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Cancer Therapy: Preclinical
Targeting the CD20 and CXCR4 Pathways in Non-HodgkinLymphoma with Rituximab and High-Affinity CXCR4Antagonist BKT140
Katia Beider1, Elena Ribakovsky1, Michal Abraham3, Hanna Wald3, Lola Weiss2, Evgenia Rosenberg1,Eithan Galun2, Abraham Avigdor1, Orly Eizenberg3, Amnon Peled2,3, and Arnon Nagler1
AbstractPurpose:Chemokine axis CXCR4/CXCL12 is critically involved in the survival and trafficking of normal
andmalignant B lymphocytes.Here,we investigated the effect of high-affinityCXCR4antagonist BKT140on
lymphoma cell growth and rituximab-induced cytotoxicity in vitro and in vivo.
Experimental Design: In vitro efficacy of BKT140 alone or in combination with rituximab was
determined in non-Hodgkin lymphoma (NHL) cell lines and primary samples from bonemarrow aspirates
of patients with NHL. In vivo efficacy was evaluated in xenograft models of localized and disseminated NHL
with bone marrow involvement.
Results: Antagonizing CXCR4 with BKT140 resulted in significant inhibition of CD20þ lymphoma cell
growth and in the induction of cell death, respectively. Combination of BKT140with rituximab significantly
enhanced the apoptosis against the lymphoma cells in a dose-dependent manner. Moreover, rituximab
induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, suggesting the possible
interaction between CD20 and CXCR4 pathways in NHL. Primary bone marrow stromal cells (BMSC)
further increased CXCR4 expression and protected NHL cells from rituximab-induced apoptosis, whereas
BKT140 abrogated this protective effect. Furthermore, BKT140 showed efficient antilymphoma activity in
vivo in the xenograft model of disseminated NHL with bone marrow involvement. BKT140 treatment
inhibited the local tumor progression and significantly reduced the number of NHL cells in the bone
marrow. Combined treatment of BKT140with rituximab further decreased the number of viable lymphoma
cells in the bone marrow, achieving 93% reduction.
Conclusions: These findings suggest the possible role of CXCR4 in NHL progression and response to
rituximab and provide the scientific basis for the development of novel CXCR4-targeted therapies for
refractory NHL. Clin Cancer Res; 19(13); 3495–507. �2013 AACR.
IntroductionNon-Hodgkin lymphoma (NHL) is a heterogeneous
group of malignancies of B cells or T cells (1). Approxi-mately 80% to85%ofNHL areB-cellmalignancies in originand more than 95% of these express surface CD20.Combining chimeric monoclonal anti-CD20 antibody
rituximab (2) with standard chemotherapy regimens is
associatedwith higher response rates and improved survivalin a subset of patients. Unfortunately, a significant percent-age of patients who initially respond to rituximab, eventu-ally relapse (3). Scientific efforts are increasingly beingfocused in developing new strategies to improve monoclo-nal antibody (mAb) activity (4).
Stromal cell–derived factor-1 (SDF-1/CXCL12; ref. 5),was initially identified as a pre-B-cell growth-stimulatingfactor (6). CXCL12 signals through CXCR4, a 7 transmem-brane, G-protein–coupled receptor, that is expressed bynormal and malignant cells of hematopoietic and nonhe-matopoietic lineage (7). Data from knockout mice indicatethat the CXCR4 receptor plays an important role in hema-topoiesis (8–11). CXCR4–CXCL12 axis is particularlyimportant in the homing and retention of hematopoieticprogenitor cells in the marrowmicroenvironment (12, 13).
There is growing evidence that CXCR4 expression andfunction in hematopoietic malignancies have a majorimpact on disease progression. CXCR4 levels are signifi-cantly elevated in B-cell chronic lymphocytic leukemia(B-CLL; ref. 14), B-cell but not T-cell acute lymphoblastic
Authors' Affiliations: 1Division of Hematology, Bone Marrow Transplan-tation andCord Blood Bank, ShebaMedical Center, Tel-Hashomer; 2Gold-yne Savad Institute of Gene Therapy, Hadassah Hebrew University Hos-pital, Jerusalem; and 3Biokine Therapeutics Ltd., SciencePark,NessZiona,Israel
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Arnon Nagler, Division of Hematology, BoneMarrow Transplantation and Cord Blood Bank, Chaim Sheba MedicalCenter, Tel-Hashomer, Israel. Phone: 972-3530-5830; Fax: 972-3530-5830; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-12-3015
�2013 American Association for Cancer Research.
ClinicalCancer
Research
www.aacrjournals.org 3495
leukemia (15, 16), multiple myeloma (17), and some acutemyeloid leukemias (AML; ref. 18). CXCR4 mediates thehoming to and engraftment of AML (19) and pre-B acutelymphoid leukemia cells to the bone marrow of nonobesediabetic/severe combined immunodeficient (NOD/SCID)mice (20). CXCR4/CXCL12 interactions not only protectCLL cells from apoptosis, but also allow the migration ofCLL cells beneath bone marrow stromal cells (BMSC),suggesting that CLL cells use this mechanism to infiltratethe marrow (21).
Microenvironment-mediated chemoresistance, whichinvolves CXCR4/CXCL12 axis, is now well recognized indifferent hematologicmalignancies, including ALL andCLL(7). However, its role in NHL is less defined. Further studiesare thus required to elucidate the critical determinants ofCXCR4-mediated resistance of malignant B-cell disorders.The knowledge of such mechanisms will guide identifica-tion of molecular targets for therapeutic interventions over-coming rituximab resistance.
Materials and MethodsCells and cell lines
The following human NHL cell lines were used: Burkittlymphoma cell lines Raji, Ramos, BL-2, and BJAB, anddiffuse large B-cell lymphoma (DLBCL) cell lines OCI-LY7,OCI-LY19, and SUDHL-4. The cells were kindly providedbylaboratory of Prof. Dina Ben-Yehuda (Hadassah HebrewUniversity Hospital, Israel). The cells were maintained inlog-phase growth inRPMI-1640medium (Biological Indus-tries) supplemented with 10% heat-inactivated fetal calf
serum (FCS), 1 mmol/L L-glutamine, 100 U/mL penicillin,and 0.01 mg/mL streptomycin (Biological Industries) in ahumidified atmosphere of 5% CO2 at 37
�C.After informed concept, primaryNHL cells were collected
from the bone marrow of 7 patients with NHL. Diagnosiswas diffuse large B-cell NHL.
Measurement of surface CD20, CD19, and CXCR4expression
The expression of CD20, CD19, and CXCR4 (clone12G5) on the surface of NHL cell lines and primary NHLcells was evaluated using specificmAbs (eBiosience).Mouseisotype control antibodies were also purchased fromeBioscience. The cells were analyzed by FACScalibur (Bec-ton Dickinson Immunocytometry Systems) using Cell-Quest software.
Cell viability assayNHL cells were seeded at 2 � 105 viable cells/1 mL per
well into a 24-well plate in triplicates in a medium supple-mented with 1% FCS and incubated with different concen-trations of 4F-benzoyl-TN14003 (BKT140; Biokine Thera-peutics Ltd.) or Rituximab (Roche, purchased from ShebaMedical Centre pharmacy) for 48 hours. Following theincubation, the cells were stained with propidium iodide(PI; Sigma-Aldrich) and the percentage of viable PI-negativecells in culture was determined by FACScalibur.
Apoptosis assaysNHL cells were plated at a density of 2� 105 viable cells/
mL in a medium supplemented with 1% FCS and culturedwith different concentrations of BKT140 and rituximab for48 hours. Apoptosis was determined by staining withAnnexin V-fluorescein isothiocyanate (FITC) and 7-ami-no-actinomycin D (7-AAD; eBioscience) according to man-ufacturer’s instructions and analyzed by flow cytometry.Caspase-3 enzymatic activity was measured using the Casp-GLOW Red Caspase-3 Staining Kit (MBL International),according to the manufacturer’s instructions.
Assessment of mitochondrial membrane potential(Dcm)
The cationic lipophilic fluorochrome DiOC6 (Sigma-Aldrich) was used to measure the Dym. Briefly, NHL cellswere cultured with different concentrations of BKT140 andrituximab for 12 hours, harvested, resuspended in fluores-cence-activated cell sorting (FACS) buffer (PBS with 0.1%FCS and 0.1% NaN3) containing 200 nmol/L DiOC6, andincubated for 15 minutes in a 37�C. The cells were thenanalyzed by FACS for the loss of DiOC6 fluorescence.
Cell-cycle analysisNHL cells were exposed in vitro to different concentra-
tions of BKT140 and rituximab for 48 hours. The cells werecollected, washed with cold PBS, and then 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 incubated at
Translational RelevanceB-cell non-Hodgkin’s lymphoma (NHL) represents
the most common malignant lymphoid neoplasm.Although anti-CD20 antibody rituximab significantlyimproved the outcome of patients with NHL, therelapsed/refractory rates are still high. Chemokine recep-tor CXCR4 and its ligand CXCL12 are critically involvedin the survival and trafficking of normal andmalignant Blymphocytes. The interaction of malignant B cells withstromal cells via CXCR4/CXCL12 signaling may providechemoresistance. Therefore, blockade of CXCR4 mayantagonize the survival and spreading of lymphomacells and restore their chemosensitivity.Our results show potent antilymphoma effect of
CXCR4-specific high-affinity antagonist BKT140 in vitroand in vivo. The BKT140-mediated antilymphoma effectsynergizes with that of rituximab. Moreover, BKT140effectively targets lymphoma cells in the bone marrowmicroenvironment, overcoming the stroma-inducedresistance to rituximab.These findings suggest the possible interaction
between CD20 and CXCR4 pathways in NHL and pro-vide the scientific basis for the development of novelcombined CXCR4-targeted therapies for refractory NHL.
Beider et al.
Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3496
37�C for 15minutes. The cells were then stainedwith 10mg/mL7-AAD in the dark for 30minutes. TheDNA contentwasdetected using FACS.
Cell migration assayMigration assay was conducted in triplicate using 5-mm
pore size Transwells (Costar). The bottom compartmentwas filled with 600 mL of 1% FCS RPMI-1640 mediumcontaining different (50, 250, and 500 ng/mL) concentra-tions of CXCL12 (PeproTech EC), and 5 � 105 cells in 100mL of 1% FCS RPMI-1640 medium were applied to the topcompartment. The amount of cells that migrated within 4hours to the bottom compartment was determined by FACSand expressed as a percentage of the input.
Coculture experimentsPrimary human BMSCswere isolated and expanded from
bone marrow aspirates of healthy donors after signedinformed consent. The mononuclear cells were plated inDulbecco’s modified Eagle medium (DMEM) with lowglucose (Biological Industries) supplemented with 15%heat-inactivated FCS, 1 mmol/L L-glutamine, 100 U/mLpenicillin, and0.01mg/mL streptomycin (Biological Indus-tries) in a humidified atmosphere of 5% CO2 at 37�C.Mediumwas refreshed once a week and adherent cells werecultured. BMSCswere plated at a density of 2� 104 cells perwell in 24-well plates and incubated overnight. The follow-ing day,NHL cellswere seeded on top of stromal cells, aloneor in combination with BKT140 and rituximab, at a densityof 2 � 105 cells/mL in a medium supplemented with 1%FCS. Following 48 hours of coincubation, nonadherentcells were collected and adherent cell fractionwas harvestedwith trypsin/EDTA. The cells were washed with PBS andanalyzed by FACS for viability (using PI exclusion) andCXCR4 expression. The cells were counterstained with anti-CD20 and NHL cells were distinguished from BMSCs aftergating on CD20þ lymphoma cells.
Murine xenograft models of human NHLNOD/SCID mice were maintained under defined flora
conditions at the Hebrew University Pathogen-Free AnimalFacility (Jerusalem, Israel). All experiments were approvedby the Animal Care Committee of the Hebrew University.NOD/SCID mice were injected subcutaneously with BJABor BL-2 cells (5 � 106/mouse) into the right flank anddeveloped clearly palpable tumors. To investigate the ther-apeutic potential of CXCR4 antagonist BKT140 on NHLdissemination and growth, mice injected with BL-2 cellswere treated with subcutaneous injections of 300 mgBKT140 or control saline, in a site different to tumorinjection. For the combination therapy studies, BL-2–bear-ing mice were treated with 300 mg of BKT140, 500 mg ofrituximab, or combination of both agents, administered byseparate subcutaneous injections.
CXCR4 immunohistochemistrySubcutaneous xenograft tumors generated by BL-2 cells
injected into NOD/SCID mice were harvested and fixed in
4% PFA. Paraffin-embedded sections (10 mmol/L) wereinitially dewaxed, rehydrated, treated with EDTA buffer,and blocked with CAS-blocking reagent (Zymed Laborato-ries) for 30 minutes at room temperature. Samples werethen incubated overnight at 4�C in a humidified chamberwith antihuman CXCR4 mAb, clone 12G5 (R&D Systems)diluted to final concentration of 10 mg/mL. Next, the sec-tions were incubated with secondary anti-mouse horserad-ish peroxidase–conjugated antibody (DakoCytomation)for 30 minutes at room temperature. 3-Amino-9-ethylcar-bazole (AEC) was used for color development, and thesections were counterstained with hematoxylin.
In situ TUNELApoptotic nuclei in control and BKT140-treated BL-2 or
BJAB xenograft tumor sections were visualized using theFluorescein In Situ Cell Death Detection Kit accordingto the manufacturer’s instructions (Roche Diagnostic). Theslides were then mounted with Permount Mounting Medi-um (Fisher Scientific) and analyzed under a fluorescentmicroscope.
ResultsCXCR4 expression in NHL cell lines and in primarylymphoma cells from patients with bone marrowinvolvement
First, we characterized the surface expression of CXCR4 ina panel of human NHL cell lines (n ¼ 7). Strong CXCR4expression was found in Burkitt lymphoma BL-2, Raji, andRamos cells, intermediate CXCR4—in DLBCL cell linesOCI-LY7, OCI-LY19, and SU-DHL-4, and low—in BJABcells, respectively (Supplementary Fig. S1A). All NHL celllines expressedhigh levels ofCD20antigen (SupplementaryFig. S1B), therefore being reliable targets for anti-CD20antibody rituximab. Primary CD20þ lymphoma cells inthe bone marrow samples from the patients also expressedhigh levels of CXCR4 (Supplementary Fig. S1C).
To assess the functionality of CXCR4 receptor expressedby lymphoma cells, we tested the in vitro chemotaxis ofNHLcells. Cell lines BL-2 and BJAB and primary lymphoma cellsfrom patients with NHL with bone marrow involvementwere allowed tomigrate in response to elevated amounts ofCXCL12. As shown in Supplementary Fig. S2A, CXCL12induced the Transwell migration of high CXCR4-expressingcells BL-2 in a dose-dependent manner, whereas lowCXCR4-expressing cells BJAB did not respond to CXCL12.Furthermore, primary lymphoma cells showed dose-depen-dentmigratory response to CXCL12. Importantly, CXCL12-induced cell migration of primary lymphoma cells corre-lated with CXCR4 cell surface expression (SupplementaryFig. S2B).
Rituximab inhibits the cell growth and induces CXCR4surface expression in NHL cells
Next, the effect of rituximab on lymphoma cell growth invitro was examined, exposing BL-2, BJAB, and Raji cells tothe elevated concentrations (10 and 100 mg/mL) of ritux-imab in vitro. Rituximab directly inhibited the cell growth
Non-Hodgkin Lymphoma and CXCR4
www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3497
and significantly diminished the number of viable cells inculture. The inhibitory effect was dose dependent. HighCXCR4-expressing cells BL-2 and Raji showed higher sen-sitivity to rituximab than low CXCR4-expressing cells BJAB(Fig. 1A). Subsequently, the effect of rituximab treatment onCXCR4 expression levels in BL-2, BJAB, and Raji cells wastested. Interestingly, statistically significant increase in cellsurface expression of CXCR4 was induced by rituximab inall 3 NHL cell lines tested. Furthermore, similar effect wasobserved in primary lymphoma cells obtained frompatients with NHL with bone marrow involvement—incu-bationwith rituximab induced surface expression ofCXCR4(Fig. 1B). Taking together, these results show CXCR4-mod-ulating effect of rituximab in NHL, showing that anti-CD20antibody rituximab decreases the viability, but increasesCXCR4 expression in NHL cells.
CXCR4 inhibitor BKT140 induces apoptosis of NHLcells and cooperates with rituximab in vitro inantilymphoma activity
Next, the effect of CXCR4 inhibitor BKT140 on NHL cellviability was assessed. BL-2, BJAB, and Raji cells wereincubated with different concentrations of BKT140 (4–100 mmol/L) during 48 hours and cell viability was testedby flow cytometry using the PI exclusionmethod. As shownin Fig. 2A and B, BKT140 significantly reduced the numberof viable cells and increased the percentage of dead cells in adose-dependent manner in all 3 NHL cell lines tested. HighCXCR4 BL-2 and Raji cells showed higher sensitivity to
BKT140-induced cell death than low CXCR4 BJAB cells.Next, the effect of combination of BKT140 with rituximabon NHL cell viability was evaluated. The cells were treatedeither with BKT140 8 mmol/L, rituximab 10 mg/mL, orcombination of both agents during 48 hours, and cellviability was measured using the PI exclusion method. Thecombined treatment with both compounds significantlyreduced the number of viable cells in culture (Fig. 2C),suggesting that BKT140 enhances the anti-NHL effect ofrituximab. Similar effect of BKT140-induced cytotoxicitywas observed in additional NHL lines tested (Ramos andOCI-LY19; data not shown) and primary NHL cells frompatients with bone marrow involvement (Fig. 2D). Todetermine the specificity of BKT140 and rituximab effects,CD20� CXCR4-positive Jurkat T cells were used, whichresponded to BKT140 treatment but were not affected byrituximab (Supplementary Fig. S3A).
BKT140 induces apoptotic cell death of NHLTo analyze the mechanism of BKT140-induced cytotox-
icity in NHL cells, we next examined phosphatidylserineexposure, a hallmark of apoptosis, using Annexin V com-binedwith the 7-AAD stainingmethod. An accumulation ofAnnexin-V–positive cells was observed following rituximabtreatment confirming early-stage apoptosis induction,whereas BKT140 increased the number of both early apo-ptotic (Annexin Vþ/7-AAD�) and late apoptotic/dead(Annexin Vþ/7-AADþ) cells. Furthermore, combinationof rituximab with BKT140 significantly increased the
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Figure 1. Effect of rituximab on NHL cell viability and CXCR4 expression. A, viability of NHL cell lines and primary lymphoma cells from bone marrowaspirates of patientswithNHL, treatedwith elevated doses of rituximab (Ritux, 10 or 100mg/mL) during 48 hours, was analyzed using the PI exclusionmethod.B, NHL cell lines and primary NHL cells were incubated in serum-reduced conditions (1% FCS) in the absence or presence of rituximab (20 mg/mL)during 48 hours, and CXCR4 cell-surface expression was evaluated and quantified by FACS. Data are represented as mean of triplicates � SD. Probabilityvalues of t test are presented (��, P < 0.01). Experiment was repeated twice. MFI, mean fluorescence intensity.
Beider et al.
Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3498
number of Annexin V/7-AAD double-positive cells, indi-cating subsequent cell death (Fig. 3A). To examine theapoptotic pathways activation, we assessed the cleavage ofcaspase 3 in NHL cells treated with BKT140 and rituximab.We first found that BKT140 treatment induced caspase-3activation in NHL cells in a dose-dependent manner. More-over, combination of BKT140 (8 mmol/L) with rituximab(100 mg/mL) significantly increased caspase-3 activation inBL-2 and Raji cells (Fig. 3B and Supplementary Fig. S3B).The effect of BKT140 and rituximab on cell-cycle progres-
sion and DNA distribution in NHL cells was then studied.Rituximab slightly increased the BL-2 cells population inG2–M phase, therefore promoting G2–M cell arrest. Incontrast, BKT140 treatment resulted in the accumulationof the cells at sub-G0–G1 phase, indicating theDNAdamageconsistent with apoptosis. Combination of BKT140 withrituximab reversed the G2–M arrest and further increased
the population in sub-G0–G1 phase, up to 54% in BL-2 andto 45% in BJAB cells, respectively, therefore further pro-moting apoptotic DNA fragmentation (Fig. 3C).
To further elucidate the mechanism by which BKT140enhances rituximab-induced apoptosis in NHL cells, weexamined the possible involvement ofmitochondria testingmitochondrial transmembrane potential (Dym) usingDiOC6 dye. Combination of BKT140 (8 mmol/L) withrituximab (100 mmol/L) significantly increased the numberof apoptotic depolarized cells in all 3 NHL cell lines tested,compared with the effect induced by each agent alone.Importantly, high CXCR4-expressing cells BL-2 and Rajishowed higher sensitivity and enhanced rate of apoptosispromoted by the combination of BKT140 and rituximabthan low CXCR4-expressing BJAB cells (Fig. 3D and Sup-plementary Fig. S3C). These results further emphasize therole of CXCR4 in BKT140-induced NHL apoptosis and
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Figure 2. BKT140 dose dependently decreases the viability of NHL cells and cooperateswith rituximab in vitro. NHL cell lines BL-2, BJAB, andRaji treatedwithelevated concentrations of BKT140 (4, 8, 20, and 40 mmol/L) during 48 hours. Viability was determined using the PI exclusion method using FACSanalysis, and percentages of viable (A) versus dead (B) cells were detected. C, the indicated NHL cell lines were coincubated in the absence or presenceof 8 mmol/L BKT140, 10 or 100 mg/mL rituximab, or combination of both agents for 48 hours. D, primary lymphoma cells from bone marrow aspiratesof patients with NHL with bone marrow involvement were incubated in the absence or presence of 8 mmol/L BKT140, 20 mg/mL rituximab, or combination ofboth agents for 48 hours. Data are represented as mean of triplicates � SD. Probability values of t test are presented (��, P < 0.01).
Non-Hodgkin Lymphoma and CXCR4
www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3499
strength the rational for CXCR4 inhibition combined withrituximab for the treatment of CXCR4-expressing B-celllymphomas.
Interaction with BMSCs elevates surface CXCR4expression on NHL cells and supports their survivaland proliferation
Previous reports have shown that stromal cells in bonemarrow microenvironment support CLL survival and pro-tect the cells from chemotherapy-induced apoptosis (22,23). To investigate the interaction between NHL cells andbone marrow microenvironment in vitro and its effect onrituximab sensitivity, lymphoma cell lines BL-2, Raji, andBJABwere treatedwith rituximab in the absence or presenceof primary BMSCs. Floating and adherent fractions of NHLcells were analyzed differentially.
First, the incorporation of lymphoma cell lines into themonolayer of stromal cells was examined. Interestingly,
high CXCR4-expressing BL-2 and intermediate CXCR4-expressing Raji cells effectively adhered and transmigratedthrough the confluent monolayer of BMSCs, producing so-called "cobble stone" structures. In contrast, low CXCR4-expressing cells BJAB mostly remained in nonadherentfloating fraction (Supplementary Fig. S4A). Then, the effectof stromal cells on NHL cell survival and proliferation inserum-reduced conditions was evaluated. Consistent withmicroscopic observations, BMSCs effectively supported thesurvival and proliferation of CXCR4-expressing cells BL-2and Raji that were in close contact with stroma. Relativeincrease in lymphoma proliferation was in correlation withCXCR4 expression levels. In contrast, the proliferation oflow CXCR4-expressing cells BJAB was not significantlyaffected by the interaction with BMSC (Fig. 4A).
Next, the effect of BMSCs on CXCR4 expression bylymphoma cells was evaluated. Primary BMSCs significant-ly increased the surface expression of CXCR4 by BL-2 and
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Figure 3. BKT140 and rituximab synergistically induce NHL cell apoptosis with mitochondrial involvement and promote caspase-3 activation. A, BL-2 cellswere incubated in the absence or presence of 8 mmol/l BKT140, 100 mg/mL rituximab, or combination of both agents for 48 hours. Apoptosis was detectedusing Annexin V-FITC and 7-AAD costaining. The percentage of early apoptotic (Annexin Vþ/7-AAD�) and late apoptotic/dead (Annexin Vþ/7-AADþ) isdisplayed. B, caspase-3 cleavage was determined by fluorescent labeling of activated caspase 3 in BL-2 and Raji cells treated with 8 mmol/L BKT140,100 mg/mL rituximab, or combination of both agents for 48 hours. C, cell-cycle analysis by 7-AAD staining was conducted on BL-2 and BJAB cellsincubated in the absence or presence of 8 mmol/L BKT140, 100 mg/mL rituximab, or combination of both for 48 hours. The subdyploid DNA peak (sub-G0–G1)represents apoptotic cell fraction. D, mitochondrial membrane potential (Dym) was determined by flow cytometry using DiOC6 staining in BL-2, BJAB, andRaji cells incubated in the absence or presence of 8 mmol/L BKT140, 100 mg/mL rituximab, or combination of both for 12 hours. Apoptotic cells showed adecrease in staining with DiOC6. Data are presented as mean of triplicates � SD (��, P < 0.01).
Beider et al.
Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3500
Raji cells. The effect was more profound in adherent cellfraction. Rituximab treatment further elevated the CXCR4levels on lymphoma cells (Fig. 4B and Supplementary Fig.S4B). These data suggest that primary BMSCs protect NHLBcells from spontaneous apoptosis, in agreement with pre-viously published findings. Moreover, the enhancing effectof primary stromal cells on CXCR4 expression by NHLcells was hereby shown for the first time, emphasizingthe role of bone marrow microenvironment in CXCR4-mediated lymphoma cell survival.
BKT140 reverses the protective effect of BMSCs andenhances rituximab-induced cell deathSubsequently, the ability of primary stromal cells to
confer the resistance of NHL cells to rituximab was exam-ined. Viability of lymphoma cell lines treated with ritux-imab for 48 hours in the absence or presence of primaryBMSCs was tested. BMSCs were able to protect lymphomacells against rituximab-induced cytotoxicity (Fig. 4C).The ability of BKT140 antagonist to overcome the stroma-
mediated resistance of NHL cells was tested next. Initially,the effects of BKT140 in combination with rituximab wereexamined in BL-2, BJAB, and Raji cells, cultured with or
without BMSCs support. Low dose of BKT140 (8 mmol/L)effectively targeted floating lymphoma cells. In contrast,lymphoma cells that were in direct contact with stroma cellswere protected from lose dose of BKT140. However, ele-vated dose of BKT140 (40 mmol/L) significantly (P < 0.01)induced cell death in bothfloating and adherent fractions ofCXCR4-expressing lymphoma cells cultured with BMSCs,abrogating the protective effect of stromal cells. Combina-tion of BKT140 with rituximab further increased the ritux-imab-mediated cell death in the presence of BMSCs (Fig.4D).
Establishment of in vivo model of disseminated NHLwith bone marrow involvement
To assess the effect of BKT140 inhibitor on lymphomadevelopment and spread in vivo, xenograft model of dis-seminated lymphoma with bone marrow involvement inmice was established. Human BL-2 cells were injectedsubcutaneously into NOD/SCID mice, developed invasivelocal tumors (at days 12–14 following the cell injection),and then specifically spread to the bone marrow (days 21–28). All animals inoculated with BL-2 cells developed localtumors and subsequent dissemination to the bonemarrow.
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Figure 4. Interaction with BMSC promotes NHL growth, elevates CXCR4 on NHL cells, and protects them from rituximab. BKT140 treatment overcomesthe protective effect of stromal cells. A, NHL cells (2 � 105/mL) were cocultured in serum-reduced conditions (1% FCS) in the absence or presence ofBMSCs for 24 and 48 hours. Viable NHL CD20þ cells in floating and adherent fractions were determined by FACS using PI exclusion. B, BL-2 cells werecultured with stromal cells in the presence or absence of rituximab (20 mg/mL) for 48 hours. CXCR4 surface expression on NHL was analyzed byFACS. C, NHL BL-2, BJAB, and Raji cells were cultured with stromal cells in the presence or absence of rituximab (20 mg/mL) for 48 hours. Viability wasdetermined using the 2,3-bis[2-methoxy-4-nitro-S-sulfophenynl]H-tetrazolium-5 carboxanilide inner salt (XTT) method. D, BL-2 cells were cultured withstromal cells in the presence or absence of rituximab (10 mg/mL), BKT140 (8 and 40 mmol/L), and combination of both agents for 48 hours. Viability of floatingand adherent NHL cells was determined by FACS using PI discrimination. Data are presented as mean � SD from triplicates (��, P < 0.01).
Non-Hodgkin Lymphoma and CXCR4
www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3501
Kinetic studies detected massive lymphoma inseminationin the bone marrow, reaching 40% to 50% of total mono-nuclear cells in themurine bonemarrowat day35 followingthe inoculation. No significant lymphoma disseminationwas observed in other hematopoietic organs, such as spleen
and liver (Fig. 5A). Importantly, subcutaneous inoculationwith low CXCR4-expressing BJAB cells resulted in localtumor developmentwithout the bonemarrow involvement(Fig. 5A) and in long-term survival: animals survived during70 to 80 days without any signs of systemic disease and
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Figure 5. In vivoNHLmodels of localized and disseminated lymphomawith bonemarrow involvement. BKT140 reduces bonemarrow spread and suppresseslocal lymphoma tumor growth. BKT140 cooperates with rituximab in bone marrow and blocks rituximab-induced CXCR4 expression in local tumor.A, BL-2 and BJAB cells (5 � 106) were subcutaneously injected into the flank of NOD/SCID mice. On day 35 following the local injection, the presenceof human lymphoma cells in themurine bonemarrow (BM) and spleenwas assessed by FACS using specific antihumanCD20 antibody (representative FACSplots are displayed). B, Kaplan–Meier survival curve of animals subcutaneously injected with BL-2 or BJAB cells, followed during 60 days, 5 mice per group.Calculation was carried out using NCSS. C, BL-2 cells (5 � 106) were subcutaneously injected into the flank of NOD/SCID mice. According to theregimen for the residual disease (early start), micewere treatedwith subcutaneous injections of 300mgBKT140 or control saline in a volume of 200 mL, startingfrom day 3 following the NHL inoculation. According to the progressive disease regimen (late start), BKT140 injections were started on day 28.Treatment schedule for both regimenswas daily injections for 5 days, followed by 2days of no drug and then 5 additional daily injections (total of 10 injections).The number of human CD20þ cells in the bonemarrow of mice was determined using specific staining and FACS analysis. Data are presented as mean� SDfrom 5 mice. D, BJAB cells (5 � 106) were injected subcutaneously into NOD/SCID mice, and animals were treated with BKT140 injections accordingto residual disease regimen (10 daily injections on days 2–15 and 1 injection 24 hours before termination). Local tumor growth wasmeasured with caliper andtumor area (length�width)was calculated. Data are shownasmean�SD, 5miceper group (��,P <0.01when comparedwith control group). E, representativeslides ofCXCR4-stained andTUNEL-stainedBJAB local tumor sections andcontrol versusBKT140-treated.Cell nuclei are visualized using40,6-diamidino-2-phenylindole (DAPI) staining. Magnification �200 and �400.
Beider et al.
Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3502
were sacrificed due to large local tumor volume. In contrast,mice injected with high CXCR4-expressing BL-2lymphomacells survived for only 38 to 45 days and succumbed tolymphoma short time after bone marrow disease develop-ment (Fig. 5B). Indirectly, these facts may indicate that thespread and subsequent growth of CXCR4-expressing BL-2cells in the bone marrow are the major causes of theanimal’s mortality.
BKT140 inhibits the spread of NHL to the BMTo examine the in vivo effect of BKT140 on the lymphoma
dissemination to the bone marrow, NOD/SCID mice weresubcutaneously inoculated with BL-2 cells and treated withdaily subcutaneous injections of BKT140 (300 mg/injec-tion), 5 days a week, during 2 weeks. Two different treat-ment regimens were tested: (i) the residual disease regimen,inwhich BKT140was administered onday 3 following BL-2inoculation and discontinued following 2 weeks. In con-trast, (ii) in the progressive disease regimen, spread of BL-2lymphoma to the bone marrow was first allowed to occur,and then BKT140 treatment was started on day 28 after theBL-2 cell inoculation. Both regimens significantly reducedthe number of human CD20þ cells in the murine bonemarrow. However, early start of BKT140 administrationreflecting the residual disease regimen was more effective,remarkably reducing the bone marrow lymphoma diseaseby 92% (P < 0.0006) comparing with the 62% reduction(P < 0.02) achieved with the late start of BKT140 injectionsin progressive disease model (Fig. 5C). Animals showedgood tolerability to the treatment regimens with BKT140,no significant modifications of body weight or side effectswere observed.To further elucidate the mechanism by which BKT140
targets NHL in the bone marrow microenvironment, weevaluated the potential effect of BKT140 on apoptosisinduction in vivo in the CD20þ BL-2 cells. Flow cytometryanalysis with Annexin V–allophycocyanin (APC) revealedthat BKT140 treatment not only reduces the number of BL-2cells in the bone marrow but rather promotes lymphomacell apoptosis in the bonemarrowmicroenvironment (Sup-plementary Fig. S4).In addition, the effect of BKT140 on lymphoma in vivo
growthwas further assessed innondisseminated lymphomamodel initiated by BJAB cells. NOD/SCID mice subcutane-ously inoculated with BJAB cells were treated daily withBKT140, starting from day 3 after cell injection, 5 days aweek, during 2 weeks, and local tumor growth was mon-itored. Significant suppression of tumor growth wasobserved in BKT140-treated animals. The median growthof BJAB tumors was delayed by 78% on day 55 and by 60%onday 60 as comparedwith vehicle control (Fig. 5D). BJAB-generated tumors expressed low but detectable levels ofCXCR4, whereas BKT140 treatment totally blocked CXCR4staining in local tumor tissues (Fig. 5E, top). Furthermore,BKT140 significantly induced apoptosis in vivo in BJABtumors, as approved by terminal deoxynucleotidyl trans-ferase–mediated dUTP nick end labeling (TUNEL) staining(Fig. 5E).
Combination of BKT140 with rituximab effectivelytargets NHL in the bone marrow microenvironment
The effect of BKT140 and combinational therapy on thedisseminated lymphoma disease was tested. BL-2–innocu-lated mice with established tumor and bone marrow dis-semination were subcutaneously injected with BKT140,rituximab, or combination of both therapeutic agents,starting from day 28. Importantly, under conditions oflarge primary tumor with established bone marrowinvolvement, rituximab treatment alone showed minimaleffect on the NHL bone marrow disease, reducing thenumber of BL-2 cells only by 20% (Fig. 6A). BKT140effectively reduced the bone marrow tumor burden by77% compared with the untreated animals (P < 0.01).However, the combination treatment of BKT140 withrituximab further decreased the number of viable lym-phoma cells in the bone marrow, achieving 93% reduc-tion (P < 0.001) comparing with the untreated control(Fig. 6A). These results clearly show the effectiveness ofthe combined treatment against NHL in the bone marrowmicroenvironment.
Mobilization of tumor cells into the blood by CXCR4antagonists was previously shown in patients with AMLand in models of AML andmultiple myeloma (24, 25). Toevaluate the effect of CXCR4 blockade on lymphoma cellrelease to the circulation, the blood of tumor-bearinganimals was collected 24 hours after the last injection ofBKT140 or rituximab at the experiment termination.Small number of human CD20þ BL-2 cells in the circu-lation was detected by flow cytometry. Interestingly, 5doses of BKT140 did not significantly affect the number ofcirculating lymphoma cells, compared with untreatedcontrol mice. In contrast to BKT140, rituximab notablyreduced the number of BL-2 cells in the blood (Fig. 6B).Furthermore, combination of BKT140 with rituximabpowerfully reduced the local tumor mass of BL-2–gener-ated diffused tumors, as indicated by body weight gain ofBL-2–inoculated animals (Fig. 6C). These data suggestthat in progressive lymphoma disease with bulk tumorand bone marrow involvement, lymphoma cells areaccessible to rituximab in the blood but not in the bonemarrow. However, BKT140 effectively targets lymphomacells in the bone marrow niche and in local tumor,whereas the combination with rituximab significantlyenhances the antitumor effect of BKT140 in vivo.
Next, the CXCR4 expression in local BL-2–producedtumors was assessed. In accordance with in vitro results,rituximab-treated BL-2 tumors exhibited increased CXCR4expression, tested by immunohistochemical analysis. Incontrast, CXCR4 staining was significantly reduced in BL-2 tumors treated with BKT140, alone or in combinationwith rituximab (Fig. 6D top). Furthermore, in vivo apoptosisinduction was detected by TUNEL in BL-2–produced localtumors following BKT140 or rituximab treatment. Howev-er, the combinational treatment resulted in profoundlyincreased apoptosis rates (Fig. 6D). These results furtherconfirman increasing effect of rituximab andblocking effectof BKT140 on CXCR4 expression in NHL cells in vivo.
Non-Hodgkin Lymphoma and CXCR4
www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3503
DiscussionCXCR4/CXCL12 axis has been implicated in the devel-
opment and progression of many hematopoietic and solidtumors (26–31). In accordance with previous reports (32,33), our data indicate that lymphoma cell lines as well asprimary lymphoma cells from patient bone marrow biop-sies express surface CXCR4 (34, 35). Interaction of lym-phoma cells with bone marrow-derived stromal cells sup-ports lymphoma cell survival and protects them fromrituximab cytotoxicity. We observed that both interactionwith primary BMSCs as well as rituximab treatment furtherincreasedCXCR4 cell surface levels on lymphoma cells. Thismay endow tumor cells with increase direct and indirectsurvival signals.
CXCR4 neutralization with small molecules such asAMD3100,AMD11070, andAMD3465were already shownto reverse the protective effect of stroma and induce che-
mosensitivity inmultiplemyeloma,mantle cell lymphoma,AML, ALL, and CLL (22, 25, 34–37).
Recently, it was reported that neutralizing mAbs forCXCR4 significantly delayed tumor growth of lymphomaNamalwa cells in NOD/SCID mice (38). Moreover, antag-onizing CXCR4 and CXCL12 significantly inhibited thegrowth of Epstein-Bar virus (EBV)-transformed B cellsinjected intraperitoneally into NOD/SCID mice and pro-longed animal survival (39). Furthermore, recently pub-lished study has shown the efficacy of cell-penetratinglipopeptide CXCR4 antagonists, called pepducins, toenhance rituximab-induced apoptosis of lymphoma cellsin vitro and in vivo (40).
Small-molecule CXCR4 inhibitors mentioned previouslywere able to abrogate stroma-mediated resistance but lackdirect cytotoxic or chemotherapy-increasing activity in theabsence of stromal cells. Here, we evaluated the effect of
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Figure 6. BKT140 cooperates with rituximab in vivo, reduces bone marrow spread, and blocks rituximab-induced CXCR4 expression in local tumor.Combinational schedule of BKT140 and rituximab for established bone marrow disease. BL-2–bearing mice were treated starting from day 28 afterlymphoma inoculationwith subcutaneous injections ofBKT140 (300mg/m), rituximab (500mg/m), or bothagents, administeredbyseparate injections.BKT140was administered on days 28, 29, 30, 32, and 33 (total of 5 injections). Rituximab was administered on days 28, 29, and 32 (total of 3 injections).Number of human CD20þ cells in the bone marrow (BM; A) and blood (B), and body weight gain of mice (C) on day 35 after BL-2 cell inoculation.Control indicates the group of age-matched mice without BL-2 injection or treatments. Data are presented as mean � SD of 7 mice per group.D, CXCR4 expression and TUNEL staining in BL-2–produced local xenografts, treated with BKT140, rituximab, or both agents. Cell nuclei are visualized withDAPI staining. Magnifications of �100, �200, and � 400 (small images) are shown.
Beider et al.
Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3504
the high-affinity CXCR4 antagonist BKT140 on NHL cellviability, interactionwithbonemarrowmicroenvironment,and rituximab responsiveness. BKT140 originally designedas a human immunodeficiency virus (HIV) entry inhibitorthrough specific binding to CXCR4 (41). BKT140 inhibitsCXCR4-mediated adhesion and migration of cells fromhematopoietic origin (42). BKT140 was found to inducemobilization of mature white blood cells (WBC), hemato-poietic progenitors, and stem cells and efficiently synergizewith granulocyte colony-stimulating factor (G-CSF) in itsability tomobilizeWBCandprogenitors (43). Furthermore,we have recently shown that BKT140 induces a CXCR4-dependent selective apoptotic cell death of hematopoieticmalignant cells such as multiple myeloma and leukemiacells (44).In the present study, we observed that CXCR4 antagonist
BKT140 alone directly induced the apoptosis of NHL celllines and primary NHL cells. Combination of BKT140 withrituximab significantly increased the cytotoxic antilym-phoma effect, synergistically inducing mitochondrial dam-age, caspase-3 activation, and subsequent apoptosis oflymphoma cells. Moreover, our data indicate that disrupt-ing the CXCR4/CXCL12 axis using CXCR4 antagonistBKT140 is an effective way to abrogate BMSC-mediatedresistance of NHL to rituximab and to target NHL in bonemarrow microenvironment. The discrepancy between theaction of BKT140 versus AMD3100 may be partiallyexplained by relatively low affinity of AMD3100 (1 vs. 84nmol/L; ref. 45).To evaluate the in vivo antilymphoma effect of BKT140, we
established a novel xenograft model of B-cell lymphomawith bone marrow involvement in mice. Human CXCR4-expressing B NHL cell line, BL-2, was subcutaneouslyimplanted into NOD/SCID mice, resulting in the develop-ment of aggressive local tumors which specifically spread tothe bone marrow. This model enables us to investigate therole ofCXCR4 indifferent aspects of lymphomaprogression.We found that CXCR4/CXCL12 drives lymphoma-spe-
cific migration to the bone marrow and mediates thestroma-induced protection from immunotherapy. BKT140treatment inhibited local tumor progression and signifi-cantly reduced tumor burden in the bone marrow. Com-bination treatment of BKT140 with rituximab furtherdecreased the number of viable lymphoma cells in the bonemarrow, achieving 93% reduction.Previous study of Mancuso and colleagues analyzed the
presence of lymphoma cells in the blood of patients withNHL with bone marrow involvement and revealed concor-dance of 95%between bonemarrow andblood. Among thediscordant cases (i.e., the presence of neoplastic B lympho-cytes in the bone marrow, but under the sensibility of thetechnique in the blood), 62% of samples were collectedafter rituximab treatment alone or in association withchemotherapy. The authors suggested that during treatmentwith rituximab, neoplastic cells may be depleted from theblood, but are still present in the bone marrow (46).These observations are supported by our current results in
a disseminated NHL model, showing the ability of ritux-
imab to decrease the number of circulating lymphoma cellsin the blood, albeit an inability to target NHL cells in thebone marrow.
The use of mobilizing agents in leukemia has a potentialto redistribute themalignant cells and therefore to aggravatethe disease course. Notably, in our in vivomodel, we did notdetect the mobilization of lymphoma cells to the bloodfollowing BKT140 treatment. In contrast, the numbers ofviable circulating lymphoma cells in the blood werereduced with BKT140. This ability to diminish the numbersof viableNHL cells in bonemarrow and in peripheral bloodmay be particularly important for in vivo purging andcollection of lymphoma-free bone marrow grafts for auto-logus transplantation in patients with NHL. Rituximab useduring mobilization procedures has been shown to beeffective in obtaining lymphoma-free peripheral bloodstem cell (PBSC ref. 47). Therefore, the integration ofBKT140 in combination with rituximab into sequentialmobilization program of patients with NHL may be arational strategy for in vivo purging and mobilization ofuncontaminated grafts.
Taken together, our findings indicate that CXCR4 con-tribute toNHLprogression and showpotent antilymphomaeffect of CXCR4-specific high-affinity antagonist BKT140in vitro and in vivo. The BKT140-mediated antilymphomaeffect synergizes with that of rituximab. Moreover, BKT140effectively targets lymphoma cells in the bone marrowmicroenvironment, overcoming the stroma-inducedresistance to rituximab. These findings suggest the possibleinteraction between CD20 and CXCR4 pathways inNHL, and provide the scientific basis for the developmentof novel combined CXCR4-targeted therapies for refractoryNHL.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: K. Beider, E. Ribakovsky, E. Galun, A. Peled,A. NaglerDevelopment of methodology: K. Beider, H. Wald, E. Rosenberg, A. Peled,A. NaglerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): K. Beider, E. Ribakovsky, M. Abraham, L. Weiss,O. Eizenberg, A. Peled, A. NaglerAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): K. Beider, O. Eizenberg, A. PeledWriting, review, and/or revision of the manuscript: K. Beider, E. Riba-kovsky, A. Avigdor, O. Eizenberg, A. Peled, A. NaglerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Beider, E. Rosenberg, O. Eizen-berg, A. PeledStudy supervision: M. Abraham, E. Galun, A. Peled, A. Nagler
Grant SupportThis work was supported (in part) by a scientific grant from Hoffman La
Roche (to A. Nagler) and Sarousy Foundation grant (to A. Nagler).The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received September 20, 2012; revised March 5, 2013; accepted April 12,2013; published OnlineFirst May 1, 2013.
Non-Hodgkin Lymphoma and CXCR4
www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3505
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Non-Hodgkin Lymphoma and CXCR4
