the sphingosine-1-phosphate modulator fty720 targets ......hanna wald2, michal abraham2, eithan...

Cancer Therapy: Preclinical

The Sphingosine-1-Phosphate ModulatorFTY720 Targets Multiple Myeloma via theCXCR4/CXCL12 PathwayKatia Beider1, Evgenia Rosenberg1, Hanna Bitner1, Avichai Shimoni1, Merav Leiba1,Maya Koren-Michowitz1, Elena Ribakovsky1, Shiri Klein2, Devorah Olam2, Lola Weiss2,Hanna Wald2, Michal Abraham2, Eithan Galun2, Amnon Peled2, and Arnon Nagler1

Abstract

Purpose: To explore the functional consequences of possiblecross-talk between the CXCR4/CXCL12 and the sphingosine-1-phosphate (S1P) pathways in multiple myeloma (MM) cells andto evaluate the effect of S1P targeting with the FTY720modulatoras a potential anti-MM therapeutic strategy.

Experimental Design and Results: S1P targeting with FTY720induces MM cell apoptosis. The combination of FTY720 with theSPHK1 inhibitor SKI-II results in synergistic inhibition of MMgrowth. CXCR4/CXCL12-enhanced expression correlates withreduced MM cell sensitivity to both FTY720 and SKI-II inhibitors,and with SPHK1 coexpression in both cell lines and primary MMbone marrow (BM) samples, suggesting regulative cross-talkbetween the CXCR4/CXCL12 and SPHK1 pathways in MM cells.FTY720 was found to directly target CXCR4. FTY720 profoundlyreduces CXCR4 cell-surface levels and abrogates the CXCR4-mediated functions of migration toward CXCL12 and signaling

pathway activation. Moreover, FTY720 cooperates with bortezo-mib, inducing its cytotoxic activity and abrogating the bortezo-mib-mediated increase in CXCR4 expression. FTY720 effectivelytargets bortezomib-resistant cells and increases their sensitivity tobortezomib, promoting DNA damage. Finally, in a recentlydeveloped novel xenograft model of CXCR4-dependent systemicMM with BM involvement, FTY720 treatment effectively reducestumor burden in the BM of MM-bearing mice. FTY720 in com-bination with bortezomib demonstrates superior tumor growthinhibition and abrogates bortezomib-inducedCXCR4 increase onMM cells.

Conclusions: Altogether, our work identifies a cross-talkbetween the S1P and CXCR4 pathways in MM cells and providesa preclinical rationale for the therapeutic application of FTY720 incombinationwithbortezomib inpatientswithMM.ClinCancerRes;23(7); 1733–47. �2016 AACR.

IntroductionMultiple myeloma (MM) is a plasma cell malignancy charac-

terized by monoclonal proliferation of B cells producing a singleimmunoglobulin and affecting hematopoiesis and osseous bone(1). Despite the recent therapeutic advances, MM remains anincurable disease, thus illustrating limitations of existing thera-pies and the urgent need for innovative approaches (2–4).

The chemokine receptor CXCR4 and its ligand CXCL12 arecritically involved in the function of normal and malignanthematopoietic cells. This axis is critical for the homing of MMcells to the protective bonemarrow (BM) niche (5); it induces theproliferation of MM cells and protects them from drug-inducedapoptosis (6, 7). CXCR4 expression onMMcells is correlatedwith

poor prognosis (8), while elevation of CXCL12 serum levels isassociated with increased osteolytic disease (9). CXCL12 is secret-ed by BM stromal cells and bone endothelium (10), as well asfrom MM cell lines and primary myeloma cells (11).

Sphingosine-1-phosphate (S1P) is a phospholipid recentlyshown to activate VLA-4–dependent myeloma cell adhesion andmigration in concert with CXCL12 (12). This suggests an inter-action between the CXCL12/CXCR4 and S1P pathways in MMpathogenesis and disease progression.

FTY720 (fingolimod) is a synthetic compound produced by themodification of myriocin, a naturally occurring substance withimmunosuppressive properties (13). FTY720 is phosphorylatedby sphingosine kinase 2 and binds to four of five S1P receptors(S1PR1, S1PR3, S1PR4, and S1PR5; ref. 14). Besides its use inrelapsing multiple sclerosis patients for immunosuppression,FTY720 was also shown to induce apoptosis of various neoplasticcells, including solid tumors (15), acute lymphoblastic leukemia(16), and chronic myelogenous leukemia (17) and MM (18).FTY720 is reportedly cytotoxic to cancer cells by inducing bothcaspase-dependent and caspase-independent apoptotic path-ways. This anticancer activity does not require phosphorylationor S1PR interaction, but depends on the restoration of proteinphosphatase 2A (PP2A) function (19, 20). Additional moleculartargets have been suggested for the unphosphorylated form ofFTY720, including cytosolic phospholipase A2 (21), proteinkinase Cd (22), ceramide synthase (23), and sphingosine kinase

1Hematology Division and CBB, Guy Weinshtock Multiple Myeloma Foundation,Chaim Sheba Medical Center, Tel-Hashomer, Israel. 2Goldyne Savad Institute ofGene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel.

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

Corresponding Author:Arnon Nagler, Chaim Sheba Medical Center, Tel-Hasho-mer, Tel-Hashomer 52621, Israel. Phone: 972-3-5305830; Fax: 972-3-5304792;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2618

�2016 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 1733

on August 13, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 3, 2016; DOI: 10.1158/1078-0432.CCR-15-2618

http://clincancerres.aacrjournals.org/

1 (SPHK1; ref. 24). In addition to apoptotic cell death induction,FTY720was shown to induce necrotic cell death and autophagy inovarian cancer cells (25).

The activity of FTY720 against MM cells needs to be morethoroughly deciphered. Here, we demonstrate a profound anti-myeloma effect of FTY720. This was achieved by inducing mito-chondrial-associated apoptosis and enhancing the cytotoxic effi-cacy of bortezomib. Furthermore, we identified CXCR4 as a noveltarget of FTY720 in MM, suggesting the mechanism by whichFTY720may restrict the tumor-promoting activities of the S1P andCXCR4/CXCL12 axes. Moreover, utilizing a recently establishedCXCR4-dependent in vivo xenograft model of MM with BMinvolvement, our data show potent preclinical anti-MM activityof FTY720, effectively targeting MM tumor load in the BM niche.

Materials and MethodsCell lines and MM patient samples

The following humanMM cell lines were obtained fromATCC:ARH77, RPMI8226, U266, NCI-H929, OPM-1, and OPM-2. TheCAG MM cell line (generated by the group at the University ofArkansas forMedical Sciences (UAMS; ref. 26)was kindly donatedby Prof. Israel Vlodavsky, Technion, Israel. Cells were maintainedin log-phase growth in RPMI 1640 medium (Biological Indus-tries) supplemented with 10% heat-inactivated fetal calfserum (FCS), 1 mmol/L L-glutamine, 100 U/mL penicillin, and0.01mg/mL streptomycin (Biological Industries) in a humidifiedatmosphere of 5% CO2 at 37�C. RPMI8226 cells were authenti-cated in 2013 by STR DNA fingerprinting using an AmpFISTRIdentifier Kit (Applied Biosystems). Other cell lines were notauthenticated, but the cells were not used for more than 20passages after being thawed from stock.

Primary MM cells were isolated from BM aspirates of consent-ing myeloma patients. The study was approved by the ethicscommittee of the Sheba Medical Center. Mononuclear cells werecollected after standard separation on Ficoll-Paque (PharmaciaBiotech). MM cells were purified (>95% purity) by CD138þ

isolation using an MACS magnetic cell sorter (Miltenyi BiotecInc.).

InhibitorsThe following chemicals were used: FTY720 and Okadaic Acid

(OA) from Cayman, SKI-II and AMD3100 from Sigma Aldrich,LY294002, SP600125, SB203580, AG490 from Calbiochem,Bortezomib, and RAD001 from LC Laboratories.

Cell line transductionIn order to stably overexpress CXCR4, the MM cell lines

RPMI8226 and CAG were transduced with the lentiviral bicis-tronic vector encoding for CXCR4 and GFP genes. This was doneusing a three-plasmid system: empty vector pHR'-CMV-GFP-WPRE or pHR'-CMV-CXCR4-IRES-GFP-WPRE; envelope codingplasmid VSV-G and a packaging construct CMVDR8.91. ForCXCR4 silencing, cells with exogenously expressed CXCR4(RPMI8226-CXCR4) were stably transduced with lentiviral vec-tors encoding for specific anti-CXCR4 short hairpin RNA (shRNA;pLKO.1-shRNA-CXCR4 TRCN, Mission TRC Sigma) using thesame envelope and packaging constructs. For CXCL12 silencing,CXCL12-expressing RPMI8826 cells were stably transduced withlentiviral vectors encoding for specific anti-CXCL12 shRNA(pLKO.1-shRNA-CXCL12 TRCN, Mission TRC Sigma).

Establishment of the bortezomib-resistant RPMI8226-CXCR4cell line

The bortezomib-resistant MM cell line RPMI8226-CXCR4-Bortwas established as described in Supplementary Methods.

Analysis of surface markersExpression levels ofCXCR4were evaluated by immune staining

with allophycocyanine (APC)-conjugated anti-CXCR4 monoclo-nal antibody (12G5 clone; eBioscience). In primary MM samples,counterstaining with anti-CD138 fluorescein (FITC)-conjugatedantibody (IQ products) was performed. The cells were analyzedby FACScalibur (Becton Dickinson Immunocytometry Systems),using the CellQuest and FlowJo software.

XTT viability assayMM cells (2 � 104 per 100 mL per well) were plated in 96-well

flat plates in quadruplicate samples, with an increasing concen-tration of FTY720 or SKI-II at various time points. To evaluate therole of CXCR4/CXCL12 autocrine signaling in MM cell growthand survival, RPMI8826 cells with silenced CXCL12 or withoverexpressed CXCR4 were plated in complete (containing10%FCS) versus serum-reduced (1%FCS)medium. Cell viabilitywas assessed using the 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide(XTT) assay (Biological Industries).

Soft-agar coloniesMM cells with native, increased or silenced CXCR4 were plated

in soft agar in 24-well plates (5,000 cells per well). Following twoweeks of incubation, colonies were counted and photographed.

Assessment of apoptosisApoptosis was determined as described in Supplementary

Methods.

Cell-cycle analysisCells were exposed in vitro to increasing concentrations of

FTY720 for 24 to 48 hours and analyzed by FACS as describedin Supplementary Methods.

Translational Relevance

Despite the progress in the development of novel therapeu-tics, multiple myeloma (MM) remains an incurable disease.The CXCR4/CXCL12 chemokine axis plays an important rolein MM pathophysiology. The sphingosine-1-phosphate (S1P)pathway is considered to be involved in MM pathogenesis aswell. In this study, we describe the potent in vitro and in vivoanti-MM effects of the S1P modulator FTY720. CXCR4 isidentified as a molecular target of FTY720, suggesting func-tional cross-talk between the CXCR4 and S1P pathways inMM. We demonstrate the ability of FTY720 to cooperate withbortezomib and target MM cells in the bone marrow (BM)niche using a novel in vivo xenograft model of CXCR4-drivenMM that recapitulate the natural BMmilieu. Finally, our in vivomodel provides a suitable tool to study the activity of novelanti-MM agents targetingMM in BMmicroenvironment. Alto-gether, our work provides the preclinical rationale for thera-peutic application of FTY720 in MM.

Beider et al.

Clin Cancer Res; 23(7) April 1, 2017 Clinical Cancer Research1734




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FTY720 suppresses MM viability and induces mitochondrial-involved apoptosis accompanied with DNA damage. A, Growth-inhibitory effect of FTY720 on MM celllines. Cells were treated with various concentrations of FTY720 for 24, 48, and 72 hours. Cell viability was evaluated using the XTT method. Decreasedviability is presented as a percentage from untreated control. IC50 of FTY720 for each MM cell line is presented. B, Apoptosis was detected using Annexin V–APC/PIstaining. The percentage of early (Annexin Vþ/PI�) and late (Annexin Vþ/PIþ) apoptotic cells was determined. C, MM cells were treated with FTY720 (3, 5, 7,and 10 mmol/L) for 24 hours and mitochondrial membrane potential (Dym) was determined using DiOC6 staining. The percentage of apoptotic cells with reducedDym and subsequent staining intensity was detected. Data are presented as mean of triplicates �STDEV (�� , P < 0.01). D, Primary BM samples from patientswith MM (n ¼ 3) were incubated with indicated concentrations of FTY720 for 24 hours. Viability was determined using 7-AAD staining and flow cytometry. Theanalysis was gated on CD138þ cells. The percentage of dead (7-AAD–positive) CD138þ MM cells is presented as mean of triplicates �STDEV (�� , P < 0.01). E,Western blot analysis of antiapoptotic proteins BCL-2 and MCL-1, apoptotic marker PARP, DNA-damage marker pH2AX and mTOR pathway target pS6 in RPMI826and CAGMM cells before and after treatment with various doses of FTY720 for 24 hours (left) or treated with fixed concentration of FTY720 (12 mmol/L) at differenttimepoints (4 hours, 8 hours, and 24 hours; right).b-Actinwas used as an internal control. Representative data fromat least two independent experiments are shown.

FTY720 Targets Multiple Myeloma via CXCR4/CXCL12

www.aacrjournals.org Clin Cancer Res; 23(7) April 1, 2017 1735




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Modulation of CXCR4 and CXCL12 expression inMM cells promotes functional responses.A,Generation of MM cells with overexpressed functional CXCR4. RPMI8226and CAG cells were stably transduced with lentiviral vector encoding for GFP (empty vector) or vector encoding for CXCR4 and GFP in order to generatethe RPMI8226-EV, CAG-EV, RPMI8226-CXCR4, and CAG-CXCR4 cell lines. CXCR4 surface expression in generated lines, assessed by flow cytometry. B,Generation of RPMI8226 cells with reduced levels of endogenously expressed CXCL12 using lentiviral transductionwith shRNA-encoding vectors. CXCL12mRNA andsecreted levels in the generated RPMI8226-shCXCL12 cell line comparing with the line transduced with an empty vector, assessed by qRT-PCR and ELISA. C,Transwell migration of cells with native or increased CXCR4 in response to CXCL12 (50–500 ng/mL) during 4 hours. To inhibit CXCR4, CXCR4-overexpressing cellswere preincubated with AMD3100 (20 mmol/L) for 30 minutes prior to migration. D, RPMI8226-EV, RPMI8226-CXCR4, CAG-EV, and CAG-CXCR4 cells wereincubated during 16 hours in serum-reduced (0.1% FCS) medium, and then stimulated with CXCL12 (250 ng/mL) for 5, 15, and 30minutes. Cell lysates were analyzedby Western blot for the levels of phosphorylated Erk1/2 and AKT proteins. b-Actin was used as an internal control. Representative data from at least twoindependent experiments are shown. E, Growth and survival of RPMI8226-EV, RPMI8226-CXCR4, and RPMI8226-shCXCL12 cells in full (10% FCS) or serum reduced(1% FCS) medium was measured by XTT at the indicated time points. Data are presented as mean of triplicates �STDEV (�� , P < 0.01).

Beider et al.





Assessment of mitochondrial membrane potential (Dcm)Effect of FTY720 treatment (elevated concentration, 24 hours)

on Dym was evaluated using DiOC6 staining as previouslydescribed (27).

Cell migration assayMigration of MM cell lines in response to various CXCL12

concentrations (5–500 ng/mL; PeproTech EC) was evaluatedusing 5-mm pore size Transwells (Costar). The quantity of cellsmigrating within four hours to the lower compartment wasdetermined by FACS and expressed as a percentage of the input.For FTY720 or SKI-II treatment, cells were pretreated (30 minutesin 37�C) with various doses of FTY720 (2.5–10 mmol/L) or SKI-II(20 mmol/L) and subjected to migration.

Immunoblot analysisTotal protein lysates (50–70 mg) were resolved by electropho-

resis in 10% SDS-PAGE and transferred onto PVDF membranes.Blots were subjected to a standard immuno-detection procedureusing specific antibodies and the ECL substrate (Biological Indus-tries). Signal was detected using a Bio-Rad image analyzer (Bio-Rad). Primary antibodies were as follows: pErk1/2 (Sigma-Aldrich), pAKT (Cell Signaling Technology), pS6 and cleavedPARP (Cell Signaling Technology), BCL-2 (Dako), pH2AX(Upstate), MCL-1 (Santa Cruz), and b-actin (Sigma-Aldrich).

Preparation of BMSCs and coculture experimentsPrimary human BM stromal cells (BMSC) were generated from

BM aspirates of healthy donor volunteers after signing aninformed consent. BMSCs were isolated by plate adherence andexpanded as previously described (28).

Murine xenograftmodels of disseminated humanMManddrugtreatment

NSG mice were maintained under defined flora conditionsat the Hebrew University Pathogen-Free Animal Facility (Jer-usalem, Israel). All experiments were approved by the AnimalCare Committee of the Hebrew University. Mice were injectedintravenously with RPMI8226-EV, RPMI8226-CXCR4, orRPMI822-CXCR4shCXCR4 human cells (5� 106/mouse). End-points were paraplegia and weight loss >10%. The mice weresacrificed on the same day the endpoint was reached. Diseasewas verified by measurement of human immunoglobulin inplasma of inoculated mice using the ELISA kit (ImmunologyConsultants Laboratory). To investigate the therapeutic poten-tial of FTY720 as a single agent, 3 days after inoculation withRPMI8226-CXCR4 cells, mice were randomized and treatedwith daily intraperitoneal (i.p.) injections of either FTY720(10 mg/kg) or control saline, during 3 weeks, for a total of15 injections. To evaluate the effect of FTY720 in combinationwith bortezomib, mice inoculated with RPMI826-CXCR4 wereinjected with a lower dose of FTY720 (i.p., 5 mg/kg) three timesper week, a total of nine injections, bortezomib (subcutane-ously, 0.25 mg/kg) twice per week, a total of six injections, orwith a combination of both agents. Animals were sacrificed 24days after tumor inoculation.

RT-PCR analysisRNA isolation and RT-PCR analysis were performed as

described in Supplementary Methods.

CXCR4 immunohistochemistryFor histologic analysis, the bone specimens from myeloma-

inoculated mice were fixed in freshly prepared 4% paraformal-dehyde, decalcified in 10% EDTA with 0.5% paraformaldehyde,and embedded in paraffin using standard procedures. CXCR4expression was evaluated by immunohistochemical staining asdescribed in Supplementary Methods.

Statistical analysesData are expressed as the mean � standard deviation (SD),

or standard error (SE). Statistical comparisons of means wereperformed by a two-tailed unpaired Student t test or the Mann-Whitney U test.

ResultsFTY720 suppresses MM cell viability and induces apoptotic celldeath with mitochondrial involvement

The viability of various human MM cell lines was examinedfollowing treatment with FTY720. As shown in Fig. 1A, FTY720effectively inhibited cell viability of all six MM cell lines testedin a time- and dose-dependent manner, with IC50 rangingbetween 2.8 and 6.3 mmol/L at 48 hours. Next, we tested themechanism of FTY720-induced cell death. A significant induc-tion of early and late apoptosis was observed in MM cells upontreatment with increasing doses of FTY720 (Fig. 1B; Supple-mentary Fig. S1A). Moreover, FTY720 promoted mitochondrialdepolarization, as demonstrated by the uptake of the mito-chondrial dye DiOC6 (Fig. 1C; Supplementary Fig. S1B).Importantly, in vitro treatment with FTY720 induced dose-dependent cell death of primary human CD138þ MM cellsisolated from BM samples from newly diagnosed patients withMM (Fig. 1D).

Furthermore, treatment with FTY720 resulted in markedreduction of expression of the antiapoptotic protein MCL-1,without a significant effect on the BCL-2 protein level in MMcell lines. FTY720 treatment markedly increased proteolyticcleavage of apoptosis-associated poly(ADP-ribose) polymerase(PARP) and elevated the levels of phosphorylated H2AX in adose- and time-dependent manner, indicating the activation ofthe latter stages of the apoptotic pathway and subsequentinduction of DNA damage (Fig. 1E).

Together, these findings indicate that FTY720 exhibits potentantiproliferative effect on MM cells and is capable of inducingapoptosis with mitochondrial involvement.

Increased expression of CXCR4 promotes a tumorigenicphenotype in MM cells

To study whether CXCR4 has a role in FTY720-mediated MMcell death, we generated a stable cell line with functional over-expression of CXCR4 (RPMI8226-CXCR4 and CAG-CXCR4), aswell as control cell lines transduced with empty lentiviral vectors(RPMI8226-EV and CAG-EV; Fig. 2A). In addition, to delineatethe role of endogenous CXCL12 expressed byMM cells, we stablysilenced CXCL12 in RPMI8226 cells using shRNA (Fig. 2B). Asexpected, enhanced expression of CXCR4 resulted in increasedmigration of MM cells toward CXCL12, while CXCR4 inhibitionwith specific antagonist AMD3100 blocked this response, dem-onstrating its specificity (Fig. 2C).

CXCR4 is known to be an important player in supportiveinteractions between tumor cells and BM stroma (29, 30).






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Beider et al.





Accordingly, MM cells with overexpressed CXCR4 demonstratedincreased susceptibility to stromal-produced signals. In addition,coculture with BMSCs supported proliferation and suppressedstarvation-induced apoptosis of CXCR4-overexpressing cellsmore effectively than that of native cells (Supplementary Fig.S2A and S2B). Importantly, CXCR4 overexpression resulted indecreased sensitivity to signal transduction inhibitors, includingLY294002 (PI3K inhibitor), SP600125 (JNK inhibitor), andSB203580 (p38 inhibitor). However, no difference in sensitivityto JAK2 inhibition (using AG490) was observed in cells witheither low or high CXCR4 expression (Supplementary Fig. S2C).These results suggest an increased activationof signaling pathwaysdownstream toCXCR4. Indeed, elevated levels of phosphorylatedAKT and Erk1/2 proteins were detected in CXCR4-overexpressingcells. Stimulation with CXCL12 further increased the phosphor-ylation of these signaling mediators (Fig. 2D). Additionally,overexpressed CXCR4 promoted the tumorigenic potential ofMMcells, supporting the clonogenic growth of CXCR4-expressingcells in soft agar (Supplementary Fig. S2D).

Upregulation of CXCR4 promoted cell growth and increasedcell survival under serum-reduced conditions, while CXCL12silencing resulted in a slower growth rate and significantlydecreased the survival upon serum deprivation, further demon-strating the importance of theCXCR4/CXCL12 axis activity inMMcell growth and survival (Fig. 2E).

Role of the CXCR4/CXCL12 axis in modulating MM cellresponse to S1P inhibitors FTY720 and SKI-II

Following the establishment of MM cell lines with increasedfunctional CXCR4 and silenced endogenous CXCL12, we eval-uated the possible role of the CXCR4/CXCL12 axis in responseof MM cells to FTY720 treatment. Of note, MM cells over-expressing CXCR4 demonstrated a decreased sensitivity toFY720-mediated cell death, while the cells with downregulatedCXCL12 were significantly more sensitive to FTY720 treatment(Fig. 3A). The effect of CXCR4 was achieved via antagonizingFTY720-mediated apoptosis induction (Fig. 3B; SupplementaryFig. S3A). Consistently, CXCR4 overexpression resulted in areduced caspase-3 activation and a decreased cytochromerelease in response to FTY720 treatment (Supplementary Fig.S3B and C). In addition, the effect of another modulator of theS1P pathway, the SPHK1 inhibitor SKI-II, was tested. SKI-IIsuppressed MM cell growth less efficiently than FTY720, dem-onstrating higher IC50 doses. However, similarly to FTY720, theCXCR4/CXCL12 expression correlated with reduced MM cellsensitivity to SKI-II-mediated inhibition (Fig. 3C). Importantly,the combination of FTY720 with SKI-II synergistically sup-pressed the viability of both native and CXCR4-overexpressing

MM cells (Fig. 3D). Levels of SPHK1 mRNA were increased inCXCR4-overexpressing cells and consistently decreased inCXCL12-silenced cells (Fig. 3E). Furthermore, the coexpressionpattern of CXCL12 and SPHK1 genes was detected in 14 BMsamples from patients with MM (Fig. 3F). Collectively, theseresults illuminate the contribution of CXCR4/CXCL12 activityto the FTY720 and SKI-II responses and suggest the regulativecross-talk between CXCR4/CXCL12 and the SPHK1 pathwaysin MM cells.

FTY720 suppresses CXCR4-induced activity and promotesCXCR4 internalization in MM cells

To explore whether FTY720 and SKI-II treatment can directlyaffect CXCR4 activity, MM cells with high functional CXCR4 werepretreated with increasing concentrations of FTY720 and sub-jected to Transwell migration in response to CXCL12. As dem-onstrated in Figure 4A, FTY720 suppressed CXCR4-mediatedmigration in a dose-dependent manner. In contrast to FTY720,a high dose of SKI-II only mildly affected the migration ofRPMI226-CXCR4 cells, having no effect on the migrationresponse of CAG-CXCR4 cells. Next, the effect of FTY720 andSKI-II on CXCR4-triggered intracellular signaling was investigat-ed. Importantly, FTY720 pretreatment completely abolishedCXCL12-mediated phosphorylation of Erk1/2 and pS6. Consis-tent with the migration results, the SKI-II treatment was noteffective in suppressing CXCL12-induced signal transduction(Fig. 4B).

Because our results showed that FTY720 blocked the CXCR4pathway activity, we investigated the effect of FTY720 on CXCR4cell-surface levels. FTY720 significantly reduced CXCR4 located atthe cell surface of RPMI8226-CXCR4, CAG-CXCR4, and CXCR4-natively expressing OPM-1 cells. Moreover, a similar effect onCXCR4 expression was observed in primary CD138þ MM cells(Fig. 4C). Incubation on ice prevented FTY720-induced relocali-zation of CXCR4, indicating that FTY720 is capable of inducingCXCR4 internalization (Fig. 4D). CXCR4 mRNA levels were notaffected by FTY720 treatment (Supplementary Fig. S4). Of note,pretreatment with the PP2A inhibitor okadaic acid (OA) did notaffect the ability of FTY720 to induce cytotoxicity, or suppressmigration of MM cells (Supplementary Fig. S5A and S5B), dem-onstrating that the FTY720-promoting effects onMM cells are notdependent on the reactivation of PP2A, thus suggesting an alter-native mechanism of action.

FTY720 cooperates with bortezomib and effectively targetsbortezomib-resistant MM cells

Bortezomib is commonly used as first-line drug combina-tions for MM patients, and the development of bortezomib

Figure 3.CXCR4/CXCL12 endogenous activity defines the sensitivity of MM cells to the S1P modulators FTY720 and SKI-II. A and B, Cells with low nativeCXCR4 (RPMI8226-EV and CAG-EV), increased CXCR4 (RPMI8226-CXCR4 and CAG-CXCR4), or silenced CXCL12 (RPMI8226-shCXCL12) were incubatedin the absence or presence of indicated concentrations of FTY720 for 48 hours. A, Cell viability was evaluated by the XTT method. B, Apoptosiswas detected using Annexin V–APC/PI staining. Percentage of live (Annexin V�/PI�), early (Annexin Vþ/PI�), and late (Annexin Vþ/PIþ) apoptoticcells was determined. C, Indicated cell lines were incubated in the absence or presence of SKI-II (2–50 mmol/L) for 48 hours. Cell viability was evaluatedby the XTT method. D, MM cells were treated with FTY720 (5 and 7 mmol/L), SKI-II (10 and 20 mmol/L), or the combination of both agents for 48 hours. Cellviability was evaluated by the XTT method. Data are presented as mean of triplicates � STDEV (�� , P < 0.01). E, Expression of SPHK1 in MM cell lines withinduced CXCR4 and silenced CXCL12, evaluated by quantitative RT-PCR. Data (RQ) are presented as mean of triplicate � STDEV; ��, P < 0.01. F, Expressionof CXCL12 and SPHK1 in BM samples from patients with MM, evaluated by quantitative RT-PCR.






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FTY720 suppresses CXCR4 activity and induces CXCR4 internalization in MM cells. A, RPMI8226-CXCR4 and CAG-CXCR4 cells were pretreated with indicateddoses of FTY720 for 30 minutes and their migratory response to CXCL12 (200 ng/mL) was measured using the Trans-well migration assay. B, Effect of FTY720 andSKI-II pretreatment (20 mmol/L for both agents, for 1 hour at 37�C) on CXCL12-induced (200 ng/mL, 30 minutes activation) intracellular signaling inRPMI8226-CXCR4, CAG-CXCR4, and primary CD138þ MM cells. Levels of phosphorylated Erk1/2 and S6 proteins, measured by Western blot, b-actin was usedas an internal control. Representative data from at least two independent experiments is shown. C, Effect of FTY720 treatment (20 mmol/L and 50 mmol/L, 1 hour,37�C) on cell-surface CXCR4 expression in RPMI8226-CXCR4, CAG-CXCR4, OPM-1, and primary CD138þ MM cells, measured by FACS. CXCR4 expressionlevel is presented as mean fluorescent intensity (MFI); data are presented as mean of triplicates �STDEV (�� , P < 0.01). D, Effect of FTY720 treatment (20 mmol/L,1-hour incubation) at 37�C or 4�C on cell levels of CXCR4, evaluated by FACS. Data are presented as mean of triplicates � STDEV (�� , P < 0.01).


Beider et al.




resistance is associated with major clinical problems duringtreatment of these patients. The mechanism of bortezomibresistance remains elusive. We observed that CXCR4-overex-pressing cells are less sensitive to bortezomib compared withparental cells. We further established bortezomib-resistant cellsby exposing RPMI8226-CXCR4 cells to gradually increasingconcentrations of bortezomib. RPMI8226-CXCR4-BortRes cellsdisplayed a significantly decreased sensitivity to the cytotoxiceffect of bortezomib (Fig. 5A). However, FTY720 treatmentinhibited cell viability irrespective of bortezomib sensitivity,demonstrating a similar dose response in both bortezomib-sensitive and -resistant lines. The combination of FTY720 withbortezomib effectively targeted MM cells with either low orhigh CXCR4 expression and resulted in increased cytotoxicitytoward bortezomib-resistant cells (Fig. 5B). Moreover, thecombination of FTY720 with bortezomib markedly increasedPARP cleavage and promoted DNA damage, as detected bypH2AX expression (Fig. 5C). A significant increase in CXCR4surface expression levels in surviving MM cells upon bortezo-mib monotherapy was detected, enabling the selection andenrichment of resistant cells with increased CXCR4. However,the combination of FTY720 with bortezomib reversed thisundesirable increase in CXCR4 expression (Fig. 5D).

These results suggest that the introduction of functional CXCR4provides prosurvival and antiapoptotic signals, antagonizingbortezomib-induced cytotoxicity. Most importantly, FTY720 wasshown to be effective in suppressing bortezomib-resistant cells,resensitizing them to bortezomib-induced cell death and abro-gating the increase in CXCR4 expression.

CXCR4 mediates stroma-provided protection of MM cells fromFTY720-induced apoptosis through the mTOR–pS6 axis

CXCR4 is known to mediate interaction with BM milieucomponents and provide chemoresistance to MM cells (6,31). Therefore, we investigated the impact of BMSCs onCXCR4-expressing MM cells' response to FTY720. Interactionwith BMSCs antagonized the FTY720-induced cytotoxicity (Fig.5E). Intracellular levels of different signaling mediators wereevaluated in order to further investigate the downstream molec-ular machinery involved in stroma-mediated resistance of MMcells with forced CXCR4 expression to FTY720. We determinedthe prosurvival mTOR pathway to be regulated by both CXCR4overexpression and interaction with BMSCs, as indicated by theincreased levels of the phosphorylated protein S6. Specifically,in the absence of BMSCs, FTY720 treatment decreased pS6levels in both native and CXCR4-overexpressing MM cells.While coculture with stromal cells effectively protectedCXCR4-expressing MM cells and prevented FTY720-inducedS6 de-phosphorylation (Fig. 5F), the combination of FTY720with the mTOR inhibitor RAD001 resulted in a significantlyincreased cell death, effectively abrogating CXCR4- and stroma-dependent resistance to FTY720 and suppressing mTOR signal-ing in MM cells (Fig. 5G and H).

In vivo modelTo investigate the in vivo functional relevance of CXCR4 in MM

dissemination and growth,MM cells with native low (RPMI8226-EV), enhanced (RPMI8226-CXCR4), or silenced (RPMI8226-CXCR4-shCXCR4) CXCR4 were intravenously injected intoNSG mice. Tumor load was assessed by flow-cytometry analysisof GFP-positive MM cells in the BM and by measuring serum

M-protein concentrations. The intravenous injection ofRPMI8226-CXCR4 cells resulted in the development of systemicMM disease specifically localized to the murine BM. As dem-onstrated in Fig. 6A, injection of CXCR4-overexpressing (but notnative or CXCR4-silenced) MM cells resulted in 100% animallethality on days 33 to 34. In mice with disseminated tumors,60% (6 of 10) of the animals developed hind leg paralysis.Kinetic studies detected massive seeding of RPMI8226-CXCR4cells in the BM, reaching 20% to 35% of total mononuclear cellsin the murine BM on day 23 following the inoculation. Nosignificant myeloma dissemination was observed in otherhematopoietic organs, such as spleen and liver (data notshown). In correlation with BM dissemination, there was acorresponding increase in human M-protein serum levels, reach-ing 12,000 pg/mL on day 23. Importantly, CXCR4 silencingtotally abolished the potential of MM cells to localize in the BMand induce systemic disease (Fig. 6B). RT-PCR analysis con-firmed the presence of human CD138-positive MM cells in theBM of mice inoculated with high-CXCR4-expressing cells butnot in those inoculated with low-CXCR4-expressing cells (Fig.6C). Finally, analysis of histological samples detected and visu-alized the presence of CXCR4-expressing MM tumors in murinefemurs and tibias (Fig. 6D). Altogether, our results demonstratethat CXCR4-overexpressing human MM cells localize in the BMof NSG mice and form disease with features consistent withthose of MM in human patients. The established preclinicalmodel of systemic myeloma with BM involvement in miceallowed us to test the antimyeloma effect of FTY720.

FTY720 targets MM and cooperates with bortezomib in vivoTo test the in vivo effect of FTY720 as a single agent, NSG mice

were inoculated with RPMI8226-CXCR4 cells and, 3 days later,were randomized into vehicle or FTY720 treatment (10 mg/kg).Mice were injected 5 days per week for 3 weeks. On day 24, thenumber of MM cells in the BM was significantly reducedin the FTY720-treated group when compared with the controlvehicle-injected group. The reduction in tumor burden was con-sistent with markedly reduced serum M-protein levels in theFTY720-treated group (Fig. 6E and F). Furthermore, animalstolerated the treatment well, and no weight loss or side effectswere observed. Next, the in vivo activity of FTY720 in combina-tion with bortezomib was evaluated. RPMI8226-CXCR4-inoc-ulated mice were treated with low-dose bortezomib (0.25 mg/kg), a reduced dose of FTY720 (5 mg/kg) or their combination.Combining both agents markedly reduced BM tumor burden(Fig. 6G). As seen in vitro, bortezomib increased CXCR4 expres-sion in surviving MM cells in the BM. The addition of FTY720 tobortezomib abrogated the undesirable bortezomib-inducedincrease in CXCR4 expression, resulting in reduced CXCR4surface levels on the remaining MM cells in vivo (Fig. 6H).Altogether, these results demonstrate that FTY720 administra-tion leads to potent in vivo reduction of MM tumor burden inthe BM microenvironment, and suggest further increased activ-ity when used in combination with bortezomib.

DiscussionDespite the introduction of effective new therapies, MM

remains an incurable malignancy in the vast majority of patients.The functional interplay between MM cells and the BM microen-vironment is believed to be a leading contributing factor by






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Beider et al.




promoting the proliferation, survival, and drug resistance of MMcells (32). These effects are mediated via pleiotropic signalingpathways and mediators. The CXCR4/CXCL12 and S1P axes arethought to be especially important forces affecting the BMmilieucellular composition.

The bioactive lipid S1P has been recognized as an importantregulator of many pathologic processes, including cancer. Abun-dant evidence indicates that S1P can stimulate growth and sur-vival of leukemia and lymphoma cells (33). S1P regulates thehematopoietic progenitor and plasma cell localization in the BM(34, 35). It was previously shown that CD138þ MM cells expressthe S1P1 receptor and that S1P enhances CXCL12-chemotacticactivity and adhesion of myeloma cells (12, 36). These findingsprompted us to consider the S1Ppathway as a therapeutic target inMM using the modulator FTY720.

We showed that FTY720 treatment resulted in a dose-depen-dent MM apoptotic cell death accompanied by a markeddecrease in mitochondrial polarization, a reduction in MCL-1 and pS6 levels, as well as an increase in PARP cleavage andpH2AX levels. These findings indicate apoptosis with subse-quent DNA damage as the prevalent cell death mechanismactivated by FTY720 in MM cells. These results are in line withthose of numerous previous reports (37). The FTY720-medi-ated downmodulation of the antiapoptotic protein MCL-1could have specific implications relevant to the pathogenesisof MM, because its critical role in the biology of MM has beenwell established (38, 39).

In agreement with several previous studies (40, 41), but incontrast to others (42, 43), we observed that cell death induced byFTY720 inMMcells was PP2A independent, as the PP2A inhibitorOA did not reverse FTY720-mediated cell death.

Because interactions between the S1P and CXCR4 pathwayswere implicated in HSC and MM migration and BM retention(12, 35, 44), we evaluated the effect of the S1P axis modulationwith FTY720 on CXCR4 expression and function in MM cells.Our results with enforced CXCR4 and reduced endogenousCXCL12 expression indicate an imperative role of CXCR4/CXCL12 axis in MM biology, showing the importance ofCXCR4 signaling for MM cell growth, motility, interaction withBMSCs and clonogenic potential. These results are in agreementwith those of previous works demonstrating the expression of

CXCR4 in MM cells and describing its role in MM migrationand BM localization (6, 45).

Enhanced CXCR4 signaling decreased the sensitivity of MMcells to apoptosis induced by SPHK1 inhibitors FTY720 andSKI-II. Further, overexpression of CXCR4 promoted the upre-gulation of SPHK1 mRNA levels while CXCL12 silencingdecreased the levels of SPHK1, suggesting regulative cross-talkbetween CXCR4/CXCL12 and SPHK1. Accordingly, wedetected a coexpression pattern of the SPHK1 and CXCL12genes in BM primary samples from patients with MM. How-ever, it was interesting to discover that FTY720, but not SKI-II,effectively suppressed known CXCR4 functions in MM cells,including the complete inhibition of CXCL12-induced migra-tion and intracellular signaling. Furthermore, FTY720 inducedCXCR4 internalization and surface downmodulation in MMcells. A high degree of variability in the expression of CXCL12and SPHK1 across MM samples suggests the possibility ofvariability in the responses of patients with MM to FTY720and SKI-II.

Partnerships between S1P and additional signaling net-works were previously recognized. It was shown that theS1P1 receptor may act in complex with tyrosine kinase recep-tors such as platelet-derived growth factor (PDGF) receptor(46) or vascular endothelial growth factor (VEGF) receptor(47). S1P1 inhibition could interrupt PDGF- and VEGF-inducedstimulation of Erk1/2 and cell migration (47, 48). Our resultssuggest that cross-talk between S1P and CXCR4 pathways in MMis an additional interaction for S1P. By identifying CXCR4 as oneof the molecular targets of FTY720, we suggest a potentialapplication of FTY720 as an agent to control CXCR4-mediatedprocesses in MM.

Resistance to bortezomib is a critical emerging issue in MMtherapy response in the clinic. We observed that an increasedCXCR4/CXCL12 expression decreases the responsiveness ofMM cells to bortezomib. In parallel, the bortezomib treatmentwas shown to upregulate CXCR4 in MM cells, therefore pos-sibly enabling an enrichment of resistant cells with increasedCXCR4. Our results are in agreement with a recently publishedwork identifying chemoresistant PCs and showing the enrich-ment of CXCR4 in minimal residual disease (MRD) subclone inMM patients (49). Of importance, we found that FTY720-

Figure 5.FTY720 cooperates with bortezomib and targets both bortezomib-sensitive and -resistant MM cells. CXCR4 mediates stroma-provided protectionof MM cells from FTY720-induced apoptosis through the mTOR–pS6 axis. A, RPMI8226-EV, RPMI8226-shCXCL12, RPMI8226-CXCR4, andRPMI8226-CXCR4-bortezomib-resistant cells were treated with indicated concentrations of bortezomib during 48 hours. Viability was evaluatedusing the XTT method. B, RPMI8226-EV, RPMI8226-CXCR4, and RPMI8226-CXCR4-bortezomib–resistant cells were incubated during 48 hours withindicated concentrations of FTY720, bortezomib, or a combination of both agents. Viability was evaluated using the XTT method. Results representthe average of triplicates �STDEV (�� , P < 0.01). C, Western blot analysis of the apoptotic marker PARP and the DNA-damage marker pH2AX inRPMI826-CXCR4, RPMI8226-CXCR4-bortezomib–resistant and OPM-1 cells before and after treatment with FTY720 (5 mmol/L), bortezomib (5 nmol/L), ortheir combination for 24 hours. b-Actin was used as the internal control. Representative data from at least two independent experiments is shown. D, Effectof treatment with FTY720 (5 mmol/L), bortezomib (5 nmol/L), or the combination of both agents during 24 hours on CXCR4 surface expression inRPMI8226-CXCR4 and OPM-1 cells, measured by FACS. The CXCR4 expression level is presented as mean fluorescent intensity (MFI); data are presented asmean of triplicates �STDEV (��, P < 0.01). E, RPMI8226-CXCR4 cells were treated with various doses of FTY720 during 48 hours in the absence orpresence of BMSCs. Proliferation was measured by FACS. Data are presented as mean of triplicates �STDEV (��, P < 0.01). F, RPMI8226-EV andRPMI8226-CXCR4 cells were treated with FTY720 (10 mmol/L) during 24 hours in the absence or presence of BMSCs. Cell lysates were analyzed byWestern blot for the levels of MCL-1 and pS6 proteins; b-actin was used as an internal control. G, Effect of treatment with FTY720 (7.5 mmol/L), RAD001(10 mmol/L), or their combination on the viability of RPMI8226-CXCR4 cells incubated in the absence or presence of BMSCs, measured by FACS. H,RPMI8226-CXCR4 cells were incubated during 24 hours with FTY720 (10 mmol/L), RAD001 (10 mmol/L), or their combination in the absence or presenceof BMSCs. Cell lysates were analyzed by Western blot for the levels of pS6 protein and b-actin was used as an internal control.






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Beider et al.




induced cytotoxicity in MM cells was irrespective of response tobortezomib because FTY20 effectively targeted bortezomib-resistant cells. Moreover, the combination of FTY20 with bor-tezomib resulted in increased cell death and elevated pH2AX inboth bortezomib-sensitive and resistant MM cells, suggestingthe role of induced DNA damage in the mechanism of com-binational treatment. Combining FTY720 with bortezomibeffectively abrogated the undesirable increase in CXCR4induced by bortezomib treatment, providing a strong rationalefor the combination treatment. This implies that FTY720 maybe a potent inducer of apoptosis independent of the protea-some pathway and distinct from proteasome inhibitor-promot-ed apoptosis.

We further confirmed that the CXCR4/CXCL12 axis is adownstream component of the S1P signaling network in MMcells. Interaction with BMSCs that provides exogenous CXCL12,and therefore activate CXCR4, partially protected CXCR4-expressing MM cells from apoptosis induced by FTY720. Thiseffect was mediated by the activation of the mTOR/pS6 path-way. Indeed, inhibition of mTOR with RAD001 overcame theprotection of BMSCs and restored the sensitivity of MM cellsto FTY720.

These results come in agreement with previous works show-ing that the mTOR pathway is activated by CXCR4 in tumorcells (50, 51). The data we present here identify mTOR as one ofthe downstream targets of the CXCR4 signaling pathway acti-vated by stromal cells, a feature that may be important forevaluating combination strategies targeting the microenviron-ment-mediated resistance. The combination of FTY720 withmTOR inhibitors may be a promising strategy for the treatmentof patients with MM with high CXCR4 expression to amelioratethe protective signals supplied by the BM niche. Recently, thesafety and activity of RAD001 (everolimus) in relapsed orrefractory MM were evaluated in a phase I clinical study andpromising clinical responses were observed (52).

Finally, the effect of FTY720 was validated in our newly estab-lished in vivo model of CXCR4-driven human MM engrafting inmurine BM. Importantly, the FTY720 treatment was effective in

targeting MM cells in the BM niche and significantly reducedtumor load. The combination of FTY720 with bortezomib dem-onstrated preferential anti-MM activity, effectively reducingtumor burden and decreasing CXCR4 levels on surviving MMcells.

Taken together, our present findings demonstrate an activat-ing interaction between S1P and CXCR4/CXCL12 signalingpathways, which is important for MM cell survival and local-ization in CXCL12-expressing protective niches. Moreover, thisis the first evidence that CXCR4 can be directly targeted withFTY720, thus limiting the tumor-promoting activities of S1Pand the CXCR4/CXCL12 axes. In addition, the mTOR pathwaywas recognized as a downstream molecular effector beinginvolved in FTY720-mediated antimyeloma activities. Finally,FTY720 was shown to be effective in targeting MM cells in arecently developed xenograft model of MM with BM involve-ment, as a single agent and in combination with bortezomib,providing a preclinical rationale for its therapeutic applicationin patients with MM.

Disclosure of Potential Conflicts of InterestM.Abraham is an employee of Biokine.Nopotential conflicts of interest were

disclosed by the other authors.

Authors' ContributionsConception and design: K. Beider, A. Shimoni, M. Leiba, S. Klein, E. Galun,A. Peled, A. NaglerDevelopment of methodology: K. Beider, H. Wald, A. PeledAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Beider, E. Rosenberg, H. Bitner, A. Shimoni,M. Koren-Michowitz, E. Ribakovsky, D. Olam, L. Weiss, M. Abraham,A. NaglerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Beider, E. Rosenberg, M. Koren-Michowitz,E. Ribakovsky, A. PeledWriting, review, and/or revision of the manuscript: K. Beider, M. Leiba,M. Koren-Michowitz, A. NaglerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. WeissStudy supervision: A. Peled

Figure 6.Development of the CXCR4-driven xenograft MM in vivo model with BM involvement. Effect of FTY720 treatment, alone, or in combination withbortezomib, effectively targeting MM tumor burden in vivo in the BM niche. A, RPMI8226-EV, RPMI8226-CXCR4, and RPMI8226-CXCR4shCXCR4 cells(5 � 106) were intravenously (i.v.) injected into NSG mice. On days 21 to 25 following the i.v injection, the presence of human MM cells in the murineBM and spleen was assessed by FACS using GFP flag detection and specific anti-human CD138 antibody. Kaplan–Meier survival curve of animalsintravenously inoculated with human MM cells with low or high CXCR4, followed during 35 days, 6 mice per group. Calculation was performed usingNCSS. B, Percent of human MM infiltrated cells in murine BM and plasma levels of human IgG in MM-inoculated and non-inoculated mice, 23 daysfollowing cell injection. Data are presented as mean � SE from 6 mice. C, The presence of human CD138 mRNA was detected in BM of inoculatedanimals by specific RT-PCR. D, RPMI8226-CXCR4 tumor mass in BM tissue slides was detected by specific anti-human CXCR4 immunohistochemicalstaining. E–F, NSG mice were inoculated with RPMI8226-CXCR4 cells, and 3 days later, daily intraperitoneal injections of either FTY720 (10 mg/kg) orvehicle, for a total 15 injections, were initiated. On day 24 following MM cell inoculation, mice were sacrificed and tumor burden was evaluated. E,Representative plots showing the detection of human GFPþ RPMI8226-CXCR4 cells in the BM of mice treated with vehicle or FTY720 injections. F,Response of tumor burden to FTY720 treatment, percentage of RPMI8226-CXCR4 MM cells in the BM and human IgG serum levels(five mice per group). Data are presented as mean �SE; �� , P < 0.01. G–H, The combination treatment with FTY720 and bortezomib. NSG mice inoculatedwith RPMI8226-CXCR4 cells were treated with subcutaneous bortezomib injections (0.25 mg/kg) twice a week, total of six injections, intraperitonealFTY720 injections (5 mg/kg) three times a week, total of nine injections, or the combination of both agents. Animals were sacrificed 24 days after tumorinoculation. G, Response of tumor burden, percentage of RPMI8226-CXCR4 MM cells in the BM and plasma levels of human IgG, five mice per group. Dataare presented as mean �SE; �� , P < 0.01. H, CXCR4 cell-surface expression on human GFPþ MM cells detected in the BM of MM-bearing control animals, orfollowing the treatment with FTY720, bortezomib or their combination (five mice per group), evaluated by FACS and presented as mean fluorescentintensity (MFI). Data are presented as mean � SE; �� , P < 0.01.






AcknowledgmentsWe wish to thank the Naor family for a generous research grant memori-

alizing their son-in-law through the "Guy Weinshtock Multiple MyelomaFoundation," in support of research in the field of MM in the Division ofHematology at Chaim Sheba Medical Center, Tel Hashomer. We thank JoshuaFein (Sheba Medical Center) and Mery Clausen (Gene Therapy Institute,Hadassah Hospital) for technical assistance.

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 November 1, 2015; revised July 20, 2016; accepted September 1,2016; published OnlineFirst October 3, 2016.

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2017;23:1733-1747. Published OnlineFirst October 3, 2016.Clin Cancer Res Katia Beider, Evgenia Rosenberg, Hanna Bitner, et al. Myeloma via the CXCR4/CXCL12 PathwayThe Sphingosine-1-Phosphate Modulator FTY720 Targets Multiple

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