanufacturer’s instructions andanalyzed by flow cytometry. The percentage of earlyapoptotic (Annexin Vþ/PI�) and late apoptotic/dead(Annexin Vþ/PIþ) cells was quantified.

Assessment of mitochondrial membrane potential(Dcm)The cationic lipophilic fluorochrome DiOC6 (Sigma-

Aldrich) was used to measure the Dcm. Briefly, CMLcells were cultured with different concentrations ofBKT140 and imatinib for 12 hours, harvested, resus-pended in FACS buffer (PBS with 0.1% FCS and 0.1%NaN3) containing 200 nmol/L DiOC6, and incubated for15minutes in 37�C. The cells were then analyzed by FACSfor the loss of DiOC6 fluorescence.

Cell-cycle analysisCML cells were exposed in vitro to increasing concen-

trations of BKT140 and imatinib for 48 hours. Cells werecollected, washed with cold PBS, and fixed with 4% ofparaformaldehyde (PFA) for 30 minutes. Fixed cells wereresuspended in staining buffer containing 0.1% saponin(Sigma-Aldrich) and 40 mg/mL RNase and were incubat-ed at 37�C for 15 minutes. Cells were then stained with 10mg/mL 7-amino-actinomycin D (7-AAD; eBioscience) indark for 30 minutes. DNA content was detected usingFACS.

Colony growth inhibitionK562 cells pretreated with BKT140 (20 mmol/L), imati-

nib (0.5 mmol/L), or combination of both agents for 24hourswere harvested, suspended in culturemedium, andplated in soft agar in 24-well plates. Following 2 weeks ofincubation, colonies were counted and photographed.

Coculture experimentsPrimary human BMSC were isolated and expanded

from bone marrow aspirates of healthy donors aftersigned informed consent as previously described (26).CML cells were labeled with 5-(and 6)-Carboxyfluores-cein diacetate succinimidyl ester (CFSE; 5 mmol/L) andseeded on the top of stromal cells, alone or in combinationwithBKT140 and/or imatinib, at a density of 2� 105 cells/mL in a medium supplemented with 1% FCS. Following48 hours of coincubation, nonadherent cells were collect-ed and adherent cell fraction (including BMSCs and CMLcells) was harvested with trypsin/EDTA. The cells werewashed with PBS and analyzed by FACS for viability(using PI exclusion) and proliferation. CFSE-unlabeledBMSCs were gated out.

BrdUrd incorporation assayTo evaluate the effect of BMSCs on CML cell prolif-

eration, CFSE-labeled K562 and LAMA-84 cells werecultured in the absence or presence of BMSCs mono-layer in a medium supplemented with 1% FCS. Follow-ing 36 hours of coculture, the cells were pulsed withbromodeoxyuridine (BrdUrd) for additional 12 hoursand analyzed for BrdUrd incorporation using APCBrdUrd Flow Kit (BD Biosciences) according to themanufacturer’s instructions. CFSE-unlabeled BMSCswere gated out. Percentage of proliferating BrdUrdþCML cells was determined.

XTT viability assayK562 and LAMA-84 cells (2 � 104 per 100 mL per well)

were plated in 1% FCS-containing medium in 96-wellflat plates in quadruplicate samples, with different con-centration of BKT140, imatinib, or a combination ofboth, in the absence or presence of BMSCs for 48 hours.Cell viability was assessed using the 2,3-bis[2-methoxy-4-nitro-S-sulfophenynl]H-tetrazolium-5- carboxanilideinner salt (XTT) assay (Biological Industries).

ELISACXCL12 levels in culture medium were quantified

using ELISA (Quantikine Human CXCL12/SDF-1 immu-noassay, R&D Systems) according to the manufacturer’sinstructions.

Immunoblot analysis and proteome arraysFor immunoblotting, total protein lysates (50–70 mg)

were resolved by electrophoresis on 10% SDS-PAGE andtransferred onto polyvinylidene difluoride membranes.Blots were subjected to a standard immunodetectionprocedure using specific antibodies and the enhanced

CML and CXCR4

www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1157




chemiluminescence substrate (Biological Industries). Sig-nal was detected using a Bio-Rad image analyzer (Bio-Rad). Primary antibodies were as follows: pErk1/2 (Sig-ma-Aldrich), pAKT (R&D Systems), pS6 and BCL-XL(Cell Signaling Technology), BCL-2 (Dako), HSP70 andHSP90 (Stressgen Biotechnologies Corp.), and b-actin(Sigma-Aldrich).

Human Phospho-Kinase Array Kit (R&D Systems) wasused to measure the activation of signal transduction inCML cells in response toCXCL12 stimulation. The humanApoptosis Array Kit (R&D Systems) was used tomeasurethe level of expression of pro- and antiapoptotic proteinsbefore and after the addition of BKT140. Relative expres-sion of proteins was determined following quantificationby the Image-Pro plus program.

Cytochrome c releaseRelease of mitochondrial cytochrome c to the cytosol of

BKT140- and imatinib-treated CML cells was measuredbyflowcytometryusing theFlowCellectCytochrome cKitwith direct fluorescein isothiocyanate (FITC)-conjugatedanti-cytochrome c antibody (Merck Millipore), accordingto the manufacturer’s instructions.

Quantification of intracellular phospho-STAT5 byflow cytometry

Cells were serum starved overnight, then incubatedeither with or without CXCL12 (200 ng/mL) for 5 and30 minutes, washed with 1� PBS, fixed with 4% PFA,permeabilizedwith ice-coldmethanol (90%final),washedand immunostained with PE-conjugated anti–phospho-STAT5 antibody or isotype control (eBioscience).

In vivo xenograft model of human CML in NOD/SCID mice

Immunodeficient nonobese diabetic/severe combinedimmunodeficient (NOD/SCID) mice (NOD/LtSzPrKdcscid/PrKdcscid; ref. 27)weremaintained under definedflora conditions at the Hebrew University Animal Path-ogen Free Facility. The Animal Care Committee of theHebrew University approved all experiments. Twentyfour hours before intravenous (i.v.) injection of cells, micewere irradiated with 375 RAD. K562LG cells (5 � 106

/mouse) were injected intravenously and monitored fortumor cell distribution 24 and 48 hours later. In vivoimaging was performed as previously described (28).

For local tumorgeneration,K562L cells (5� 106/mouse)were injected subcutaneously (s.c.) into the left flank.Tumor growth was followed using caliper measurement.

BKT140 was administered subcutaneously 400 mg permouse per injection, in a site different to tumor injection.Imatinib was injected 10mg/kg i.p. Control animals weretreated with saline.

Statistical analysesData are expressed as the mean � SD or SE. Statistical

comparisons of means were performed by a two-tailedunpaired Student’s t test.

ResultsHigh CXCR4 expression promotes in vitro survivalandproliferation, butnotmigration, of theK562 cellsin response to CXCL12

We and others have demonstrated the role of CXCR4 inaberrant migration and adhesion of primary CML cells(19, 29). However, BCR-ABL–positive CML cell linesexpress low cell-surface and mRNA levels of CXCR4. Tostudy the in vitro and in vivo roles of CXCL12/CXCR4 inCML, we generated a K562 cell line that expresses highlevels of CXCR4, similar to the levels observed in primaryCMLCD34þ cells. For in vivomonitoring of tumor growthand migration, K562 cells were first stably transducedwith a retrovirus carrying the luciferase (luc) gene(K562L). FACS analysis demonstrated that more than80% of K562LG-CXCR4 cells and more than 72% of thecontrol YTS-CXCR4 cells expressed high levels of CXCR4on their cell surface (Supplementary Fig. S1A and S1B).

To investigate the possible role of CXCR4 in mediatingCMLmigration in response to CXCL12, K562 or K562LG-CXCR4 cells were tested for their migration potential.Both cell lines migrated poorly in response to CXCL12.In contrast, Ph- cells overexpressingCXCR4 (YTS-CXCR4)increased their migration in response to CXCL12 by 8-folds comparedwith YTS cells not expressing the receptorCXCR4.

These results indicate functional activity of the trans-duced CXCR4 receptor in YTS cells, while its signaling forchemotaxis is impaired in K562LG-CXCR4 cells (Supple-mentary Fig. S1C).

Next, we generated single-cell clones with high andstable level of CXCR4 expression, comparable withCXCR4 expression by primary CML CD34þ cells (Sup-plementary Fig. S1D and S1E). Neither K562 nor K562LG-CXCR4.86 cells expressed CXCR7 (Supplementary Fig.S1F). K562 cells with high cell-surface CXCR4 (K562LG-CXCR4.86) demonstrated dose-dependent in vitro prolif-eration response to CXCL12 stimulation that was highercompared with native K562 cells. Proliferative rate ofK562LG-CXCR4.86 in response to CXCL12 was compa-rable with that of primary CD34þCML cells (Supplemen-tary Fig. S1G).

CXCR4 signaling in CML cellsTo delineate the signaling pathways activated by

CXCR4 in CML cells, K562 and K562LG-CXCR4.86cells were stimulated with CXCL12 and protein phos-phorylation was analyzed by the Phospho-kinase Prote-ome Profiler Array. In high CXCR4-expressing cells,CXCL12 treatment resulted in profound activation ofsurvival pathways, including increase in phosphorylatedextracellular signal-regulated kinase (Erk)-1/2, AKT,STAT5, STAT3, S6K, RSK1, and WNK1. In contrast, lowCXCR4-expressing K562 cells poorly responded toCXCL12 stimulation, mainly by a delayed increase inpAKT (following 30 minutes of treatment; Fig. 1A).

Toverify the results anddetermine the level of signalingmediators’ activation in response to CXCR4 stimulation,

Beider et al.





FACS and immunoblot analyses were performed.CXCL12 treatment increased the levels of pSTAT5 inCXCR4-expressing K562 cells, as confirmed by phso-pho-flowanalysis (Fig. 1B). Furthermore, rapidphosphor-ylation of Erk1/2 and AKT occurred as early as 5 minutesin K562LG-CXCR4.86 cells, and started diminishing30 minutes posttreatment. Conversely, in K562 cells,CXCL12 resulted in very weak Erk1/2 and delayed (post30 minutes) AKT activation (Fig. 1C). Interestingly,increased phosphorylation of the mTOR target S6 wasdetected in CXCR4-expressing K562 cells. Together, thesefindings indicate that CXCR4 activates multiple prosur-vival signaling pathways in CML cells.

CXCR4-specific high-affinity antagonist BKT140inhibits proliferation of K562 cellsThe enhanced proliferative response of K562LG-

CXCR4.86 cells to CXCL12 stimulation prompted us tostudy the effect of the CXCR4 antagonist BKT140 (Sup-

plementary Fig. S2) on CML cell survival and prolifera-tion. K562 cells with low and high levels of CXCR4 weretreated with increased concentrations of BKT140 for 48hours. BKT140 treatment reduced viability and corre-spondingly increased cell death, in a dose-dependentmanner, in both low- and high CXCR4-expressing K562cells. Notably, K562LG-CXCR4.86 cells were significantlymore sensitive (P < 0.01) to low concentrations of BKT140(4 and 8 mmol/L) than native K562 cells (Fig. 2A).

Because CXCL12 stimulated the growth of CXCR4-expressing K562 cells and has been shown to mediatechemoresistance of leukemic cells in bonemarrowmilieu,we examined whether BKT140 can overcome the protec-tive effect of exogenous CXCL12. Importantly, CXCL12treatment did not inhibit BKT140-induced cytotoxicityagainst K562LG-CXCR4.86 cells (Fig. 2B).

These results illustrate the antiproliferative potential ofBKT140 in CML cells and indicate a role of CXCR4 inBKT140-induced anti-CML effect.

Figure 1. CXCR4 stimulationactivates multiple signalingpathways in CML cells. A, humanphospho-kinase proteome profilerarray was used to screen foractivation of specific signalingpathways in CML cells followingCXCR4 stimulation. K562 andK562LG-CXCR4.86 cells wereserum starved overnight andexposed to CXCL12 (200 ng/mL)for 5 and 30 minutes. Cells werelysed and relative level ofphosphorylated kinases wasanalyzed. B, intracellular levels ofphospho-STAT5 in K562LG-CXCR4.86 cells in response toCXCL12 activation was monitoredby flow cytometry. Columns, meanfluorescent intensity (MFI) ofpSTAT5-PE staining inunstimulated versus CXCL12-stimulated cells. Results representthe average of triplicates � SD(��,P <0.01). C, K562 andK562LG-CXCR4.86 cells were serumstarved overnight and treated withCXCL12 (200 ng/mL) as in A.Expression of the indicatedproteins was analyzed byimmunoblotting. Blots werestripped and reprobed with anti–b-actin antibody to ensure equalloading and transfer of protein(50 mg each lane).

CML and CXCR4





Figure 2. BKT140 demonstrates potent a cytotoxic activity against CML cells and cooperates with imatinib in vitro. A, K562 and K562LG-CXCR4.86 cellswere cultured in serum-reduced medium (1%) with different concentrations of BKT140 (4, 8, and 20 mmol/L) for 48 hours. Cell viability was determinedusing PI-exclusion and FACS count. Decrease in viability is presented as percentage from untreated control. Percentage of dead (PI-positive) cells wasdetermined. B, K562LG-CXCR4.86 cells were cultured with CXCL12 (500 ng/mL), BKT140 (8 and 20 mmol/L), or combination of both for 48 hours.Number of viable cells in culture was determined by FACS. Data represent the mean of triplicates�SD (��, P < 0.01). C–F, K562 and LAMA-84 cells werecultured in serum-reduced medium (1% FCS) in the absence or presence of BKT140 (8 mmol/L), imatinib (0.1 and 0.2 mmol/L), or combination of bothagents for 48 hours. C, viability was determined using PI exclusion by FACS. Data represent the mean of triplicates �SD (��, P < 0.01). D, representativeimages of untreated or treated CML cells following 48 hours of incubations depict the presence of apoptotic cells. E and F, apoptosis was detectedusing Annexin V-FITC/PI staining. Percentage of early (Annexin Vþ/PI�) and late (Annexin Vþ/PIþ) apoptotic cells was determined. Data are presented asmean of triplicates �SD (��, P < 0.01). G and H, K562 cells were treated with imatinib, BKT140, or combination of both agents for 12 hours andmitochondrial membrane potential (Dcm) was determined using DiOC6 staining. Percentage of apoptotic cells with reduced Dcm and subsequentstaining intensity was detected. Data are presented as mean of triplicates �SD (��, P < 0.01).

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BKT140 cooperates with imatinib in vitro andeffectively inhibits CML growthTo further elucidate the role of CXCR4 and its inhib-

itor BKT140 on CML growth in vitro, BKT140 wascombined with the TKI imatinib and their effect onCML viability was evaluated. CML cells, K562 andLAMA-84, were treated with low concentrations of ima-tinib (0.1 or 0.5 mmol/L) either alone or in combinationwith BKT140 (8 mmol/L) for 48 hours. Treatment of K562cellswith single-agent imatinib (0.1 mmol/L) or BKT140 (8mmol/L) did not significantly alter cell viability, whereasthe combination of both agents promoted significant celldeath. LAMA-84 cells demonstrated a higher sensitivityto imatinib and BKT140 single therapy, but similarly toK562, the combination of both agents significantlyincreased cell death in culture (Fig. 2C). This cytotoxiceffect of combinatory treatment with imatinib andBKT140 against CML cells was also demonstrated inmicroscopic evaluation of the cells in culture (Fig. 2D).These results suggest that BKT140 enhances the anti-CML effect of imatinib.

BKT140 treatment induces mitochondrial-dependent apoptosis in CML cellsTo analyze the mechanism of BKT140-induced cytotox-

icity in CML cells, we next examined phosphatidylserineexposure, a hallmark of apoptosis, using Annexin Vcombined PI staining method. An accumulation ofAnnexin-V–positive cells was observed following imati-nib treatment confirming early-stage apoptosis induction,while BKT140 increased the number of both early apo-ptotic (Annexin Vþ/PI�) and late apoptotic/dead(Annexin Vþ/PIþ) cells (Fig. 2E). Moreover, combinationof imatinib with BKT140 significantly increased the num-ber of Annexin V/PI double-positive cells, indicatingsubsequent cell death (Fig. 2E and F).Next, we questioned whether the combined proapop-

totic effect of imatinib and BKT140 is mediated throughthe mitochondria using DiOC6 staining. Mitochondrialmembrane permeabilization is an important marker ofintrinsic and extrinsic pathways of apoptosis induction(30). We measured the effects of BKT140 and imatinibon the mitochondrial membrane potential (Dcm) inK562 cells. Each agent alone promoted modest mito-chondrial depolarization. Remarkably, the combinationof BKT140 with imatinib significantly reduced the Dcm

and increased the percentage of apoptotic cells to 68%(Fig. 2G and H). These results correspond with theresults obtained from viability assays and detect apo-ptotic pathway with mitochondrial involvement.

BKT140 reduces clonogenic potential of K562 cellsand targets primary CML CD34þ cells in vitroNext, we assessed the effect of BKT140 on the clono-

genic potential and colony formation ability of K562cells. Figure 3A demonstrates that BKT140 alone marked-ly inhibited soft agar colony growth, reducing the numberand size of colonies compared with untreated cells (P <

0.01). Combination of BKT140 with imatinib further sup-pressed colony formation, achieving 97% suppressioncompared with control (P < 0.01; Fig. 3B). We also deter-mined that the effect of BKT140 treatment on primaryCML CD34þ cells procured from newly diagnosedpatients with CML. BKT140 alone decreased the percent-age of viable CML CD34þ cells in culture in a dose-dependent manner. However, cotreatment with imatinibfurther promoted cell viability loss (Fig. 3C and D). Thesefindings demonstrate the ability of BKT140 to inhibit theclonogenic potential of CML cells and possibly to targetCML stem cells.

Effect of BKT140 treatment on apoptotic signaling inCML cells

To investigate the involvement of apoptotic signalingmolecules following BKT140 treatment, expression levelof apoptosis-related proteins was analyzed using anti-body array. For this, K562 and LAMA-84 cells weretreated with BKT140 (20 mmol/L) for 24 hours, celllysates were prepared, and apoptosis antibody arraywas performed. Our results show that a number ofapoptotic signaling proteins were modulated followingtreatment with BKT140 (Fig. 4A). Among the proapop-totic proteins, increase in catalase, TRAIL R2/DR5, andSMAC/Diablo was detected in both cell lines. Increasein proapoptotic Bad and Bax was determined in LAMA-84 cells. On the other hand, several antiapoptotic pro-teins were downregulated by BKT140, including BCL-2,BCL-XL, and XIAP. Interestingly, significant reductionin hypoxia-inducible factor-1a and HSP70 level wasdetected in both cell lines treated with BKT140. Toconfirm these results, effect of BKT140 treatment onchaperone protein levels in CML cells was evaluatedby immunoblotting. As demonstrated in Fig. 4B,BKT140 reduced the levels of HSP70 and HSP90 in bothK562 and LAMA-84 cells in a time- and dose-dependentmanner. Moreover, reduction in BCL-2 and BCL-XLlevels was observed in K562 cells treated with highconcentrations of BKT140 (Fig. 4B).

In addition, BKT140 dose dependently induced cyto-chrome c release, a late apoptotic event associated withloss of mitochondrial integrity. Combination of low-doseBKT140 (8 mmol/L) with imatinib significantly increasedimatinib-induced release of cytochrome c (Fig. 4C). Thesedata suggest that BKT140 triggers mitochondrial altera-tions, followed by cytochrome c release and apoptosis.

Interaction of CML cells with bone marrow stromalcells

To study the cross-talk between CML cells and thenonmalignant bone marrow counterparts, an in vitrococulture system was established. For this purpose,K562 and LAMA-84 cells were seeded on a pre-estab-lished monolayer of primary BMSC. Microscopic obser-vation revealed that K562 cells remained mainly in thefloating fraction, whereas LAMA-84 cells incorporatedinto the BMSCs monolayer and produced so called

CML and CXCR4





"cobblestone" structures (Fig. 5A). Generated BMSCs pro-duced detectable levels of CXCL12, confirmed by ELISA(Fig. 5B).

Effect of stromal cells on CML proliferation in serum-reduced conditions was evaluated using BrdUrd incor-poration method. Population of CFSE-labeled CML cellswas distinguished from stromal cells (Fig. 5C). BMSCssignificantly (P < 0.01) increased the percentage ofproliferating K562 and LAMA-84 cells (Fig. 5D). Imati-nib increased the surface expression of CXCR4 inLAMA-84 cells (Fig. 5E), demonstrating the undesirableupregulating effect of TKIs on CXCR4, that can poten-tially promote the CML–stroma protective interactions.

Next, we tested the effect of BMSCs on imatinib sensi-tivity. In accordance with previous reports, incubationwith stromal cells significantly reduced the sensitivity of

K562 cells to imatinib and completely blocked its inhib-itory effect in LAMA-84 cells, therefore demonstrating thestromal-provided protection. We then used BKT140 tostudy whether the sensitivity of CML cells to imatinibcan be restored by inhibition of the CXCR4 axis. Cellviability was tested using the XTT method. Interestingly,low concentration (8 mmol/L) of single-agent BKT140 inthe presence of BMSC had no significant effect on K562viability, and even increased the proliferation of LAMA-84 cells. However, a combination of BKT140with imatinibor usage of higher concentration of BKT140 (20 mmol/L)reversed the protective effect of BMSCs and significantlyimpaired CML proliferation (Fig. 5F).

These results suggest the ability of CXCR4 antagonistBKT140 to disrupt the interaction between CML andBMSCs, restoring the sensitivity to imatinib.

Figure 3. BKT140 suppressescolony formation and targetsprimary CML CD34þ cells in vitro.A and B, following pretreatmentwith BKT140 (20 mmol/L), imatinib(0.5 mmol/L), or combination ofboth agents for 24 hours, K562cells were plated in triplicates insoft agar and colonies werephotographed and counted after14 days. Chart shows mean values�SD and P value of twoindependent experiments. C andD, mononuclear cells from bonemarrow sample of patients withpatient in chronic phase wereincubated in the absence orpresence of BKT140 (8 and 20mmol/L), imatinib (0.5 mmol/L), orcombination of both agents for 48hours. Percent of viable CD34þ

cells was determined by 7-AADcounterstain and FACS analysis.7-AAD–positive dead cells weregated out. Data are presentedas mean of triplicates �SD(��, P < 0.01).

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BKT140 releases BMSC-inducedCML cell dormancyand effectively targets CML cells in the presence ofsupportive BMSCsTo evaluate the mechanisms involved in the anti-

CML effects induced by imatinib and BKT140, we testedthe cell cycle of K562 cells treated with each agentalone or in combination, in the absence or presence ofBMSCs. Figure 5G demonstrates representative histo-grams of DNA content in K562 cells cultured for48 hours in serum-deprived conditions. In the absenceof stroma, each agent alone reduced the percentageof proliferating (G2–MþS phase) cells. Combination ofimatinib with BKT140 further significantly decreasedthe percentage of cycling cells (Fig. 5H, a). Coculture

with BMSCs slightly increased the percentage ofK562 cells in G2–MþS phases, (from 22% withoutBMSCs to 30% in the presence of BMSCs, respectively).Imatinib treatment did not significantly affect thepercentage of cycling cells in the presence of stroma,whereas BKT140 slightly increased the percentage ofcycling cells. Importantly, the combination of imatinibwith BKT140 considerably decreased the percentageof proliferating cells even in the presence of BMSCs(Fig. 5H, b). These results could reflect the fact thatblockade of CXCR4/CXCL12 axis can overcomestroma-mediated CML cell quiescence and make thecells more susceptible to the antiproliferative effect ofimatinib.

Figure 4. Effect of BKT140treatment on apoptotic signalingpathways. A, K562 and LAMA-84were treated with 20 mmol/LBKT140 for 24 hours. The cellswere lysed, and the expression ofseveral pro- and antiapoptoticproteins was measured asdescribed in the Materials andMethods section. The relativeexpression of these proteinscompared with that of cells nottreated with BKT140 is presented.The controls are considered as 1(dotted line). B, K562 and LAMA-84cells were incubated in serum-reduced medium (1% FCS) in theabsence or presence of variousconcentrations of BKT140, for 24and 48 hours. Expression of theindicated proteins was monitoredby immunoblotting. Blots werestripped and reprobed with anti–b-actin antibody to ensure equalloading and transfer of protein(50 mg each lane). C, K562 andLAMA-84 cells were incubated inserum-reduced medium (1% FCS)in the absence or presence ofBKT140 (8 mmol/L), imatinib (0.25mmol/L), or combination of bothagents for 48 hours. Release ofmitochondrial cytochrome c tocytosol was measured by specificimmunolabeling and flowcytometry analysis. Decrease instaining intensity represents therelease of cytochrome c frommitochondria to cytosol.Percentage of low fluorescent cellpopulation compared with controlcells was quantified. Data arepresented as mean of triplicates�SD (��, P < 0.01).

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Figure 5. BMSCs support the survival and proliferation of CML cells. K562 and LAMA-84 cells were prestained with CFSE and cocultured in serum-reducedconditions (0.1%) for 72 hours in the absence or presence of BMSCs. A, representative images of CML cells on the monolayer of BMSCs are presented(original magnification of �400). B, CXCL12 levels in conditioned medium produced by BMSCs, measured by ELISA. C, the analysis was gated onCFSE-labeled cells. Representative plots of unlabeled BMSCS and CFSE-labeled K562 cells incubated in the absence or presence of BMSCs are depicted.D, proliferation of CFSE-stained K562 and LAMA-84 cells was determined using BrdUrd incorporation method. (Continued on the following page.)

Beider et al.





BMSCs enhance the imatinib-induced increase inBCL6 in a CXCR4-dependent mannerRecently, a new mechanism for imatinib resistance in

CML cells was reported. Proto-oncogenic transcriptionfactor BCL6 was found to be elevated upon imatinibtreatment and was required for the survival of CML-initiating cells (31). Because bone marrow stroma pro-tects CML cells from imatinib-induced apoptosis,we studied whether BCL6 is involved in the stroma-mediated protection. Corresponding with previouslyreported data, imatinib increased BCL6 expression inboth CML cell lines. Moreover, BCL6 mRNA levelswere even further increased by imatinib treatment inthe presence of BMSCs, suggesting the involvementof BCL6 in stroma-mediated resistance to TKIs. Toevaluate the possible regulatory role of CXCR4 in ima-tinib-mediated upregulation of BCL6, we assessed thelevels of BCL6 in CXCR4-transduced K562 cells.K562LG-CXCR4.86 cells expressed significantly higherlevels of BCL6 in comparison with the native K562, andimatinib treatment in coculture of K562LG-CXCR4.86and BMSCs further elevated BCL6 levels in the CXCR4-expressing K562 cells. Notably, BKT140 treatment effec-tively abrogated the imatinib-induced increase in BCL6in K562 as well as in K562LG-CXCR4.86 and LAMA-84cells (Fig. 5I).

BKT140 effectively targets K562 CML cells in vivoTo assess the in vivo activity of BKT140, a CML xeno-

graft model was established. Luciferase-expressing K562cells were injected intravenously or subcutaneously intoNOD/SCID mice and monitored using a CCCD camera.Intravenously injected K562L cells did not produce sys-temic disease and no spread to the vital organs wasdetected. In contrast, subcutaneously injected K562L cells(5 � 106) produced fast-growing locally invasive tumors,without systemic spread to the spleen or bone marrow(data not shown).Therefore, we used the local tumor model to evaluate

the effect of BKT140, alone or in combination with ima-tinib. Three days following cell inoculation, animals werestarted on treatment with BKT140 (400 mg/mouse), ima-tinib (10 mg/kg), or a combination of both agents inaccordance to the schedule presented in Fig. 6A. Imatinibdose was chosen based on previous report showing thatdaily treatment with 10 mg/kg partially inhibited tumorgrowth of BCR-ABL–transformed 32D cells (32). In accor-dance, imatinib treatment partially inhibited the growthof K562 xenograft, achieving 52% suppression in tumor

size. Importantly, BKT140 single-agent treatment wasable to significantly reduce tumor burden by 46%. Signif-icantly, imatinib and BKT140 combined treatment essen-tially abrogated tumor growth, resulting in 95% reductionin tumor size and weight (Fig. 6B and C). Histologicevaluation of hematoxylin and eosin (H&E)–stainedtumor sections demonstrated that combination of BKT140with imatinib promoted extensive necrotic tissue damagein treated tumors (Fig. 6D).

These results demonstrate the potent in vivo effect ofBKT140 in tumor burden reduction of CML, and reinforcethe role of CXCR4/CXCL12 axis in CML growth andprogression in vivo.

DiscussionIn this study,we investigated the role of CXCR4 inCML

progression. Using a lentiviral transduction method,CXCR4 was exogenously elevated in low-expressingK562 cell line to levels comparable with primary CD34þ

CML cells. Our data indicate impaired migration abilityand support a prosurvival role of CXCR4 signaling inCML.CXCR4 is expressed by a broad range of normal andmalignant hematopoietic cells and regulates chemotaxis,cell survival and proliferation in CXCL12-expressingniches (33–37). Consistent with previously reported data,our results demonstrate that CXCR4 is important for CMLsurvival and growth.

Awell-known aspect of BCR–ABL transformation is itsability to activatemultiple signaling pathways that lead toproliferation, reduced growth factor dependence andapoptosis, and abnormal interaction with extracellularmatrix and stroma. However, these pathways can be alsotriggered by external microenvironmental signals, inBCR-ABL–independent manner. Here, we show thatCXCR4 stimulation in CML cells results in profoundactivation of numerous signaling pathways, resulting inphosphorylation of Erk1/2, AKT, p70S6K, STAT3, andSTAT5. Mitogen-activated protein kinase, phosphoino-sitide 3-kinase, and JAK-STAT signaling are known to beinvolved in BCR-ABL–independent TKI resistance andrepresent targeting opportunities in CML (38). For exam-ple, STAT5was shown tomediate imatinib resistance andwas upregulated in leukemic cells from imatinib-resistantpatients (39). Similarly, CML cell lines cultured with HS5stromal cells were protected from imatinib-induced apo-ptosis by STAT3 upregulation (40). Here, we provideevidence for a mechanistic role of CXCR4 in activationof prosurvival signaling pathways in CML that was notpreviously described.

(Continued.) Data represent themeanof triplicates�SD (��,P <0.01). E, CXCR4expression byK562 andLAMA-84 cells cultured in the absenceor presence ofimatinib. F, CML cells were treated with imatinib (0.2 mmol/L for K562 or 0.1 mmol/L for LAMA-84), BKT140 (8 mmol/L), or combination of both agentsin the absence or presence of BMSCs for 48 hours. Viability was determined using XTT method. Data represent the mean of triplicates �SD (��, P < 0.01).G and H, K562 cells were incubated with imatinib (0.2 mmol/L), BKT140 (8 mmol/L), or combination of both agents in the absence or presence of BMSCs for48 hours and cell-cycle analysis was performed using 7-AAD staining. Percentage of cycling cells (in G2–M and S phases) is presented as mean oftriplicates �SD (��, P < 0.01). I, K562, K562LG-CXCR4.86 and LAMA-84 cells were pretreated with imatinib, BKT140, or combination of both agents for24 hours in the absence or presence of BMSCs and subjected to qPCR analysis. Expression of BCL6 was evaluated. Data represent the mean of triplicatesfrom two separate experiments �SD, all normalized against b2-microglobulin expression (��, P < 0.01).

CML and CXCR4





A large body of evidence, including our previouswork,indicates that the p210BCR-ABL oncoprotein impairs thefunction of the CXCR4/CXCL12 pathway in CML cells(19). Using leukemia MO7e cells, Geay and colleaguesdemonstrated that p210BCR-ABL inhibits the chemotacticresponse to CXCL12 and downregulates CXCR4 expres-sion (21). Chen and colleagues demonstrated thatp210BCR-ABL alters CXCL12-mediated adhesion throughb2-integrin (41). The mechanism underlying BCR-ABL–mediated inhibition of CXCR4 function in CML cellsinvolves the Src-related kinase Lyn. It was found thatBCR-ABL constitutively activates Lyn in CML cells,resulting in Lyn becoming unresponsive to CXCL12-

mediated signaling andpromoting the egress of immaturecells from the bone marrow (20). Importantly, it wasdemonstrated that TKI treatment restores CXCR4 expres-sion and CXCR4-mediated migration toward the bonemarrow niche, therefore promoting stroma-mediatedresistance of CML cells to TKI (22).

In agreement with previously reported data demon-strating an upregulation of CXCR4 in K562 and KBM-5cells upon imatinib treatment, our results show a similarincrease in CXCR4 surface levels in LAMA-84 cells, there-fore supporting a protective role for CXCR4-mediatedstroma interaction. Indeed, we found that adhesion toBMSCs increased the proliferation of CML cells and

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Figure 6. BKT140 displays anti-CML activity and cooperates withimatinib in vivo in xenograft model.K562L cells (5 � 106 per mouse)were injected subcutaneously intothe flank of NOD/SCID mice.Treatment was started 72 hoursfollowing cell inoculation.BKT140 (400 mg per injectionsubcutaneously.), imatinib(10 mg/kg, i.p.), or combination ofboth were administered accordingto the schedule, total of 11injections. Control mice wereinjected with saline. A, treatmentschedule. B, representativeimages of harvestedsubcutaneous tumors. C, tumorsize (length � width, cm2) andtumor weight (g), presentedas mean � SD of 4 mice(��, P < 0.01; Student t test).D, representative images ofH&E–stained section of K562L-produced xenograft tumors,magnification of �200 and �400.

Beider et al.





protected them from imatinib-induced apoptosis. Fur-thermore, previous work has demonstrated that adherentsubpopulations of CML cell lines retained increasedmalignant properties (16).On the basis of this data, we hypothesized that in the

presence of BMSCs, CXCR4 blockade with the specificantagonist BKT140 could restore CML cells’ sensitivity toimatininb. Indeed, CXCR4 inhibition is a recently devel-oped strategy to enhance the antitumor activity of che-motherapy drugs via interference with stroma-mediateddrug resistance in different solid and hematologic can-cers, including CML. CXCR4 inhibition with the small-molecule inhibitor AMD3100 (plerixafor) was found tobe effective in combination with TKIs both in vitro andin vivo. Thus, pretreatmentwithAMD3100 in vitro increas-ed the imatinib-induced apoptosis of stroma-protectedBV173 lymphoid BCR-ABL-positive cells in culture, andsignificantly reduced the repopulating CML content inbonemarrow and spleen of SCIDmice (42). Anotherworkdemonstrated that combination of AMD3100 with sec-ond-generation TKI nilotinib restored the sensitivity ofstroma-protected human K562 and KU812F CML cellsin vitro. In addition, AMD3100 enhanced nilotinib-induc-ed reduction of 32D.p210 cell-produced tumor in vivo in amurine model of CML (43). However, AMD3100 did notdemonstrate single-agent activity against CML cells andhad no additive effect to TKI-induced apoptosis of CMLcells cultured without BMSCs.We previously demonstrated a role for the CXCR4

antagonist BKT140 inmobilization ofHSCs from the bonemarrow (44). In addition, BKT140was shown tomediate apotent cytotoxic activity against various hematologicmalignancies, including multiple myeloma (MM), acutepromyelocytic leukemia (APL), and non-Hodgkin lym-phoma (NHL) cells, respectively (24, 26). In the currentwork, we were able to show that BKT140 treatmentinduced a modest inhibition of CML cell growth in vitro.Combining BKT140 with imatinib significantly increasedthe anti-CML effect, inducing cell apoptosis with mito-chondrial involvement. Furthermore, BKT140 treatmentameliorated the BMSC-mediated resistance to imatiniband effectively targeted CML cells when combined withimatinib.Importantly, this report provides the first evidence

illuminating the molecular mechanism of BKT140-trri-gered apoptosis. BKT140 treatment increased the level oftheH2O2-specific scavenger catalase,while simultaneous-ly downregulated the chaperone proteins HSP70 andHSP90 in CML. These changes suggest the involvementof cellular chaperones in BKT140-mediated cell death.Chaperones were shown to be important regulators ofapoptosis. HSPs protect cells from stress-induced apopto-sis and have been shown to interact with different keyapoptotic proteins and to block caspases activation (45).Furthermore, a proapoptotic role of catalase via inhibitionofHSP70, promotion of cytochrome c release and caspase-3 activation, was demonstrated in leukemic U937 cell line(46). Chaperones are also implicated in CML pathogen-

esis. Disruption of chaperone function by histone deace-tylase inhibitors was shown to enhance TKI activity (47).

Our findings of mitochondrial destabilization withsubsequent release of cytochrome c and SMAC/Diablofollowing BKT140 treatment may represent secondaryapoptotic events. Nevertheless, deeper characterizationof the mechanism underlying BKT140-induced apoptosisshould be performed.

Interestingly, single-agent BKT140 treatment of CMLcells in the presence of stroma increased the percentage ofcycling cells. Interaction with BMSCs is particularlyimportant in CML. It was suggested that BMSCs canpreserve the proliferative capacity and renewal ability ofCML cells by inhibition of cell-cycle progression andmediation of antiapoptotic signals, thus protecting CMLprogenitors and leukemia stem cells fromTKIs action andpreventing full CML elimination (48–50). The increase inproportion of cycling cells upon BKT140 treatment in thepresence of BMSCs may actually indicate that BKT140could release the CML cells from BMSC-induced quies-cent state. Thus, interference with BMSCs-associatedCML cell quiescence using CXCR4 blockade may be oneof the mechanisms by which BKT140 restores sensitivityto TKI treatment.

Our results identify BCL6 as another factor that maybe involved in the stroma-provided protection fromimatinib-induced apoptosis. Recent works have dem-onstrated that upregulation of BCL6 is induced by TKIs,mediates resistance to TKIs, and enables the survival ofCML stem cells and Phþ ALL cells (31, 51). In accor-dance with these reports, we show that imatinibincreased expression of BCL6 in K562 and LAMA-84cells. Furthermore, we found that imatinib treatment inthe presence of BMSCs further elevated BCL6 mRNAlevels, indicating a role for stroma in BCL6-mediatedTKI resistance. Moreover, CXCR4-transduced K562cells demonstrated a higher basal, as well as TKI-induced levels of BCL6, suggesting the possible role ofCXCR4 in BCL6 regulation in CML cells. Importantly,BKT140 treatment effectively mitigated the imatinib-induced increase in BCL6 levels in the absence as wellas the presence of BMCSs. Although the mechanisms bywhich BMSCs affect the imatinib-induced increase inCML cells in BCL6 levels require further studies, ourdata indicate the importance of CXCR4/CXCL12 axis inthis process and further substantiate that CXCR4 isrational target for combinational therapy in CML.

Collectively, our results demonstrate the importantrole of CXCR4 in activation of major downstream reg-ulators of proliferation and survival responses in CMLcells, and suggest that CXCR4 is an important mediatorin CML–stromal protective interactions. CXCR4 inhibi-tion with BKT140 efficiently cooperated with imatinib,overcoming the protective effect of the bone marrowstroma. BKT140 treatment reduced the stroma-mediat-ed dormancy and restored elevated BCL6 levels tobaseline levels, therefore resensitizing CML cells toimatinib and effectively promoting CML cell death.

CML and CXCR4





Moreover, BKT140 demonstrated single-agent anti-CML activity in vivo and significantly potentiated ima-tinib activity against K562-produced tumors. Theseresults provide the rational basis for CXCR4-targetedtherapy in combination with TKI to override drugresistance and eliminate residual disease.

Disclosure of Potential Conflicts of InterestM. Koren-Michowitz has honoraria from speakers’ bureau and is a

consultant/advisory board member of Novartis. A. Peled is employedas CSO, has ownership interest (including patents) in, and is a consul-tant/advisory board member for, and a shareholder in Biokine Ther-apeutics. A. Nagler is a medical consultant for Biokine Therapeutics,Ltd. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: K. Beider, M. Darash-Yahana, M. Koren-Micho-witz, M. Abraham, O. Wald, A. Peled, A. NaglerDevelopment of methodology: K. Beider, M. Darash-Yahana, O. Blaier,M. Abraham, H. Wald, O. Wald, A. Peled, A. Nagler

Acquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): K. Beider, M. Darash-Yahana, O. Blaier,M. Koren-Michowitz, A. NaglerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K. Beider, M. Darash-Yahana, O. Blaier,M. Koren-Michowitz, O. Eizenberg, A. PeledWriting, review, and/or revision of themanuscript:K. Beider,M.Darash-Yahana,M. Koren-Michowitz, E. Galun, O. Eizenberg, A. Peled, A. NaglerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. BeiderStudy supervision: A. Peled, A. Nagler

Grant SupportThis work was supported (in part) by scientific grant from Guy

Weinshtock Foundation (to A. Nagler), Sarousy Foundation grant (to A.Nagler), and the Israeli Jack Craps foundation for funding this research.

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 May 22, 2013; revised January 17, 2014; accepted January 27,2014; published OnlineFirst February 6, 2014.

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CML and CXCR4





2014;13:1155-1169. Published OnlineFirst February 6, 2014.Mol Cancer Ther Katia Beider, Merav Darash-Yahana, Orly Blaier, et al.

In Vivo and VitroInOvercomes the Protective Effect of Stroma and Targets CML

Combination of Imatinib with CXCR4 Antagonist BKT140

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Combination of Imatinib with CXCR4 Antagonist BKT140 ...mct.aacrjournals.org/content/molcanther/13/5/1155.full.pdfin the bone marrow niche (12–14). Furthermore, stroma-derived CXCL12