a Promotes Dissemination of Plasma Cells in Multiple ... · multiple myeloma patients, malignant PCs continually migrate and repopulate multiple sites throughout the bone marrow (2).

Molecular and Cellular Pathobiology

HIF-2a Promotes Dissemination of Plasma Cells inMultiple Myeloma by Regulating CXCL12/CXCR4and CCR1Kate Vandyke1,2,3, Mara N. Zeissig1,2, Duncan R. Hewett1,2, Sally K. Martin1,2,Krzysztof M. Mrozik1,2, CheeMan Cheong1,2, Peter Diamond1, L. Bik To3,4, Stan Gronthos2,5,Daniel J. Peet6, Peter I. Croucher7,8, and Andrew C.W. Zannettino1,2,3,9

Abstract

Disease progression and relapse in multiple myeloma isdependent on the ability of the multiple myeloma plasmacells (PC) to reenter the circulation and disseminate through-out the bone marrow. Increased bone marrow hypoxia isassociated with increased recirculation of multiple myelomaPCs. Accordingly, we hypothesized that during chronic hyp-oxia, activation of HIF-2a may overcome the bone marrowretention signal provided by stromal-derived CXCL12, therebyenabling dissemination of multiple myeloma PCs. Here wedemonstrate that HIF-2a upregulates multiple myeloma PCCXCL12 expression, decreasing migration toward CXCL12 andreducing adhesion to mesenchymal stromal cells in vitro. Wealso found that HIF-2a strongly induced expression of the

chemokine receptor CCR1 in multiple myeloma PCs. CCR1activation potently induces multiple myeloma PC migrationtoward CCL3 while abrogating the multiple myeloma PCmigratory response to CXCL12. In addition, increased CCR1expression by multiple myeloma PCs conferred poor prognosisin newly diagnosed multiple myeloma patients and was asso-ciated with an increase in circulating multiple myeloma PCs inthese patients. Taken together, our results suggest a role forhypoxia-mediated CCR1 upregulation in driving the egress ofmultiple myeloma PCs from the bone marrow. TargetingCCR1 may represent a novel strategy to prevent disseminationand overt relapse inmultiple myeloma. Cancer Res; 77(20); 5452–63.�2017 AACR.

IntroductionMultiple myeloma is an incurable hematologic cancer that is

responsible for an estimated 80,000 deaths worldwide, each year(1).Multiplemyeloma is characterized by the clonal proliferationofmalignant plasma cells (PC)within the bonemarrow.While, insome cases, malignant PCs establish at a single site, forming asolitary plasmacytoma, in the majority of cases, the transformedPC will disseminate throughout the bone marrow, leading to the

development of the asymptomatic precursor disease monoclonalgammopathy of undetermined significance (MGUS; ref. 2). In asmall proportion of MGUS patients (1% per year), intrinsicgenetic changes, coupled with extrinsic factors, lead to the pro-liferationof themultiplemyelomaPCs and their dissemination tosites throughout the skeleton (3). Ultimately, multiple myelomaPC proliferation and dissemination results in hypercalcemia,renal insufficiency, anemia, and osteolytic bone disease, featurescharacteristic of progressive disease (4).

Dissemination and repopulation throughout the bonemarrowis key, not only duringmultiplemyelomadisease progression, butalso in the outgrowth of resistant multiple myeloma PCs duringrelapse following therapy. Evidence suggest that inmost, if not allmultiple myeloma patients, malignant PCs continually migrateand repopulate multiple sites throughout the bone marrow (2).Circulating multiple myeloma PCs are observed in over two-thirds of newly diagnosed multiple myeloma patients and area predictor of response to therapy, with increased numbers ofcirculating multiple myeloma PCs representing an independentindicator of poor prognosis and rapid progression (5–8).

The homing of multiple myeloma PCs from the peripheralcirculation to the bone marrow is critically dependent on thechemokine (C-X-Cmotif) ligand CXCL12 (also known as stromalcell–derived factor-1; SDF-1), the ligand for CXCR4. CXCL12 ishighly expressed by bone marrow mesenchymal stromal cells(BMSC; refs. 9, 10) and acts as potent attractor for the homing ofboth normal and malignant PCs into the bone marrow (11, 12).In addition, CXCL12 is crucial in the subsequent retention ofmultiplemyelomaPCs in the bonemarrow, stimulating increased

1Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health andMedical Sciences, University of Adelaide, Adelaide, Australia. 2Cancer Theme,South Australian Health and Medical Research Institute, Adelaide, Australia. 3SAPathology, Adelaide, Australia. 4Haematology and Bone Marrow TransplantUnit, Royal Adelaide Hospital, Adelaide, Australia. 5Mesenchymal Stem CellLaboratory, Adelaide Medical School, Faculty of Health and Medical Sciences,University of Adelaide, Adelaide, Australia. 6School of Biological Sciences,Faculty of Sciences, University of Adelaide, Adelaide, Australia. 7Bone BiologyDivision, Garvan Institute of Medical Research, Sydney, Australia. 8St Vincent'sClinical School, Faculty of Medicine, University of New South Wales, Sydney,Australia. 9Centre for Cancer Biology, University of South Australia, Adelaide,Australia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Andrew C.W. Zannettino, Myeloma Research Labora-tory, AdelaideMedical School, Faculty of Health andMedical Sciences, Universityof Adelaide, North Terrace, Adelaide, SA 5000, Australia. Phone: 618-812-8490;Fax: 618-8222-3139; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0115

�2017 American Association for Cancer Research.

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adhesion of multiple myeloma PCs to bonemarrow stromal cellsand promoting multiple myeloma PC survival and resistance totherapeutics (13, 14).

While the mechanisms involved in multiple myeloma PChoming to the bone marrow are relatively well characterized, themechanisms responsible for drivingmultiplemyelomaPCs egressfrom the bone marrow remain to be elucidated. Emerging datasuggest that the elevated bone marrow hypoxia observed inmultiple myeloma (15–17) is a potential microenvironmentalstimulus that drives multiple myeloma PC dissemination (18).Recent studies suggest bone marrow hypoxia strongly correlateswith both tumor burden and numbers of circulating multiplemyeloma PCs in mouse models of multiple myeloma, suggestingthat hypoxic multiple myeloma PCs may be preferentially mobi-lized to theperipheral blood (18). Thismay, at least in part, be dueto decreased adhesion and a reduced chemotactic response tobone marrow stromal cells under hypoxia (18). A similar asso-ciation between tumor hypoxia and increased metastasis has alsobeen seen in patients with solid tumors and in animal models ofmetastasis (17).

Exposure to hypoxia induces gene expression through thestabilization and increased expression of the hypoxia-inducibletranscription factors HIF-1a and HIF-2a (17). Our previousstudies suggest that HIF-2a plays a critical role in multiplemyeloma disease progression, through binding to the promoterand driving expression of CXCL12 (15). In addition to beinghighly expressed by bonemarrow stromal cells, CXCL12 is highlyexpressed by multiple myeloma PCs (19) and multiple myelomaPC–derived CXCL12 plays an important role in driving osteolysis(19, 20) and angiogenesis (15) in myeloma patients.

In the studies presented here, we investigated whether upre-gulation of CXCL12 expression in multiple myeloma PCs, inresponse to HIF-2a expression, abrogates the chemoattractiontowards CXCL12. Notably, we identified the chemokine (C-C)receptor CCR1 as being strongly upregulated by chronic hypoxiain multiple myeloma PCs. Our data suggest that the interplaybetween CCR1 upregulation and inactivation of CXCR4 signaling(the CCR1/CXCR4 axis) drives the egress of multiple myelomaPCs from the bone marrow, leading to disease dissemination.

Materials and MethodsPatient samples

Ethical approval for this study was obtained from the RoyalAdelaide Hospital Institutional Ethics Review Committee (RAHapproval no. 030206, 131132, and 110304) and all patientsprovided written, informed consent, in accordance with theDeclaration of Helsinki. Posterior superior iliac spine bone mar-row aspirates (n ¼ 27) and peripheral blood (n ¼ 82) werecollected from 82 randomly selected patients [median age: 68years (range 35–84); male:female ratio 1.2:1] with symptomaticmultiple myeloma who presented at the Royal Adelaide Hospital(Adelaide, Australia). All multiple myeloma patients fulfilled thediagnostic criteria for active multiple myeloma (4), were newlydiagnosed, and had not received previous therapy. Bone marrowmononuclear cells (BMMNC) andperipheral bloodmononuclearcells (PBMNC)were isolated by density gradient centrifugation asdescribed previously (15) and were cryopreserved prior to use.Peripheral blood and bone marrow plasma samples were collect-ed and stored as described previously (19). BMSCs were isolatedby plastic adherence from bone chips recovered from bone

marrow aspirates isolated from the posterior iliac crest of healthyadult human donors and were used at passage 6. Survival wascalculated from the date of bonemarrow or blood collection untildeath, by any cause. Patients who were lost to follow up werecensored at the date of last contact.

Cell cultureUnless otherwise specified, all cell culture reagents were

sourced from Sigma-Aldrich and all media were supplementedwith 2 mol/L L-glutamine, 100 U/mL penicillin, 100 mg/mLstreptomycin, 1 mmol/L sodium pyruvate, and 10 mmol/LHEPES buffer. BMSCs were maintained as described previously(21).HMCLsRPMI-8226, LP-1, andU266were obtained from theATCC between 2000 and 2003; STR authentication was notconducted on these lines as they were obtained directly from theATCC. OPM2 cells were provided by Prof. Andrew Spencer(Monash University, Melbourne, Australia) in 2004; this cell linewas authenticated in 2016 using STR analysis performed by theMolecular Genetics Laboratory, SA Pathology, using anAmpFLSTR Identifiler PCR Amplification Kit (Thermo FisherScientific). HMCLs were last mycoplasma tested in May 2016using a MycoAlert Mycoplasma Detection Kit (Lonza). HMCLswere maintained in RPMI1640 medium with 10% FCS (ThermoFisher Scientific) and supplements. All experiments were con-ductedwithin 4weeks of thawing of cells. Cell lineswere routinelycultured in a humidified environment with 5% carbon dioxide at37�C. Hypoxic culture conditions (<1%oxygen) were establishedusing an anaerobic sachet (Oxoid). Where indicated, cells weretreated with the CCR1 inhibitor BX471 (Sigma Aldrich) in0.0004%DMSO, or 0.0004%DMSO alone, or with recombinanthuman (rh)CXCL12 (100 ng/mL; R&D Systems) or rhCCL3(100 ng/mL; PeproTech). Generation of RPMI-8226 and LP-1cell lines overexpressing CXCL12, HIF-1a, or HIF-2a is describedin Supplementary Methods and in refs. 15 and 20. Stable shRNA-mediated HIF-1a and HIF-2a knockdown in LP-1 cells wasconducted as described previously (15). CRISPR/Cas9-mediatedknockout of CCR1 in U266 cells was conducted as described inSupplementary Methods and in ref. 22.

CXCL12 ELISAPeripheral blood and bone marrow plasma and conditioned

media CXCL12 levels were determined using a commercialCXCL12 immunoassay (R&D Systems) according to the manu-facturer's recommendations, as described previously (19).

Real-time PCRTotal RNAwas isolated using TRIzol (Thermo Fisher Scientific)

and cDNA was synthesized using Superscript III (Life Technolo-gies). Real-timePCRwasperformedusing aRotor-Gene6000PCRmachine (Qiagen) using primers for human HIF2A (forward, 50-CTCTCCTCAGTTTGCTCTGAAAA-30; reverse, 50-GTCGCAGG-GATGAGTGAAGT-30), CXCL12 (forward, 50-ATGCCCATGCC-GATTCTTCG-30; reverse, 50-GTCTGTTGTTGTTCTTCAGCC-30),CXCR4 (forward, 50-CAGCAGGTAGCAAAGTGACG-30; reverse,50-GTAGATGGTGGGCAGGAAGA-30), CCR1 (forward, 50-AA-GCCCCAGAAACAAAGACTTC-30; reverse, 50-TGCATCCCCAT-AGTCAAACTCT-30), CCL3 (forward, 50-CTGGTTTCAGACTT-CAGAAGGAC-30; reverse, 50-GTAGTCAGCTATGAAATTCTGT-GG-30), B2M (forward, 50-AGGCTATCCAGCGTACTCCA-30; re-verse, 50-TCAATGTCGGATGGATGAAA-30), and ACTB (forward,

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50-GATCATTGCTCCTCCTGAGC-30; reverse, 50-GTCATAGTCC-GCCTAGAAGCAT-30) as described previously (15). Changes ingene expression were calculated relative to ACTB or B2M usingthe 2–DDCt method (23).

Flow cytometryCell surface expression of CXCR4 and CCR1 on HMCL was

assessed by staining 2 � 105 cells per test with an anti-CCR1mouse mAb (clone 53504; R&D Systems), an anti-CXCR4mousemAb (clone 44714; R&D Systems), or an in-house IgG2B isotypecontrol antibody (1A6.11), and a PE-conjugated goat anti-mousesecondary antibody (Southern Biotech) and were analyzed on aFACSCanto II flow cytometer (BDBiosciences). Cell surface CCR1andCXCR4 expression onmultiplemyelomaPCswas assessed onviable CD38þþ/CD138þ/CD45lo/CD19�multiple myeloma PCsby multicolor flow cytometry (LSRFortessa; BD Biosciences).Briefly, 3 � 105 mononuclear cells per test were stained with ananti-CCR1 or anti-CXCR4 antibody, as detailed above, or noprimary antibody [fluorescence minus one (FMO) control] fol-lowed by a PE-conjugated goat anti-mouse secondary antibody(Southern Biotech). Cells were subsequently blockedwith humangamma globulin prior to staining with CD38-PE-Cy7 (HIT2;BioLegend), CD138-AlexaFluor-647 (B-B4; Serotec) CD45-FITC(J.33; Beckman Coulter), and CD19-Brilliant Violet 421 (HIB19;BioLegend) antibodies. Cells were stained with the viability dyehydroxystilbamidine (FluoroGold; Invitrogen, Life Technologies)immediately before analysis. Viable, single cells were gated on thebasis of FSC and SSC characteristics and FluoroGold negativityand multiple myeloma PCs were identified as CD38-bright andCD138-positive cells, with contaminating cells excluded by sub-sequently gating on the CD45-low and CD19-negative popula-tion. Aminimumof 100 cells was required to fulfil these criteria tobe identified as a multiple myeloma PC population. A minimumof 5� 104 total nucleated cells were analyzed per test; 1 � 105 to1 � 106 total cells were analyzed per patient. CCR1 and CXCR4expression was quantitated as the change in the median fluores-cence intensity (DMFI), defined as the difference in MFI betweenthe CCR1- or CXCR4-stained sample and the FMO control(patient samples) or isotype control (cell lines).

Transwell migration assaysMigration assays were performed as described previously

(24). Briefly, cells (1 � 105) were washed once in RPMI1640with 1% FCS and were seeded in transwells in RPMI1640 with1% FCS and supplements in triplicate and cell migrationtoward chemoattractant (10% FCS or cytokines) or RPMI1640with 1% FCS (untreated controls) was assessed after 18 hoursby microscopy as described previously (24). Percentage cellmigration is represented normalized to the untreated controlsor appropriate control cells. Where indicated, cells were pre-treated with rhCXCL12 or rhCCL3 (100 ng/mL) for 72 hoursor BX471 (100 nmol/L; Sigma Aldrich) for 24 hours and werewashed once in RPMI1640 with 1% FCS prior to seedingmigration assays.

Adhesion assaysAdhesion assays were performed as described previously (24).

Briefly, pooled BMSCs from three independent normal donorswere plated 96-well plates at 8 � 103 cells/well and allowed toadhere overnight. RPMI-8226 cells (1 � 105 cells/well) were

overlaid onto the BMSCs and allowed to adhere for 15 minutes.Wells were then washed gently 3 times with Hank Buffered SaltSolution containing 5% FCS to remove nonadherent cells and thenumber of adherent cells enumerated as described previously(24). The percentage cell adhesion was calculated relative to totalcell input.

Actin remodeling assaysTo assess filamentous actin (F-actin) remodeling in response to

CXCL12 treatment, human multiple myeloma cells were washedtwice in serum-free media and were stimulated with rhCXCL12(100 ng/mL), for the indicated times, in triplicates. Cells wereimmediately fixed in 2% paraformaldehyde (pH 8.0) for 15 min-utes at room temperature. Cells were washed three times in PBScontaining 0.2% saponin and 5% FCS and stained with Alexa-fluor-680-phalloidin (200 U/mL; Life Technologies) for 30 min-utes on ice. Cells were then washed three times and phalloidinstaining was quantitated using a LSRFortessa flow cytometer (BDBiosciences).

Gene expression profilingRNA was extracted from LP-1-pRUF and LP-1-HIF2A cell lines

using TRIzol and the RNA was further purified using a silica gel–basedmembrane column (RNeasy kit;Qiagen). RNA labeling andhybridization to the Affymetrix Human Gene 1.0 ST Array andsubsequent data analysis was performed by the Adelaide Micro-array Centre (Adelaide, Australia). Microarray data have beendeposited in the GEO database (GSE102235). Probesets specificto noncoding RNA (miR, SNORNA, LNC, andpseudogenes)wereexcluded from further analysis. Gene set enrichment analysis(GSEA; ref. 25) was conducted using the Molecular SignaturesDatabase v5.0 (26).

Publicly available microarray dataMicroarray analysis of publicly available data was conducted

as described (24). For analysis of CXCL12 and CCR1 expressionin CD138-selected bone marrow PCs from newly diagnosedMGUS, multiple myeloma or plasma cell leukemia (PCL)patients or normal controls, two independent microarray data-sets were used: E-GEOD-16122 (normal, n ¼ 5; MGUS, n ¼ 11;multiple myeloma, n ¼ 133; PCL, n ¼ 9; ref. 27) and E-MTAB-363 (normal, n ¼ 5; MGUS, n ¼ 5; multiple myeloma, n ¼ 155;ref. 28). Analysis of overall survival in multiple myelomapatients stratified on the basis of CD138þ bone marrow PCgene expression at diagnosis was carried out using the datasetE-TABM-1138 (n ¼ 142; ref. 29). Correlative analysis of CCR1and CXCL12 gene expression in CD138-selected bone marrowPC from newly diagnosed multiple myeloma patients wasperformed using E-MTAB-363, E-TABM-1138, E-GEOD-16122, and E-MTAB-317 (n ¼ 226; ref. 30). E-MTAB-363,E-MTAB-317, and E-TABM-1138 were conducted on AffymetrixGeneChip Human Genome U133 plus 2.0 arrays; E-GEOD-16122 was conducted on U133A arrays. Raw microarray data(CEL files) were downloaded from ArrayExpress (EMBL-EBI) orGene Expression Omnibus (GEO; NCBI) and were normalizedby RMA using the bioconductor package affy (31) and R(version 3.03) and log2 transformed. Gene expression levelswere assessed in a panel of human multiple myeloma cell lines(n ¼ 66) using publicly available RNA-Seq data, downloadedfrom www.keatslab.org (32).

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Figure 1.

HIF-2a or CXCL12 overexpression in the human multiple myeloma cell line RPMI-8226 decreases migration toward CXCL12, adhesion to mesenchymal stromal cells,and cell surface CXCR4 expression.A,Upregulation of CXCL12mRNA and proteinwas confirmed in RPMI-8226-HIF2A cells by real-time PCR and ELISA. B,Adhesionof RPMI-8226-HIF2A and LP-1-HIF2A cells to bone marrow mesenchymal stromal cells was assessed. Adhesion is expressed relative to the vector control cells,following normalization to the total number of cells seeded. C, Migration of RPMI-8226-HIF2A and RPMI-8226-CXCL12 cells toward 100 ng/mL rhCXCL12 wasassessed in a transwell assay.Migration is expressed relative to the vector control cells, following subtraction of thebackground (no chemoattractant).D,Adhesion ofRPMI-8226-CXCL12 and LP-1-CXCL12 cells to bone marrow mesenchymal stromal cells was assessed. Adhesion is expressed relative to the vector control cells,following normalization to the total number of cells seeded. Cell surface CXCR4 expression was assessed by flow cytometry on RPMI-8226-CXCL12 and vectorcontrols (E), RPMI-8226 cells following 72-hour culture with 100 ng/mL rhCXCL12 or media alone (F), or RPMI-8226 cells overexpressing HIF-2a and vector controls(G), following staining with an anti-CXCR4 antibody or isotype control. Graphs depict the mean þ SEM of three biological replicates (A) or of three or moreindependent experiments (B–G). � , P < 0.05, paired t test. E–G, Representative histograms show CXCR4 expression on CXCL12-overexpressing (E), CXCL12-treated(F), or HIF-2alpha-overexpressing (red; G) or control (black) cells; representative isotype controls are shown in gray.






Statistical analysesUnless otherwise described, statistical analysis was performed

using GraphPad Prism (version 5.02; GraphPad Software). Dif-ferences in gene expression between groups in microarray experi-ments were assessed using linear models for microarray dataanalysis (LIMMA; ref. 33) in MultiExperiment Viewer (MeV;ref. 34). Correlations were assessed using Pearson correlationcoefficients. Overall survival was assessed using Kaplan–Meiercurves; comparisons between groups were made using the log-rank (Mantel–Cox) test and theMantel–Haenszel hazard ratio. Assurvival in the CXCL12 expression subgroups did not meet theproportional hazards assumption, each variable was divided intotwo groups (overall survival �104 weeks or >104 weeks) prior toconducting univariate analyses. For time-course and CCR1 inhib-itor experiments, groups were compared using two-way ANOVAwith Sidak multiple comparison test. For analysis of CCR1expression on peripheral blood and bone marrow multiple mye-lomaPCs, asCCR1expressionwasundetectable in some cases, thenonparametricWilcoxonmatchedpairs signed rank test was used.For all other experiments, groups were compared using paired orunpaired Student t tests, as described.Differenceswere consideredto be statistically significant when the P < 0.05.

ResultsCXCL12 expression desensitizes multiple myeloma PCs toexogenous CXCL12

We have previously demonstrated that HIF-2a and hypoxiastrongly upregulate the expression of CXCL12 in multiple mye-loma PCs (15). Overexpression of HIF-2a in the HMCL LP-1(LP-1-HIF2A) and RPMI-8226 (RPMI-8226-HIF2A; refs. 15, 20)was confirmed using real-time PCR (Supplementary Fig. S1A) andwas found to induce CXCL12 mRNA and protein expression(Fig. 1A; Supplementary Fig. S1B and S1C).

We hypothesized that HIF-2a–mediated upregulation ofendogenous CXCL12may inhibitmultiplemyeloma PC responseto exogenous CXCL12. HIF-2a overexpression significantlyreduced the adhesion of RPMI-8226-HIF2A or LP-1-HIF2A cellsto BMSCs (P < 0.05; Fig. 1B). LP-1 cells did not migrate under anycondition used here, precluding assessment of the effects ofHIF-2a and CXCL12 on migration in this cell line. However, inRPMI-8226 cells, there was a significant decrease in the migrationof RPMI-8226-HIF2A cells toward CXCL12 (P < 0.05; Fig. 1C)despite an increase in the migration of RPMI-8226-HIF2A cellstoward 10% FCS (P < 0.05; Supplementary Fig. S1D).

Figure 2.

Expression of the chemokine receptor CCR1 is upregulated by hypoxia and HIF-2a in human multiple myeloma cell lines. CCR1 expression wasassessed by real-time PCR in LP-1 cells following overexpression (A) or shRNA-mediated knockdown (B) of HIF-2a or culture under normoxic or hypoxic(<1% O2) conditions for 6–48 hours (C). D, CCR1 expression was assessed by real-time PCR in RPMI-8226 cells following overexpression of HIF-2a.Upregulation of CCR1 protein in RPMI-8226 cells following overexpression of HIF-2a (E) or hypoxic culture for 72 hours (F) was confirmed by flow cytometryfollowing staining with an anti-CCR1 antibody or isotype control. Expression was normalized to the vector control cells following subtraction of the isotypecontrol (DMFI). Graphs depict mean þ SEM of three biological replicates (A, B, and D) or three or more independent experiments (C, E, and F). � , P < 0.05,paired t test (A, B, and D–F) or two-way ANOVA with Sidak multiple comparison test (C).

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CXCL12 was overexpressed in HMCLs, as described previously(Supplementary Fig. S1E and S1F; refs. 15, 20). Similar to theeffects of HIF-2a overexpression, CXCL12 overexpression inRPMI-8226 cells abrogated the chemotactic response to CXCL12(P < 0.05; Fig. 1C). In addition, pretreatment of RPMI-8226 cellswith CXCL12 for 72 hours decreased migration toward CXCL12(P<0.05; Supplementary Fig. S2A). Thiswas also demonstrated inthe HMCL OPM2 (P < 0.05; Supplementary Fig. S2A). Adhesionof RPMI-8226 and LP-1 cells to BMSCs was also decreasedfollowing overexpression of CXCL12 (P < 0.05; Fig. 1D) but wasunaffected by exogenous CXCL12 in RPMI-8226 and LP-1 cells(P¼ 0.30 and P¼ 0.17, respectively; Supplementary Fig. S2B). Incontrast, exogenous CXCL12 increased adhesion in OPM2 cells(P < 0.05; Supplementary Fig. S2B).

Hypoxia reduces cell surface expression of CXCR4 in multiplemyeloma PCs

Overexpression of CXCL12, or treatment with CXCL12,decreased the cell surface expression of CXCR4 in HMCL(Fig. 1E and F; Supplementary Fig. S2C and S2D), consistent

with previous studies (11). In addition, cell surface CXCR4protein was downregulated in response to HIF-2a overexpres-sion (Fig. 1G; Supplementary Fig. S2E) or by hypoxicculture (Supplementary Fig. S2F) in RPMI-8226 and LP-1 cells.CXCR4 mRNA levels were also decreased in LP-1-HIF2A,but not RPMI-8226-HIF2A cells (Supplementary Fig. S2G;P < 0.05).

The chemokine receptor CCR1 is upregulated by HIF-2aTo investigate other mechanisms whereby hypoxia may drive

dissemination in multiple myeloma, we used microarrays toassess the effects of HIF-2a overexpression on gene expressionin the LP-1 cell line. A total of 79 genes were upregulated, and 83genes were downregulated, by more than 2-fold in LP-1-HIF2Acells (Supplementary Table S1). Importantly, the most highlyupregulated gene was chemokine (C-C) receptor 1 (CCR1;6.5-fold upregulated), the receptor for C-C chemokine ligand 3[CCL3; also known as macrophage inflammatory protein-1a(MIP-1a); Supplementary Table S1]. GSEA of the upregulatedgenes found that the top-most significant hallmark genesets was

Figure 3.

CCL3 treatment reduces migration toward CXCL12 and interferes with CXCR4 signaling. A, Migration of RPMI-8226-HIF2A and RPMI-8226-pRUF cellstoward 100 ng/mL rhCCL3 was assessed in a transwell assay. Results are expressed relative to the vector control cells. B, RPMI-8226 cells were seeded inthe top chamber of a transwellwith 100ng/mL rhCCL3ormedia aloneandmigration toward 100ng/mL rhCXCL12 ormedia alonewas assessed. Results are expressedrelative to the untreated control. C, RPMI-8226 and OPM2 cells were treated with 100 ng/mL rhCCL3 or media alone for 72 hours, cells were washed, and migrationtoward 100 ng/mL rhCXCL12 was assessed in a transwell assay. Migration is expressed relative to the untreated control cells following subtraction of the "nochemoattractant" controls. D, RPMI-8226 or OPM2 cells were treated with 100 ng/mL rhCCL3 or media alone for 72 hours, cells were seeded on a bone marrowmesenchymal stromal cell monolayer, and percent cell adhesion, relative to total cell input, was assessed after 15 minutes. Results are expressed relative tothe untreated controls. E, Expression of CXCR4 protein was determined by flow cytometry following staining with an anti-CXCR4 antibody, after 72 hours of culturewith 100 ng/mL rhCCL3 or media alone. Expression is shown normalized to the untreated controls following subtraction of the isotype control (DMFI). F, RPMI-8226cells were cultured with 100 ng/mL rhCCL3 or media alone for 72 hours, washed, and stimulated with 100 ng/mL rhCXCL12 for the indicated times. Cells wereimmediately fixed and F-actin was quantitated by flow cytometry following staining with AlexaFluor-680-phalloidin. Expression is shown as MFI, normalizedto baseline levels. Graphs depict the mean þ SEM of three independent experiments (A–D and F) or two or more independent experiments (E). � , P < 0.05, pairedt test (A and C–E), one-way ANOVA with Dunnett posttest (B) or two-way ANOVA with Sidak multiple comparison test (F).






the hypoxia hallmark set (false discovery rate q value ¼ 2.06 �10�6; Supplementary Table S2).

HIF-2a overexpression and knockdown in LP-1 resulted in arespective 23-fold upregulation and a 1.5-fold reduction in CCR1expression (Fig. 2A andB). Induction ofCCR1 expressionwas alsoconfirmed following 24 and 48 hours of hypoxic culture (<1%O2; Fig. 2C). In contrast, overexpression or knockdown of HIF-1ahad no effect on CCR1 expression in LP-1 cells (SupplementaryFig. S3A and S3B). Despite expressing detectable, low levels ofCCR1 mRNA (Supplementary Fig. S3C and S3D), LP-1 cells didnot express cell surface CCR1 protein (Supplementary Fig. S3E),or migrate in response to CCL3 (Supplementary Fig. S3F). RPMI-8226 and OPM2 expressed the highest CCR1 mRNA and cellsurface protein (Supplementary Fig. S3C–S3E) and readilymigrated in response to CCL3 in vitro (Supplementary Fig.S3F). In RPMI-8226 cells, overexpression of HIF-2a increasedCCR1mRNAandprotein levels (Fig. 2DandE).Cell surfaceCCR1expression was also increased following hypoxic culture in RPMI-8226 cells (Fig. 2F). In addition, hypoxic induction of CCR1 geneexpression at 24 hours was confirmed in OPM2 and U266 celllines (Supplementary Fig. S3G).

CCL3 inhibits the migratory response to CXCL12We hypothesized that upregulation of CCR1 in response to

HIF-2a could increase the migratory response of multiple mye-

loma cells toCCL3. In support of this, overexpressionofHIF-2a inRPMI-8226 cells increased migration toward CCL3 (Fig. 3A).

CCL3/CCR1 signaling can lead to internalization and/or inac-tivation of CXCR4, as has been shown for other cell types (35, 36).In keepingwith this,we found that either incubationwithCCL3 inthe top chamber of a transwell (Fig. 3B), or CCL3 pretreatment for3 days (Fig. 3C), reduced the migration of RPMI-8226 andOPM2cells toward CXCL12. However, CCL3 treatment had no effect onthe adhesion of RPMI-8226 or OPM2 cells to BMSCs (Fig. 3D) orthe expression of cell surface CXCR4 protein in a panel of humanmultiple myeloma cell lines (Fig. 3E). In contrast, CXCL12 over-expression or pretreatment had no inhibitory effects onmigrationtoward CCL3 or cell surface expression of CCR1 in humanmultiple myeloma cell lines (Supplementary Fig. S4A–S4C).Furthermore, we examined the effects of CCL3 pretreatment for72 hours on F-actin remodeling (37). CCL3 treatment preventedthe induction of F-actin remodeling in response to CXCL12treatment in RPMI-8226 cells (Fig. 3F).

The HMCL U266 was evaluated as it expresses high levels ofCXCR4 on the cell surface (Fig. 4A), but does not migrate towardCXCL12 in vitro (Fig. 4B). We hypothesized that the abundantendogenous production of CCL3 by these cells (Fig. 4C; ref. 38)may prevent their response to CXCL12. In support of this, eitherknockout of CCR1 or pretreatment of the cells with a CCR1inhibitor, BX471, restored the migratory response of U226 cells

Figure 4.

CCR1 inhibition or knockout restores the migratory response of U266 cells to CXCL12. A, Cell surface CXCR4 expression was assessed by flow cytometry on apanel of human multiple myeloma cell lines following with an anti-CXCR4 or isotype control antibody. Expression is shown following subtraction ofthe isotype control (DMFI). B,Migration of humanmultiple myeloma cell lines toward 100 ng/mL rhCXCL12 was assessed in a transwell assay. C, Expression of CCL3was assessedby real-timePCR in a panel of humanmultiplemyelomacell lines.D,CCR1was knockedout inU266 cells using theCRISPR-Cas9 systemandmigration ofCCR1 knockout (CCR1 KO) or vector control cells toward 100 ng/mL rhCXCL12 or media alone was assessed in a transwell assay. E, U266 cells were treatedwith 100 nmol/L BX471 or 0.0004%DMSO vehicle control for 24 hours andmigration toward 100 ng/mL rhCXCL12 or media alone was assessed in a transwell assay.Graphs depict the mean þ SEM of three or more independent experiments (A, B, D, and E) or three biological replicates (C). � , P < 0.05, paired t test (B) ortwo-way ANOVA with Sidak multiple comparison test (D and E).

Vandyke et al.





toward CXCL12 (Fig. 4D and E). In contrast, the migration ofOPM2 cells toward CXCL12 was unaffected by BX471 treatment(Supplementary Fig. S4D).

Cell surface CXCR4 expression is decreased in bone marrow–resident multiple myeloma PCs in multiple myeloma patients

CXCR4 protein expression was assessed by flow cytometry onviable PCs in the bone marrow and peripheral blood of 16 newlydiagnosedmultiplemyeloma patients. Peripheral bloodmultiplemyeloma PCs were detectable by flow cytometry in 11 of 16(68.8%) patients. CXCR4 was expressed on both bone marrowand peripheral blood multiple myeloma PCs in all patients forwhommatched peripheral blood and bonemarrow samples wereavailable (Supplementary Fig. S5A). Notably, expression ofCXCR4 was significantly lower in bone marrow–resident thanperipheral blood multiple myeloma PCs (P ¼ 0.0051; Fig. 5A).However, there was no association between either bone marrowor peripheral blood CXCR4 expression and bone marrow PCburden (bone marrow CXCR4: r ¼ �0.086; P ¼ 0.80; peripheralblood CXCR4: r ¼ �0.23, P ¼ 0.49, Pearson correlation) orperipheral blood multiple myeloma PC number (bone marrowCXCR4: r¼0.13,P¼0.70; peripheral bloodCXCR4: r¼�0.0001,P ¼ 1.00, Pearson correlation).

Elevated serum or multiple myeloma PC CXCL12 expression isassociated with poor early survival in multiple myelomapatients

Here, we hypothesized that high local levels of CXCL12 in thebone marrow (19) result in a downregulation of cell surfaceCXCR4. In support of this, plasma CXCL12 protein levels were

significantly higher in the bone marrow than in the peripheralblood of 11 newly diagnosed multiple myeloma patients withactive disease (P < 0.05; Fig. 5B), with higher CXCL12 proteinlevels in the bone marrow than the peripheral blood in 8 of 11(72.7%) patients.

Importantly, we found that increasedmultiplemyelomaPCsorperipheral blood plasma CXCL12 levels were associated withearly poor survival outcomes (Fig. 5C and D). Above medianCXCL12, either at themRNA level (Fig. 5C), or protein level in theperipheral plasma of patients (Fig. 5D), was associated with aninitial survival disadvantage within two years of diagnosis[mRNA, P ¼ 0.0236; HR ¼ 2.63 (95% CI: 1.14–6.06); protein,P ¼ 0.0274, HR ¼ 3.148 (95% CI: 1.14–8.72)].

Expression of CCR1 in multiple myeloma PCs of patientscorrelates with circulating multiple myeloma PC numbers

In silico analysis of CCR1 expression in publicly availablemicroarray data from newly diagnosed patients with MGUS,multiple myeloma, or PCL and normal controls found that CCR1expression was elevated (>95% CI of normal) in 55.5% (86/155;E-MTAB-363) and 20.3% (27/133; E-GEOD-16122) of multiplemyeloma patients, and was significantly elevated in 55.6% (5/9;E-GEOD-16122: Padj ¼ 0.0047; LIMMA) of PCL patients (Fig. 6Aand B).Moreover, abovemedianCCR1 expression was associatedwith poor prognosis in newly diagnosed multiple myelomapatients in an independent dataset [P ¼ 0.025; HR ¼ 2.3 (95%CI: 1.1–4.9); n ¼ 142 patients; E-TABM-1138; Fig. 6C]. In addi-tion, in support of our data, expression of CCR1 positivelycorrelated with CXCL12 expression in multiple myeloma PCsfrom newly diagnosed multiple myeloma patients in four

Figure 5.

Multiple myeloma PC CXCR4expression is increased, and CXCL12levels are decreased, in the peripheralblood (PB) of patients with newlydiagnosed multiple myelomacompared with bone marrow (BM).A, CXCR4 expression (DMFI) is shownfor bone marrow and peripheral bloodmultiple myeloma PCs from 11 multiplemyeloma patients for whom pairedbone marrow and peripheral bloodsamples were available. B, Levels ofCXCL12 protein in paired peripheralblood and bone marrow plasmasamples from newly diagnosedmultiple myeloma patients (n ¼ 11) asassessed by ELISA. � , P < 0.05, paired ttest. C, Kaplan–Meier plots of overallsurvival are shown for newlydiagnosed multiple myeloma patientsstratified on the basis of medianCD138þ plasma cell CXCL12expression, as derived frommicroarraydataset E-TABM-1138 (n ¼ 142patients). D, Kaplan–Meier plots ofoverall survival are shown for newlydiagnosed multiple myeloma patientssubdivided on the basis of medianperipheral blood plasma CXCL12protein levels, as determined by ELISA(n ¼ 66 patients).






independent datasets (E-MTAB-363: r ¼ 0.34, P < 0.0001;T-TABM-1138: r ¼ 0.36, P < 0.0001; E-GEOD-16122: r ¼ 0.57,P < 0.0001; E-MTAB-317: r ¼ 0.51, P < 0.0001; Pearsoncorrelation).

Furthermore, CCR1 protein was expressed on viable bonemarrow multiple myeloma PCs in 13 of 16 (81%) patients(Supplementary Fig. S5B), as assessed by flow cytometry.Expression of CCR1 was significantly lower in peripheral bloodmultiple myeloma PCs than in bone marrow multiple myelo-ma PCs in patients with paired bone marrow and peripheralblood samples (n ¼11; P ¼ 0.031; Fig. 6D). In addition, bonemarrow infiltration with multiple myeloma PCs positivelycorrelated with bone marrow multiple myeloma PC expressionof CCR1 (r ¼ 0.78; P ¼ 0.0046; Fig. 6E). CCR1 expression onperipheral blood multiple myeloma PCs also positively corre-lated with the number of circulating multiple myeloma PCs(r ¼ 0.70; P ¼ 0.016; Fig. 6F), and there was a trend towardincreased bone marrow multiple myeloma PC expression ofCCR1 and increased numbers of circulating multiple myelomaPCs (r ¼ 0.56; P ¼ 0.070).

DiscussionThe homing and retention of leukocytes and tumor cells

within the bone marrow, and their subsequent mobilizationfrom the bone marrow, is regulated by the altered expressionof chemokine receptors. The trafficking of normal and malig-nant PCs from the peripheral blood to the bone marrow isdriven by the CXCL12 gradient generated by high productionof CXCL12 by BMSCs (9). Disruption of this CXCL12 gra-dient is required to release leukocytes and hematopoieticprecursor cells into the peripheral blood (39, 40). Similarly,egress of multiple myeloma PCs from the bone marrow isdependent on overcoming this CXCL12/CXCR4 retentionsignal, with decreased CXCR4 expression in bone marrowmultiple myeloma PCs being associated with increasedtumor dissemination and poor prognosis (41, 42). Further-more, treatment with the CXCR4 inhibitor AMD3100 leadsto mobilization of multiple myeloma cells from the bonemarrow to the peripheral blood in a mouse model ofmultiple myeloma (14).

Figure 6.

Elevated CCR1 expression is associated with poor prognosis and increased numbers of circulating multiple myeloma (MM) PCs. In silico analysis wasperformed on publicly available datasets analyzing gene expression in CD138þ plasma cells isolated from MGUS (n ¼ 11) and multiple myeloma (n ¼ 133)patients and healthy controls (n ¼ 5; E-GEOD-16122; A) and MGUS (n ¼ 5), multiple myeloma (n ¼ 155), and PCL (n ¼ 9) patients, and healthy controls (n ¼ 5;E-MTAB-363; B). Scatter dot plots show median and interquartile range. � , P < 0.05, relative to normal controls, LIMMA. C, Kaplan–Meier plots of overallsurvival are shown for newly diagnosed multiple myeloma patients stratified on the basis of median CD138þ plasma cell CCR1 expression, derived from microarraydataset E-TABM-1138 (n¼ 142 patients).D, CCR1 expression (DMFI) is shown for bonemarrow and peripheral bloodmultiple myeloma PCs from 11 multiple myelomapatients. �, P < 0.05 relative to bone marrow, Wilcoxon matched pairs signed rank test. CCR1 expression on bone marrow (E) and peripheral blood (F)multiple myeloma PCs plotted against bone marrow multiple myeloma PC burden (E) and peripheral blood multiple myeloma PC burden (F), as indicated. rand P values are shown for Pearson correlation analyses.

Vandyke et al.





Here, we demonstrate that increased HIF-2a–mediated expres-sion of CXCL12 in PCs desensitizes cells to CXCL12 in vitro,decreasing migration and adhesion to stroma. This is consistentwith previous findings demonstrating that high endogenousCXCL12 expression could lead to homologous desensitizationof CXCR4, downregulating cell surface CXCR4 expression anddecreasing response to CXCL12 (11). In addition, we found thatcell surface CXCR4 is strongly downregulated in response toHIF-2a or sustained hypoxic culture in humanmultiplemyelomacell lines, suggesting that chronic hypoxia may be sufficient todownregulate response to CXCL12. While short-term hypoxiamay increase CXCR4 expression (as shown previously in ref. 15at 24 hours and less), sustained hypoxia, as shown here, candownregulate CXCR4 protein expression, at least in part throughincreased expression of CXCL12.

Our in vitro data suggest that, in response to hypoxia, CXCL12overexpression and subsequent CXCR4 downregulation in mul-tiple myeloma PCs may decrease responsiveness to CXCL12,facilitating egress from the bone marrow. However, while wefound that increasedCXCL12expressionwas associatedwith earlypoor survival outcomes for patients, we found no associationbetween expression of CXCR4 on peripheral blood or bonemarrow multiple myeloma PCs and the number of circulatingmultiple myeloma cells. This strongly suggests that mechanismsother than regulation of cell surface CXCR4 levels may be moreimportant in regulating multiple myeloma PC dissemination inpatients. Using microarray, we identified that the most highlyupregulated gene following HIF-2a overexpression in LP-1 cellswas the chemokine receptor CCR1. CCR1 has previously beensuggested to play an important role inmultiplemyelomawith thelevels of the CCR1 ligand CCL3 being elevated in the peripheralblood of multiple myeloma patients with advanced disease(43, 44). Notably, CCL3 acts as a potent stimulus for the migra-tion of multiple myeloma cells in vitro (45).

Here, we show that HIF-2a increases CCR1 expression, increas-ing migration toward CCL3 and abrogating the chemotacticresponse to CXCL12 in HMCL. This provides a novel meanswhereby hypoxia may increase PC dissemination in patients withmultiple myeloma. While the mechanisms responsible for thisremain unclear, CCL3/CCR1 signaling has been shown to haveCXCR4-dependent and -independent roles in mobilization of

hematopoietic progenitor cells from the bone marrow. In addi-tion, CCL3/CCR1 signaling may lead to heterologous desensiti-zation of CXCR4, as has been shown for other cell types (35, 36).

Our data show that CCL3 stimulation does not affect cellsurface CXCR4 expression, suggesting that the effects of CCL3are downstream of CXCR4. It is possible that CXCR4 and CCR1may heterodimerize, as has been shown for CXCR4 and CCR5(46). However, as CCL3 downregulates cell surface CCR1 (datanot shown) without affecting cell surface CXCR4 levels, thiswould suggest that stable CCR1–CXCR4 heterodimers are notpresent. In addition, CCL3 signaling through CCR1 may activatekinases that modulate activation of CXCR4 downstream targets,thereby altering downstream signaling (47). In support of this, wefound that the abrogation of CXCL12-mediated migration byCCL3 was associated with decreased F-actin remodeling inresponse to CXCL12.

In addition to CCR1, CCL3 is also the ligand for the chemokinereceptor CCR5. However, the effects of CCL3 observed here aredue to CCR1 and not CCR5 as the cell lines used do not expressdetectable CCR5 (45, 48, 49). Furthermore, we demonstrated thateither knockout of CCR1 or treatment with an inhibitor specificfor CCR1, but not CCR5, restored themigratory response of U266cells to CXCL12, further supporting the role of CCR1 in thisprocess.

We also demonstrated that CCR1 gene expression is associatedwith poor prognosis in multiple myeloma patients and there is asignificant positive association between peripheral blood multi-ple myeloma PC numbers and the expression of CCR1 on eitherperipheral blood or bone marrow multiple myeloma PCs. Incontrast, cell surface CXCR4 expression was not associated withcirculating multiple myeloma tumor cell numbers, suggestingthat CCR1 may be more important than CXCR4 in determiningthe numbers of peripheral blood multiple myeloma PCs. As thenumbers of patients included in flow cytometry studies wereinsufficient to conduct multivariate analysis, it is unclear whetherthe adverse survival effect ofCCR1 expression is dependent on theincrease in peripheral blood multiple myeloma PCs; future stud-ies in this area are warranted.

CXCR4plays a key role in thehomingofmultiplemyelomaPCsfrom the peripheral blood to the bone marrow (11, 12, 45) andreactivation of CXCR4when in the peripheral blood is required to

Egress

Tumor growthCXCR4

CCR1

CCR1

CXCR4

MM PC

Blood vessel

Hypoxic MM PCNormoxic MM PC

HIF-2Hypoxia

Bone marrow

++++ CXCR4+CCR1

+ CXCR4++++CCR1

BMSC

Homing

Figure 7.

Proposed mechanism of hypoxic regulation ofCXCR4 and CCR1 and subsequent plasma celldissemination in multiple myeloma. IncreasedCCR1 expression in multiple myeloma (MM) PCsin response to bone marrow hypoxia and HIF-2aactivation overrides the BMSC-derived CXCL12retention signal and triggers egress of multiplemyeloma PCs to the peripheral circulation. Asubsequent increase in CXCR4 receptorexpression and resensitization to CXCL12 inmultiple myeloma PCs in the peripheralcirculation allows homing to other bone marrowsites.






enable subsequent homing of the cells in the circulation tosecondary sites in the bone marrow. Our data show that CCR1expression is decreased, and CXCR4 expression increased, inmultiple myeloma tumor cells in the peripheral blood whencompared with the bone marrow. While the mechanisms respon-sible for this remain to be fully elucidated,wehypothesize that theincreased oxygen tension in the peripheral blood compared withthe bonemarrow (17, 50, 51) may be sufficient to decrease CCR1expression, allowing resensitization to CXCL12. In addition, ourdata suggest that CXCL12 levels are lower in the peripheral bloodthan in the bone marrow in patients with high CXCL12, support-ing previous findings (11, 18). Decreased local CXCL12 concen-trations in the peripheral blood may therefore lead to increasedCXCR4 levels and allow multiple myeloma PCs to subsequentlyrehome to the bone marrow.

Taken together, our studies point to a potential role for hypoxi-cally mediated changes in the CCR1/CXCR4 axis that drive egressof multiple myeloma PCs from the bone marrow (Fig. 7). Wesuggest that increased CCR1 expression inmultiple myeloma PCsin response to hypoxia overrides the stroma-derived CXCL12retention signal and triggers egress to the peripheral bood. Futurestudies are required to determine whether the desensitization ofCXCR4 in response to CXCL12 and CCL3 is involved in themobilization and dissemination of multiple myeloma tumorcells in vivo. Moreover, identification of the factor(s) involved inthe recirculation and dissemination process inmultiple myelomais key in the development of therapeutic strategies that limit overtrelapse.We suggest that further studies arewarranted to determinewhether therapeutic targeting of CCR1 is a potential strategy forpreventing multiple myeloma cell dissemination to limit diseaseprogression and relapse.

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

Authors' ContributionsConception and design: K. Vandyke, D.R. Hewett, L.B. To, D.J. Peet,A.C.W. ZannettinoDevelopment of methodology: K. Vandyke, P. Diamond, D.J. PeetAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Vandyke, M.N. Zeissig, S.K. Martin, K.M. Mrozik,C.M. Cheong, P. DiamondAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Vandyke, K.M. Mrozik, L.B. To, S. GronthosWriting, review, and/or revision of the manuscript: K. Vandyke, M.N. Zeissig,D.R. Hewett, S.K. Martin, C.M. Cheong, S. Gronthos, D.J. Peet, P.I. Croucher,A.C.W. ZannettinoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.B. ToStudy supervision: K. Vandyke, D.R. Hewett, A.C.W. Zannettino

AcknowledgmentsBiospecimens were provided by the South Australian Cancer Research

Biobank (SACRB), which is supported by the Cancer Council SA Beat CancerProject, Medvet Laboratories Pvt. Ltd., and the Government of South Australia.The authorswould like to thank JoGardiner for providing clinical followupdatafrom the Royal Adelaide Hospital Paraproteinaemia Database, which is sup-ported by funding from Celgene and the Royal Adelaide Hospital HaematologyPrivate Practise Fund.

Grant SupportThis research was supported in part by a Young Investigator Project Grant

from the Cancer Australia Priority-driven Collaborative Cancer ResearchScheme, funded by Cure Cancer Australia (APP1100358 to K. Vandyke),a National Health & Medical Research Council Project Grant (APP626911 toA.C.W. Zannettino, S. Gronthos, D.J. Peet, L.B. To), and a grant from the RoyalAdelaide Hospital Contributing Haematologists' Committee to K. Vandyke,L.B. To, A.C.W. Zannettino, D.R. Hewett.

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

Received January 18, 2017; revised May 11, 2017; accepted August 18, 2017;published OnlineFirst August 30, 2017.

References1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.

Cancer incidence and mortality worldwide: sources, methods and majorpatterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86.

2. Ghobrial IM. Myeloma as a model for the process of metastasis: implica-tions for therapy. Blood 2012;120:20–30.

3. Dutta AK, Hewett DR, Fink JL, Grady JP, Zannettino ACW. Cutting edgegenomics reveal new insights into tumour development, disease progres-sion and therapeutic impacts in multiple myeloma. Br J Haematol2017;178:196–208.

4. Rajkumar SV,DimopoulosMA, PalumboA, Blade J,Merlini G,MateosMV,et al. International Myeloma Working Group updated criteria for thediagnosis of multiple myeloma. Lancet Oncol 2014;15:e538–48.

5. Nowakowski GS, Witzig TE, Dingli D, Tracz MJ, Gertz MA, Lacy MQ, et al.Circulating plasma cells detected by flow cytometry as a predictor ofsurvival in 302 patients with newly diagnosed multiple myeloma. Blood2005;106:2276–9.

6. Witzig TE, GertzMA, Lust JA, Kyle RA, O'FallonWM, Greipp PR. Peripheralblood monoclonal plasma cells as a predictor of survival in patients withmultiple myeloma. Blood 1996;88:1780–7.

7. Dingli D,Nowakowski GS,Dispenzieri A, LacyMQ,Hayman SR, RajkumarSV, et al. Flow cytometric detection of circulating myeloma cells beforetransplantation in patients with multiple myeloma: a simple risk stratifi-cation system. Blood 2006;107:3384–8.

8. BianchiG,KyleRA, LarsonDR,Witzig TE,KumarS,DispenzieriA, et al.Highlevels of peripheral blood circulating plasma cells as a specific risk factor forprogression of smoldering multiple myeloma. Leukemia 2013;27:680–5.

9. Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA. A highlyefficacious lymphocyte chemoattractant, stromal cell-derived factor 1(SDF-1). J Exp Med 1996;184:1101–9.

10. Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, Gronthos S.Stromal-derived factor-1 promotes the growth, survival, and develop-ment of human bone marrow stromal stem cells. Blood 2005;105:3793–801.

11. Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM, et al.Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migra-tion and homing in multiple myeloma. Blood 2007;109:2708–17.

12. Roccaro AM, Mishima Y, Sacco A, Moschetta M, Tai YT, Shi J, et al. CXCR4regulates extra-medullarymyeloma through epithelial-mesenchymal-tran-sition-like transcriptional activation. Cell Rep 2015;12:622–35.

13. Sanz-Rodriguez F, Hidalgo A, Teixido J. Chemokine stromal cell-derivedfactor-1alpha modulates VLA-4 integrin-mediated multiple myeloma celladhesion to CS-1/fibronectin and VCAM-1. Blood 2001;97:346–51.

14. Azab AK, Runnels JM, Pitsillides C, Moreau AS, Azab F, Leleu X, et al.CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myelomacells with the bone marrow microenvironment and enhances their sensi-tivity to therapy. Blood 2009;113:4341–51.

15. Martin SK, Diamond P,Williams SA, To LB, Peet DJ, Fujii N, et al. Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression inmultiple myeloma plasma cells. Haematologica 2010;95:776–84.

16. Giatromanolaki A, Bai M, Margaritis D, Bourantas KL, Koukourakis MI,Sivridis E, et al. Hypoxia and activated VEGF/receptor pathway in multiplemyeloma. Anticancer Res 2010;30:2831–6.


Vandyke et al.




17. Martin SK, Diamond P, Gronthos S, Peet DJ, Zannettino AC. The emergingrole of hypoxia, HIF-1 and HIF-2 in multiple myeloma. Leukemia2011;25:1533–42.

18. Azab AK, Hu J, Quang P, Azab F, Pitsillides C, Awwad R, et al. Hypoxiapromotes dissemination of multiple myeloma through acquisition of epi-thelial to mesenchymal transition-like features. Blood 2012;119:5782–94.

19. Zannettino ACW, Farrugia AN, Kortesidis A, Manavis J, To LB, Martin SK,et al. Elevated serum levels of stromal-derived factor-1alpha are associatedwith increased osteoclast activity and osteolytic bone disease in multiplemyeloma patients. Cancer Res 2005;65:1700–9.

20. Diamond P, Labrinidis A, Martin SK, Farrugia AN, Gronthos S, To LB, et al.Targeted disruption of the CXCL12/CXCR4 axis inhibits osteolysis in amurine model of myeloma-associated bone loss. J Bone Miner Res2009;24:1150–61.

21. Fitter S, Dewar AL, Kostakis P, To LB, Hughes TP, Roberts MM, et al. Long-term imatinib therapy promotes bone formation in CML patients. Blood2008;111:2538–47.

22. Aubrey BJ, Kelly GL, Kueh AJ, Brennan MS, O'Connor L, Milla L, et al. Aninducible lentiviral guide RNA platform enables the identification oftumor-essential genes and tumor-promoting mutations in vivo. Cell Rep2015;10:1422–32.

23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods2001;25:402–8.

24. Cheong CM, Chow AW, Fitter S, Hewett DR, Martin SK, Williams SA, et al.Tetraspanin 7 (TSPAN7) expression is upregulated in multiple myelomapatients and inhibits myeloma tumour development in vivo. Exp Cell Res2015;332:24–38.

25. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA,et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci U S A2005;102:15545–50.

26. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P,Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics2011;27:1739–40.

27. Agnelli L, Mosca L, Fabris S, Lionetti M, Andronache A, Kwee I, et al. A SNPmicroarray and FISH-based procedure to detect allelic imbalances inmultiple myeloma: an integrated genomics approach reveals a wide genedosage effect. Genes Chromosomes Cancer 2009;48:603–14.

28. Reme T, Hose D, Theillet C, Klein B. Modeling risk stratification in humancancer. Bioinformatics 2013;29:1149–57.

29. Shaughnessy JD Jr, Qu P, Usmani S, Heuck CJ, Zhang Q, Zhou Y, et al.Pharmacogenomics of bortezomib test-dosing identifies hyperexpressionof proteasome genes, especially PSMD4, as novel high-risk feature inmyeloma treated with Total Therapy 3. Blood 2011;118:3512–24.

30. Hose D, Reme T, Hielscher T, Moreaux J, Messner T, Seckinger A, et al.Proliferation is a central independent prognostic factor and target forpersonalized and risk-adapted treatment in multiple myeloma. Haema-tologica 2011;96:87–95.

31. Gautier L, Cope L, Bolstad BM, Irizarry RA. affy–analysis of AffymetrixGeneChip data at the probe level. Bioinformatics 2004;20:307–15.

32. Yellapantula V, Allen K, Jelinek DF, Bergsagel L, Keats JJ. The comprehen-sive genomic characterisation of all commercially and non-commerciallyavailable multiple myeloma cell lines. Blood 2013;122:1914-.

33. Smyth GK. Linear models and empirical bayes methods for assessingdifferential expression in microarray experiments. Stat Appl Genet MolBiol 2004;3:Article3.

34. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free,open-source system for microarray data management and analysis. Bio-techniques 2003;34:374–8.

35. Bernardini G, Sciume G, Bosisio D, Morrone S, Sozzani S, Santoni A. CCL3and CXCL12 regulate trafficking of mouse bone marrow NK cell subsets.Blood 2008;111:3626–34.

36. Honczarenko M, Le Y, Glodek AM, Majka M, Campbell JJ, Ratajczak MZ,et al. CCR5-binding chemokines modulate CXCL12 (SDF-1)-inducedresponses of progenitor B cells in human bone marrow through heterol-ogous desensitization of the CXCR4 chemokine receptor. Blood2002;100:2321–9.

37. Drury LJ, Ziarek JJ, Gravel S, Veldkamp CT, Takekoshi T, Hwang ST, et al.Monomeric and dimeric CXCL12 inhibit metastasis through distinctCXCR4 interactions and signaling pathways. Proc Natl Acad Sci U S A2011;108:17655–60.

38. Vallet S, Raje N, Ishitsuka K, Hideshima T, Podar K, Chhetri S, et al.MLN3897, anovel CCR1 inhibitor, impairs osteoclastogenesis and inhibitsthe interaction of multiple myeloma cells and osteoclasts. Blood2007;110:3744–52.

39. Kim CH, Broxmeyer HE. In vitro behavior of hematopoietic progenitorcells under the influence of chemoattractants: stromal cell-derived factor-1,steel factor, and the bone marrow environment. Blood 1998;91:100–10.

40. Dar A, Schajnovitz A, Lapid K, Kalinkovich A, Itkin T, Ludin A, et al. Rapidmobilization of hematopoietic progenitors by AMD3100 and catechola-mines is mediated by CXCR4-dependent SDF-1 release from bonemarrowstromal cells. Leukemia 2011;25:1286–96.

41. Bao L, Lai Y, Liu Y, Qin Y, Zhao X, Lu X, et al. CXCR4 is a good survivalprognostic indicator in multiple myeloma patients. Leuk Res 2013;37:1083–8.

42. Stessman HA, Mansoor A, Zhan F, Janz S, Linden MA, Baughn LB, et al.Reduced CXCR4 expression is associated with extramedullary diseasein a mouse model of myeloma and predicts poor survival in multiplemyeloma patients treated with bortezomib. Leukemia 2013;27:2075–7.

43. Terpos E, Politou M, Szydlo R, Goldman JM, Apperley JF, Rahemtulla A.Serum levels of macrophage inflammatory protein-1 alpha (MIP-1alpha)correlate with the extent of bone disease and survival in patients withmultiple myeloma. Br J Haematol 2003;123:106–9.

44. Tsirakis G, Roussou P, Pappa CA, Kolovou A, Vasilokonstantaki C, Mimi-nas I, et al. Increased serum levels of MIP-1alpha correlate with bonedisease and angiogenic cytokines in patients with multiple myeloma. MedOncol 2014;31:778.

45. Moller C, Stromberg T, JuremalmM, Nilsson K, Nilsson G. Expression andfunction of chemokine receptors in human multiple myeloma. Leukemia2003;17:203–10.

46. Contento RL, Molon B, Boularan C, Pozzan T, Manes S, Marullo S, et al.CXCR4-CCR5: a couple modulating T cell functions. Proc Natl Acad SciU S A 2008;105:10101–6.

47. Bennett LD, Fox JM, Signoret N. Mechanisms regulating chemokine recep-tor activity. Immunology 2011;134:246–56.

48. Oba Y, Lee JW, Ehrlich LA, Chung HY, Jelinek DF, Callander NS, et al. MIP-1alpha utilizes both CCR1 and CCR5 to induce osteoclast formation andincrease adhesion of myeloma cells to marrow stromal cells. Exp Hematol2005;33:272–8.

49. Durig J, Schmucker U, Duhrsen U. Differential expression of chemokinereceptors in B cell malignancies. Leukemia 2001;15:752–6.

50. Chow DC, Wenning LA, Miller WM, Papoutsakis ET. Modeling pO(2)distributions in the bone marrow hematopoietic compartment. I. Krogh'smodel. Biophys J 2001;81:675–84.

51. Spencer JA, Ferraro F, Roussakis E, Klein A, Wu J, Runnels JM, et al. Directmeasurement of local oxygen concentration in the bone marrow of liveanimals. Nature 2014;508:269–73.






2017;77:5452-5463. Published OnlineFirst August 30, 2017.Cancer Res Kate Vandyke, Mara N. Zeissig, Duncan R. Hewett, et al. Myeloma by Regulating CXCL12/CXCR4 and CCR1

Promotes Dissemination of Plasma Cells in MultipleαHIF-2

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a Promotes Dissemination of Plasma Cells in Multiple ... · multiple myeloma patients, malignant PCs continually migrate and repopulate multiple sites throughout the bone marrow (2).

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