HIF-2A Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1 Kate Vandyke1,2,3, Mara N

Published OnlineFirst August 30, 2017; DOI: 10.1158/0008-5472.CAN-17-0115

Cancer Molecular and Cellular Pathobiology Research

HIF-2a Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1 Kate Vandyke1,2,3, Mara N. Zeissig1,2, Duncan R. Hewett1,2, Sally K. Martin1,2, Krzysztof M. Mrozik1,2, Chee Man 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 is chemokine receptor CCR1 in multiple myeloma PCs. CCR1 dependent on the ability of the multiple myeloma plasma activation potently induces multiple myeloma PC migration cells (PC) to reenter the circulation and disseminate through- toward CCL3 while abrogating the multiple myeloma PC out the bone marrow. Increased bone marrow hypoxia is migratory response to CXCL12. In addition, increased CCR1 associated with increased recirculation of multiple myeloma expression by multiple myeloma PCs conferred poor prognosis PCs. Accordingly, we hypothesized that during chronic hyp- in newly diagnosed multiple myeloma patients and was asso- oxia, activation of HIF-2a may overcome the bone marrow ciated with an increase in circulating multiple myeloma PCs in retention signal provided by stromal-derived CXCL12, thereby these patients. Taken together, our results suggest a role for enabling dissemination of multiple myeloma PCs. Here we hypoxia-mediated CCR1 upregulation in driving the egress of demonstrate that HIF-2a upregulates multiple myeloma PC multiple myeloma PCs from the bone marrow. Targeting CXCL12 expression, decreasing migration toward CXCL12 and CCR1 may represent a novel strategy to prevent dissemination reducing adhesion to mesenchymal stromal cells in vitro.We and overt relapse in multiple myeloma. Cancer Res; 77(20); 5452–63. also found that HIF-2a strongly induced expression of the 2017 AACR.

Introduction development of the asymptomatic precursor disease monoclonal gammopathy of undetermined signiﬁcance (MGUS; ref. 2). In a Multiple myeloma is an incurable hematologic cancer that is small proportion of MGUS patients (1% per year), intrinsic responsible for an estimated 80,000 deaths worldwide, each year genetic changes, coupled with extrinsic factors, lead to the pro- (1). Multiple myeloma is characterized by the clonal proliferation liferation of the multiple myeloma PCs and their dissemination to of malignant plasma cells (PC) within the bone marrow. While, in sites throughout the skeleton (3). Ultimately, multiple myeloma some cases, malignant PCs establish at a single site, forming a PC proliferation and dissemination results in hypercalcemia, solitary plasmacytoma, in the majority of cases, the transformed renal insufﬁciency, anemia, and osteolytic bone disease, features PC will disseminate throughout the bone marrow, leading to the characteristic of progressive disease (4). Dissemination and repopulation throughout the bone marrow 1Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and is key, not only during multiple myeloma disease progression, but Medical Sciences, University of Adelaide, Adelaide, Australia. 2Cancer Theme, also in the outgrowth of resistant multiple myeloma PCs during South Australian Health and Medical Research Institute, Adelaide, Australia. 3SA relapse following therapy. Evidence suggest that in most, if not all Pathology, Adelaide, Australia. 4Haematology and Bone Marrow Transplant multiple myeloma patients, malignant PCs continually migrate 5 Unit, Royal Adelaide Hospital, Adelaide, Australia. Mesenchymal Stem Cell and repopulate multiple sites throughout the bone marrow (2). Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, Circulating multiple myeloma PCs are observed in over two- University of Adelaide, Adelaide, Australia. 6School of Biological Sciences, Faculty of Sciences, University of Adelaide, Adelaide, Australia. 7Bone Biology thirds of newly diagnosed multiple myeloma patients and are Division, Garvan Institute of Medical Research, Sydney, Australia. 8St Vincent's a predictor of response to therapy, with increased numbers of Clinical School, Faculty of Medicine, University of New South Wales, Sydney, circulating multiple myeloma PCs representing an independent Australia. 9Centre for Cancer Biology, University of South Australia, Adelaide, indicator of poor prognosis and rapid progression (5–8). Australia. The homing of multiple myeloma PCs from the peripheral Note: Supplementary data for this article are available at Cancer Research circulation to the bone marrow is critically dependent on the Online (http://cancerres.aacrjournals.org/). chemokine (C-X-C motif) ligand CXCL12 (also known as stromal Corresponding Author: Andrew C.W. Zannettino, Myeloma Research Labora- cell–derived factor-1; SDF-1), the ligand for CXCR4. CXCL12 is tory, Adelaide Medical School, Faculty of Health and Medical Sciences, University highly expressed by bone marrow mesenchymal stromal cells of Adelaide, North Terrace, Adelaide, SA 5000, Australia. Phone: 618-812-8490; (BMSC; refs. 9, 10) and acts as potent attractor for the homing of Fax: 618-8222-3139; E-mail: [email protected] both normal and malignant PCs into the bone marrow (11, 12). doi: 10.1158/0008-5472.CAN-17-0115 In addition, CXCL12 is crucial in the subsequent retention of 2017 American Association for Cancer Research. multiple myeloma PCs in the bone marrow, stimulating increased

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CCR1, CXCL12, and Hypoxia in MM Dissemination

adhesion of multiple myeloma PCs to bone marrow stromal cells marrow aspirates isolated from the posterior iliac crest of healthy and promoting multiple myeloma PC survival and resistance to adult human donors and were used at passage 6. Survival was therapeutics (13, 14). calculated from the date of bone marrow or blood collection until While the mechanisms involved in multiple myeloma PC death, by any cause. Patients who were lost to follow up were homing to the bone marrow are relatively well characterized, the censored at the date of last contact. mechanisms responsible for driving multiple myeloma PCs egress from the bone marrow remain to be elucidated. Emerging data Cell culture suggest that the elevated bone marrow hypoxia observed in Unless otherwise specified, all cell culture reagents were multiple myeloma (15–17) is a potential microenvironmental sourced from Sigma-Aldrich and all media were supplemented stimulus that drives multiple myeloma PC dissemination (18). with 2 mol/L L-glutamine, 100 U/mL penicillin, 100 mg/mL Recent studies suggest bone marrow hypoxia strongly correlates streptomycin, 1 mmol/L sodium pyruvate, and 10 mmol/L with both tumor burden and numbers of circulating multiple HEPES buffer. BMSCs were maintained as described previously myeloma PCs in mouse models of multiple myeloma, suggesting (21). HMCLs RPMI-8226, LP-1, and U266 were obtained from the that hypoxic multiple myeloma PCs may be preferentially mobi- ATCC between 2000 and 2003; STR authentication was not lized to the peripheral blood (18). This may, at least in part, be due conducted on these lines as they were obtained directly from the to decreased adhesion and a reduced chemotactic response to ATCC. OPM2 cells were provided by Prof. Andrew Spencer bone marrow stromal cells under hypoxia (18). A similar asso- (Monash University, Melbourne, Australia) in 2004; this cell line ciation between tumor hypoxia and increased metastasis has also was authenticated in 2016 using STR analysis performed by the been seen in patients with solid tumors and in animal models of Molecular Genetics Laboratory, SA Pathology, using an metastasis (17). AmpFLSTR Identifiler PCR Amplification Kit (Thermo Fisher Exposure to hypoxia induces gene expression through the Scientific). HMCLs were last mycoplasma tested in May 2016 stabilization and increased expression of the hypoxia-inducible using a MycoAlert Mycoplasma Detection Kit (Lonza). HMCLs transcription factors HIF-1a and HIF-2a (17). Our previous were maintained in RPMI1640 medium with 10% FCS (Thermo studies suggest that HIF-2a plays a critical role in multiple Fisher Scientific) and supplements. All experiments were con- myeloma disease progression, through binding to the promoter ducted within 4 weeks of thawing of cells. Cell lines were routinely and driving expression of CXCL12 (15). In addition to being cultured in a humidified environment with 5% carbon dioxide at highly expressed by bone marrow stromal cells, CXCL12 is highly 37C. Hypoxic culture conditions (<1% oxygen) were established expressed by multiple myeloma PCs (19) and multiple myeloma using an anaerobic sachet (Oxoid). Where indicated, cells were PC–derived CXCL12 plays an important role in driving osteolysis treated with the CCR1 inhibitor BX471 (Sigma Aldrich) in (19, 20) and angiogenesis (15) in myeloma patients. 0.0004% DMSO, or 0.0004% DMSO alone, or with recombinant In the studies presented here, we investigated whether upre- human (rh)CXCL12 (100 ng/mL; R&D Systems) or rhCCL3 gulation of CXCL12 expression in multiple myeloma PCs, in (100 ng/mL; PeproTech). Generation of RPMI-8226 and LP-1 response to HIF-2a expression, abrogates the chemoattraction cell lines overexpressing CXCL12, HIF-1a, or HIF-2a is described towards CXCL12. Notably, we identified the chemokine (C-C) in Supplementary Methods and in refs. 15 and 20. Stable shRNA- receptor CCR1 as being strongly upregulated by chronic hypoxia mediated HIF-1a and HIF-2a knockdown in LP-1 cells was in multiple myeloma PCs. Our data suggest that the interplay conducted as described previously (15). CRISPR/Cas9-mediated between CCR1 upregulation and inactivation of CXCR4 signaling knockout of CCR1 in U266 cells was conducted as described in (the CCR1/CXCR4 axis) drives the egress of multiple myeloma Supplementary Methods and in ref. 22. PCs from the bone marrow, leading to disease dissemination. CXCL12 ELISA Materials and Methods Peripheral blood and bone marrow plasma and conditioned Patient samples media CXCL12 levels were determined using a commercial Ethical approval for this study was obtained from the Royal CXCL12 immunoassay (R&D Systems) according to the manu- Adelaide Hospital Institutional Ethics Review Committee (RAH facturer's recommendations, as described previously (19). approval no. 030206, 131132, and 110304) and all patients provided written, informed consent, in accordance with the Real-time PCR Declaration of Helsinki. Posterior superior iliac spine bone mar- Total RNA was isolated using TRIzol (Thermo Fisher Scientific) row aspirates (n ¼ 27) and peripheral blood (n ¼ 82) were and cDNA was synthesized using Superscript III (Life Technolo- collected from 82 randomly selected patients [median age: 68 gies). Real-time PCR was performed using a Rotor-Gene 6000 PCR 0 years (range 35–84); male:female ratio 1.2:1] with symptomatic machine (Qiagen) using primers for human HIF2A (forward, 5 - 0 0 multiple myeloma who presented at the Royal Adelaide Hospital CTCTCCTCAGTTTGCTCTGAAAA-3 ; reverse, 5 -GTCGCAGG- 0 0 (Adelaide, Australia). All multiple myeloma patients fulfilled the GATGAGTGAAGT-3 ), CXCL12 (forward, 5 -ATGCCCATGCC- 0 0 0 diagnostic criteria for active multiple myeloma (4), were newly GATTCTTCG-3 ; reverse, 5 -GTCTGTTGTTGTTCTTCAGCC-3 ), 0 0 diagnosed, and had not received previous therapy. Bone marrow CXCR4 (forward, 5 -CAGCAGGTAGCAAAGTGACG-3 ; reverse, 0 0 0 mononuclear cells (BMMNC) and peripheral blood mononuclear 5 -GTAGATGGTGGGCAGGAAGA-3 ), CCR1 (forward, 5 -AA- 0 0 cells (PBMNC) were isolated by density gradient centrifugation as GCCCCAGAAACAAAGACTTC-3 ; reverse, 5 -TGCATCCCCAT- 0 0 described previously (15) and were cryopreserved prior to use. AGTCAAACTCT-3 ), CCL3 (forward, 5 -CTGGTTTCAGACTT- 0 0 Peripheral blood and bone marrow plasma samples were collect- CAGAAGGAC-3 ; reverse, 5 -GTAGTCAGCTATGAAATTCTGT- 0 0 0 ed and stored as described previously (19). BMSCs were isolated GG-3 ), B2M (forward, 5 -AGGCTATCCAGCGTACTCCA-3 ;re- 0 0 by plastic adherence from bone chips recovered from bone verse, 5 -TCAATGTCGGATGGATGAAA-3 ), and ACTB (forward,

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50-GATCATTGCTCCTCCTGAGC-30; reverse, 50-GTCATAGTCC- overlaid onto the BMSCs and allowed to adhere for 15 minutes. GCCTAGAAGCAT-30) as described previously (15). Changes in Wells were then washed gently 3 times with Hank Buffered Salt gene expression were calculated relative to ACTB or B2M using Solution containing 5% FCS to remove nonadherent cells and the –DDC the 2 t method (23). number of adherent cells enumerated as described previously (24). The percentage cell adhesion was calculated relative to total Flow cytometry cell input. Cell surface expression of CXCR4 and CCR1 on HMCL was assessed by staining 2 105 cells per test with an anti-CCR1 Actin remodeling assays mouse mAb (clone 53504; R&D Systems), an anti-CXCR4 mouse To assess filamentous actin (F-actin) remodeling in response to mAb (clone 44714; R&D Systems), or an in-house IgG2B isotype CXCL12 treatment, human multiple myeloma cells were washed control antibody (1A6.11), and a PE-conjugated goat anti-mouse twice in serum-free media and were stimulated with rhCXCL12 secondary antibody (Southern Biotech) and were analyzed on a (100 ng/mL), for the indicated times, in triplicates. Cells were FACSCanto II flow cytometer (BD Biosciences). Cell surface CCR1 immediately fixed in 2% paraformaldehyde (pH 8.0) for 15 min- and CXCR4 expression on multiple myeloma PCs was assessed on utes at room temperature. Cells were washed three times in PBS þþ þ viable CD38 /CD138 /CD45lo/CD19 multiple myeloma PCs containing 0.2% saponin and 5% FCS and stained with Alexa- by multicolor flow cytometry (LSRFortessa; BD Biosciences). fluor-680-phalloidin (200 U/mL; Life Technologies) for 30 min- Briefly, 3 105 mononuclear cells per test were stained with an utes on ice. Cells were then washed three times and phalloidin anti-CCR1 or anti-CXCR4 antibody, as detailed above, or no staining was quantitated using a LSRFortessa flow cytometer (BD primary antibody [fluorescence minus one (FMO) control] fol- Biosciences). lowed by a PE-conjugated goat anti-mouse secondary antibody (Southern Biotech). Cells were subsequently blocked with human Gene expression profiling gamma globulin prior to staining with CD38-PE-Cy7 (HIT2; RNA was extracted from LP-1-pRUF and LP-1-HIF2A cell lines BioLegend), CD138-AlexaFluor-647 (B-B4; Serotec) CD45-FITC using TRIzol and the RNA was further purified using a silica gel– (J.33; Beckman Coulter), and CD19-Brilliant Violet 421 (HIB19; based membrane column (RNeasy kit; Qiagen). RNA labeling and BioLegend) antibodies. Cells were stained with the viability dye hybridization to the Affymetrix Human Gene 1.0 ST Array and hydroxystilbamidine (FluoroGold; Invitrogen, Life Technologies) subsequent data analysis was performed by the Adelaide Micro- immediately before analysis. Viable, single cells were gated on the array Centre (Adelaide, Australia). Microarray data have been basis of FSC and SSC characteristics and FluoroGold negativity deposited in the GEO database (GSE102235). Probesets specific and multiple myeloma PCs were identified as CD38-bright and to noncoding RNA (miR, SNO RNA, LNC, and pseudogenes) were CD138-positive cells, with contaminating cells excluded by sub- excluded from further analysis. Gene set enrichment analysis sequently gating on the CD45-low and CD19-negative popula- (GSEA; ref. 25) was conducted using the Molecular Signatures tion. A minimum of 100 cells was required to fulfil these criteria to Database v5.0 (26). be identified as a multiple myeloma PC population. A minimum of 5 104 total nucleated cells were analyzed per test; 1 105 to Publicly available microarray data 1 106 total cells were analyzed per patient. CCR1 and CXCR4 Microarrayanalysisofpubliclyavailabledatawasconducted expression was quantitated as the change in the median fluores- as described (24). For analysis of CXCL12 and CCR1 expression cence intensity (DMFI), defined as the difference in MFI between in CD138-selected bone marrow PCs from newly diagnosed the CCR1- or CXCR4-stained sample and the FMO control MGUS, multiple myeloma or plasma cell leukemia (PCL) (patient samples) or isotype control (cell lines). patients or normal controls, two independent microarray datasets were used: E-GEOD-16122 (normal, n ¼ 5; MGUS, n ¼ 11; Transwell migration assays multiple myeloma, n ¼ 133; PCL, n ¼ 9; ref. 27) and E-MTAB- Migration assays were performed as described previously 363 (normal, n ¼ 5; MGUS, n ¼ 5; multiple myeloma, n ¼ 155; (24). Briefly, cells (1 105) were washed once in RPMI1640 ref. 28). Analysis of overall survival in multiple myeloma þ with 1% FCS and were seeded in transwells in RPMI1640 with patients stratified on the basis of CD138 bone marrow PC 1% FCS and supplements in triplicate and cell migration gene expression at diagnosis was carried out using the dataset toward chemoattractant (10% FCS or cytokines) or RPMI1640 E-TABM-1138 (n ¼ 142; ref. 29). Correlative analysis of CCR1 with 1% FCS (untreated controls) was assessed after 18 hours and CXCL12 gene expression in CD138-selected bone marrow by microscopy as described previously (24). Percentage cell PC from newly diagnosed multiple myeloma patients was migration is represented normalized to the untreated controls performed using E-MTAB-363, E-TABM-1138, E-GEOD- or appropriate control cells. Where indicated, cells were pre- 16122, and E-MTAB-317 (n ¼ 226; ref. 30). E-MTAB-363, treated with rhCXCL12 or rhCCL3 (100 ng/mL) for 72 hours E-MTAB-317, and E-TABM-1138 were conducted on Affymetrix or BX471 (100 nmol/L; Sigma Aldrich) for 24 hours and were GeneChip Human Genome U133 plus 2.0 arrays; E-GEOD- washed once in RPMI1640 with 1% FCS prior to seeding 16122 was conducted on U133A arrays. Raw microarray data migration assays. (CEL files) were downloaded from ArrayExpress (EMBL-EBI) or Gene Expression Omnibus (GEO; NCBI) and were normalized Adhesion assays by RMA using the bioconductor package affy (31) and R Adhesion assays were performed as described previously (24). (version 3.03) and log2 transformed. Gene expression levels Briefly, pooled BMSCs from three independent normal donors were assessed in a panel of human multiple myeloma cell lines were plated 96-well plates at 8 103 cells/well and allowed to (n ¼ 66) using publicly available RNA-Seq data, downloaded adhere overnight. RPMI-8226 cells (1 105 cells/well) were from www.keatslab.org (32).

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CCR1, CXCL12, and Hypoxia in MM Dissemination

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 CXCL12 mRNA and protein was conﬁrmed in RPMI-8226-HIF2A cells by real-time PCR and ELISA. B, Adhesion of 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 was assessed in a transwell assay. Migration is expressed relative to the vector control cells, following subtraction of the background (no chemoattractant). D, Adhesion of RPMI-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 vector controls (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 more independent 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.

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Statistical analyses Results Unless otherwise described, statistical analysis was performed using GraphPad Prism (version 5.02; GraphPad Software). Dif- CXCL12 expression desensitizes multiple myeloma PCs to ferences in gene expression between groups in microarray experi- exogenous CXCL12 a ments were assessed using linear models for microarray data We have previously demonstrated that HIF-2 and hypoxia analysis (LIMMA; ref. 33) in MultiExperiment Viewer (MeV; strongly upregulate the expression of CXCL12 in multiple mye- a ref. 34). Correlations were assessed using Pearson correlation loma PCs (15). Overexpression of HIF-2 in the HMCL LP-1 coefficients. Overall survival was assessed using Kaplan–Meier (LP-1-HIF2A) and RPMI-8226 (RPMI-8226-HIF2A; refs. 15, 20) fi curves; comparisons between groups were made using the log- was con rmed using real-time PCR (Supplementary Fig. S1A) and rank (Mantel–Cox) test and the Mantel–Haenszel hazard ratio. As was found to induce CXCL12 mRNA and protein expression survival in the CXCL12 expression subgroups did not meet the (Fig. 1A; Supplementary Fig. S1B and S1C). a– proportional hazards assumption, each variable was divided into We hypothesized that HIF-2 mediated upregulation of two groups (overall survival 104 weeks or >104 weeks) prior to endogenous CXCL12 may inhibit multiple myeloma PC response a fi conducting univariate analyses. For time-course and CCR1 inhib- to exogenous CXCL12. HIF-2 overexpression signi cantly itor experiments, groups were compared using two-way ANOVA reduced the adhesion of RPMI-8226-HIF2A or LP-1-HIF2A cells P < with Sidak multiple comparison test. For analysis of CCR1 to BMSCs ( 0.05; Fig. 1B). LP-1 cells did not migrate under any expression on peripheral blood and bone marrow multiple mye- condition used here, precluding assessment of the effects of a loma PCs, as CCR1 expression was undetectable in some cases, the HIF-2 and CXCL12 on migration in this cell line. However, in fi nonparametric Wilcoxon matched pairs signed rank test was used. RPMI-8226 cells, there was a signi cant decrease in the migration P < For all other experiments, groups were compared using paired or of RPMI-8226-HIF2A cells toward CXCL12 ( 0.05; Fig. 1C) unpaired Student t tests, as described. Differences were considered despite an increase in the migration of RPMI-8226-HIF2A cells P < to be statistically significant when the P < 0.05. toward 10% FCS ( 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 was assessed 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 cytometry following staining with an anti-CCR1 antibody or isotype control. Expression was normalized to the vector control cells following subtraction of the isotype control (DMFI). Graphs depict mean þ SEM of three biological replicates (A, B,andD) or three or more independent experiments (C, E, and F). , P < 0.05, paired t test (A, B,andD–F) or two-way ANOVA with Sidak multiple comparison test (C).

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CCR1, CXCL12, and Hypoxia in MM Dissemination

CXCL12 was overexpressed in HMCLs, as described previously with previous studies (11). In addition, cell surface CXCR4 (Supplementary Fig. S1E and S1F; refs. 15, 20). Similar to the protein was downregulated in response to HIF-2a overexpres- effects of HIF-2a overexpression, CXCL12 overexpression in sion (Fig. 1G; Supplementary Fig. S2E) or by hypoxic RPMI-8226 cells abrogated the chemotactic response to CXCL12 culture (Supplementary Fig. S2F) in RPMI-8226 and LP-1 cells. (P < 0.05; Fig. 1C). In addition, pretreatment of RPMI-8226 cells CXCR4 mRNA levels were also decreased in LP-1-HIF2A, with CXCL12 for 72 hours decreased migration toward CXCL12 but not RPMI-8226-HIF2A cells (Supplementary Fig. S2G; (P < 0.05; Supplementary Fig. S2A). This was also demonstrated in P < 0.05). the HMCL OPM2 (P < 0.05; Supplementary Fig. S2A). Adhesion of RPMI-8226 and LP-1 cells to BMSCs was also decreased The chemokine receptor CCR1 is upregulated by HIF-2a following overexpression of CXCL12 (P < 0.05; Fig. 1D) but was To investigate other mechanisms whereby hypoxia may drive unaffected by exogenous CXCL12 in RPMI-8226 and LP-1 cells dissemination in multiple myeloma, we used microarrays to (P ¼ 0.30 and P ¼ 0.17, respectively; Supplementary Fig. S2B). In assess the effects of HIF-2a overexpression on gene expression contrast, exogenous CXCL12 increased adhesion in OPM2 cells in the LP-1 cell line. A total of 79 genes were upregulated, and 83 (P < 0.05; Supplementary Fig. S2B). genes were downregulated, by more than 2-fold in LP-1-HIF2A cells (Supplementary Table S1). Importantly, the most highly Hypoxia reduces cell surface expression of CXCR4 in multiple upregulated gene was chemokine (C-C) receptor 1 (CCR1; myeloma PCs 6.5-fold upregulated), the receptor for C-C chemokine ligand 3 Overexpression of CXCL12, or treatment with CXCL12, [CCL3; also known as macrophage inﬂammatory protein-1a decreased the cell surface expression of CXCR4 in HMCL (MIP-1a); Supplementary Table S1]. GSEA of the upregulated (Fig. 1E and F; Supplementary Fig. S2C and S2D), consistent genes 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 cells toward 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 in the top chamber of a transwell with 100 ng/mL rhCCL3 or media alone and migration toward 100 ng/mL rhCXCL12 or media alone was assessed. Results are expressed relative 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 migration toward 100 ng/mL rhCXCL12 was assessed in a transwell assay. Migration is expressed relative to the untreated control cells following subtraction of the "no chemoattractant" 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 marrow mesenchymal stromal cell monolayer, and percent cell adhesion, relative to total cell input, was assessed after 15 minutes. Results are expressed relative to the untreated controls. E, Expression of CXCR4 protein was determined by flow cytometry following staining with an anti-CXCR4 antibody, after 72 hours of culture with 100 ng/mL rhCCL3 or media alone. Expression is shown normalized to the untreated controls following subtraction of the isotype control (DMFI). F, RPMI-8226 cells 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 were immediately fixed and F-actin was quantitated by flow cytometry following staining with AlexaFluor-680-phalloidin. Expression is shown as MFI, normalized to 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, paired t test (A and C–E), one-way ANOVA with Dunnett posttest (B) or two-way ANOVA with Sidak multiple comparison test (F).

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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 a panel of human multiple myeloma cell lines following with an anti-CXCR4 or isotype control antibody. Expression is shown following subtraction of the isotype control (DMFI). B, Migration of human multiple myeloma cell lines toward 100 ng/mL rhCXCL12 was assessed in a transwell assay. C, Expression of CCL3 was assessed by real-time PCR in a panel of human multiple myeloma cell lines. D, CCR1 was knocked out in U266 cells using the CRISPR-Cas9 system and migration of CCR1 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 treated with 100 nmol/L BX471 or 0.0004% DMSO vehicle control for 24 hours and migration 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,andE) or three biological replicates (C). , P < 0.05, paired t test (B)or two-way ANOVA with Sidak multiple comparison test (D and E).

the hypoxia hallmark set (false discovery rate q value ¼ 2.06 loma cells to CCL3. In support of this, overexpression of HIF-2a in 10 6; Supplementary Table S2). RPMI-8226 cells increased migration toward CCL3 (Fig. 3A). HIF-2a overexpression and knockdown in LP-1 resulted in a CCL3/CCR1 signaling can lead to internalization and/or inac- respective 23-fold upregulation and a 1.5-fold reduction in CCR1 tivation of CXCR4, as has been shown for other cell types (35, 36). expression (Fig. 2A and B). Induction of CCR1 expression was also In keeping with this, we found that either incubation with CCL3 in conﬁrmed following 24 and 48 hours of hypoxic culture (<1% the top chamber of a transwell (Fig. 3B), or CCL3 pretreatment for O2; Fig. 2C). In contrast, overexpression or knockdown of HIF-1a 3 days (Fig. 3C), reduced the migration of RPMI-8226 and OPM2 had no effect on CCR1 expression in LP-1 cells (Supplementary cells toward CXCL12. However, CCL3 treatment had no effect on Fig. S3A and S3B). Despite expressing detectable, low levels of the adhesion of RPMI-8226 or OPM2 cells to BMSCs (Fig. 3D) or CCR1 mRNA (Supplementary Fig. S3C and S3D), LP-1 cells did the expression of cell surface CXCR4 protein in a panel of human not express cell surface CCR1 protein (Supplementary Fig. S3E), multiple myeloma cell lines (Fig. 3E). In contrast, CXCL12 over- or migrate in response to CCL3 (Supplementary Fig. S3F). RPMI- expression or pretreatment had no inhibitory effects on migration 8226 and OPM2 expressed the highest CCR1 mRNA and cell toward CCL3 or cell surface expression of CCR1 in human surface protein (Supplementary Fig. S3C–S3E) and readily multiple myeloma cell lines (Supplementary Fig. S4A–S4C). migrated in response to CCL3 in vitro (Supplementary Fig. Furthermore, we examined the effects of CCL3 pretreatment for S3F). In RPMI-8226 cells, overexpression of HIF-2a increased 72 hours on F-actin remodeling (37). CCL3 treatment prevented CCR1 mRNA and protein levels (Fig. 2D and E). Cell surface CCR1 the induction of F-actin remodeling in response to CXCL12 expression was also increased following hypoxic culture in RPMI- treatment in RPMI-8226 cells (Fig. 3F). 8226 cells (Fig. 2F). In addition, hypoxic induction of CCR1 gene The HMCL U266 was evaluated as it expresses high levels of expression at 24 hours was conﬁrmed in OPM2 and U266 cell CXCR4 on the cell surface (Fig. 4A), but does not migrate toward lines (Supplementary Fig. S3G). CXCL12 in vitro (Fig. 4B). We hypothesized that the abundant endogenous production of CCL3 by these cells (Fig. 4C; ref. 38) CCL3 inhibits the migratory response to CXCL12 may prevent their response to CXCL12. In support of this, either We hypothesized that upregulation of CCR1 in response to knockout of CCR1 or pretreatment of the cells with a CCR1 HIF-2a could increase the migratory response of multiple mye- inhibitor, BX471, restored the migratory response of U226 cells

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CCR1, CXCL12, and Hypoxia in MM Dissemination

Figure 5. Multiple myeloma PC CXCR4 expression is increased, and CXCL12 levels are decreased, in the peripheral blood (PB) of patients with newly diagnosed multiple myeloma compared with bone marrow (BM). A, CXCR4 expression (DMFI) is shown for bone marrow and peripheral blood multiple myeloma PCs from 11 multiple myeloma patients for whom paired bone marrow and peripheral blood samples were available. B, Levels of CXCL12 protein in paired peripheral blood and bone marrow plasma samples from newly diagnosed multiple myeloma patients (n ¼ 11) as assessed by ELISA. , P < 0.05, paired t test. C, Kaplan–Meier plots of overall survival are shown for newly diagnosed multiple myeloma patients stratiﬁed on the basis of median CD138þ plasma cell CXCL12 expression, as derived from microarray dataset E-TABM-1138 (n ¼ 142 patients). D, Kaplan–Meier plots of overall survival are shown for newly diagnosed multiple myeloma patients subdivided on the basis of median peripheral blood plasma CXCL12 protein levels, as determined by ELISA (n ¼ 66 patients).

toward CXCL12 (Fig. 4D and E). In contrast, the migration of significantly higher in the bone marrow than in the peripheral OPM2 cells toward CXCL12 was unaffected by BX471 treatment blood of 11 newly diagnosed multiple myeloma patients with (Supplementary Fig. S4D). active disease (P < 0.05; Fig. 5B), with higher CXCL12 protein levels in the bone marrow than the peripheral blood in 8 of 11 Cell surface CXCR4 expression is decreased in bone marrow– (72.7%) patients. resident multiple myeloma PCs in multiple myeloma patients Importantly, we found that increased multiple myeloma PCs or CXCR4 protein expression was assessed by flow cytometry on peripheral blood plasma CXCL12 levels were associated with viable PCs in the bone marrow and peripheral blood of 16 newly early poor survival outcomes (Fig. 5C and D). Above median diagnosed multiple myeloma patients. Peripheral blood multiple CXCL12, either at the mRNA level (Fig. 5C), or protein level in the myeloma PCs were detectable by flow cytometry in 11 of 16 peripheral plasma of patients (Fig. 5D), was associated with an (68.8%) patients. CXCR4 was expressed on both bone marrow initial survival disadvantage within two years of diagnosis and peripheral blood multiple myeloma PCs in all patients for [mRNA, P ¼ 0.0236; HR ¼ 2.63 (95% CI: 1.14–6.06); protein, whom matched peripheral blood and bone marrow samples were P ¼ 0.0274, HR ¼ 3.148 (95% CI: 1.14–8.72)]. available (Supplementary Fig. S5A). Notably, expression of CXCR4 was significantly lower in bone marrow–resident than Expression of CCR1 in multiple myeloma PCs of patients peripheral blood multiple myeloma PCs (P ¼ 0.0051; Fig. 5A). correlates with circulating multiple myeloma PC numbers However, there was no association between either bone marrow In silico analysis of CCR1 expression in publicly available or peripheral blood CXCR4 expression and bone marrow PC microarray data from newly diagnosed patients with MGUS, burden (bone marrow CXCR4: r ¼0.086; P ¼ 0.80; peripheral multiple myeloma, or PCL and normal controls found that CCR1 blood CXCR4: r ¼0.23, P ¼ 0.49, Pearson correlation) or expression was elevated (>95% CI of normal) in 55.5% (86/155; peripheral blood multiple myeloma PC number (bone marrow E-MTAB-363) and 20.3% (27/133; E-GEOD-16122) of multiple CXCR4: r ¼ 0.13, P ¼ 0.70; peripheral blood CXCR4: r ¼0.0001, myeloma patients, and was significantly elevated in 55.6% (5/9; P ¼ 1.00, Pearson correlation). E-GEOD-16122: Padj ¼ 0.0047; LIMMA) of PCL patients (Fig. 6A and B). Moreover, above median CCR1 expression was associated Elevated serum or multiple myeloma PC CXCL12 expression is with poor prognosis in newly diagnosed multiple myeloma associated with poor early survival in multiple myeloma patients in an independent dataset [P ¼ 0.025; HR ¼ 2.3 (95% patients CI: 1.1–4.9); n ¼ 142 patients; E-TABM-1138; Fig. 6C]. In addi- Here, we hypothesized that high local levels of CXCL12 in the tion, in support of our data, expression of CCR1 positively bone marrow (19) result in a downregulation of cell surface correlated with CXCL12 expression in multiple myeloma PCs CXCR4. In support of this, plasma CXCL12 protein levels were from newly diagnosed multiple myeloma patients in four

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Vandyke et al.

Figure 6. Elevated CCR1 expression is associated with poor prognosis and increased numbers of circulating multiple myeloma (MM) PCs. In silico analysis was performed 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 overall survival are shown for newly diagnosed multiple myeloma patients stratiﬁed on the basis of median CD138þ plasma cell CCR1 expression, derived from microarray dataset E-TABM-1138 (n ¼ 142 patients). D, CCR1 expression (DMFI) is shown for bone marrow and peripheral blood multiple myeloma PCs from 11 multiple myeloma patients. , 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. r and P values are shown for Pearson correlation analyses.

independent datasets (E-MTAB-363: r ¼ 0.34, P < 0.0001; Discussion T-TABM-1138: r ¼ 0.36, P < 0.0001; E-GEOD-16122: r ¼ 0.57, The homing and retention of leukocytes and tumor cells P < 0.0001; E-MTAB-317: r ¼ 0.51, P < 0.0001; Pearson within the bone marrow, and their subsequent mobilization correlation). from the bone marrow, is regulated by the altered expression Furthermore, CCR1 protein was expressed on viable bone of chemokine receptors. The trafficking of normal and malig- marrow multiple myeloma PCs in 13 of 16 (81%) patients nant PCs from the peripheral blood to the bone marrow is (Supplementary Fig. S5B), as assessed by flow cytometry. driven by the CXCL12 gradient generated by high production Expression of CCR1 was significantly lower in peripheral blood of CXCL12 by BMSCs (9). Disruption of this CXCL12 gra- multiple myeloma PCs than in bone marrow multiple myelo- dient is required to release leukocytes and hematopoietic ma PCs in patients with paired bone marrow and peripheral precursor cells into the peripheral blood (39, 40). Similarly, blood samples (n ¼11; P ¼ 0.031; Fig. 6D). In addition, bone egress of multiple myeloma PCs from the bone marrow is marrow infiltration with multiple myeloma PCs positively dependent on overcoming this CXCL12/CXCR4 retention correlated with bone marrow multiple myeloma PC expression signal, with decreased CXCR4 expression in bone marrow of CCR1 (r ¼ 0.78; P ¼ 0.0046; Fig. 6E). CCR1 expression on multiple myeloma PCs being associated with increased peripheral blood multiple myeloma PCs also positively corre- tumor dissemination and poor prognosis (41, 42). Further- lated with the number of circulating multiple myeloma PCs more, treatment with the CXCR4 inhibitor AMD3100 leads (r ¼ 0.70; P ¼ 0.016; Fig. 6F), and there was a trend toward to mobilization of multiple myeloma cells from the bone increased bone marrow multiple myeloma PC expression of marrow to the peripheral blood in a mouse model of CCR1 and increased numbers of circulating multiple myeloma multiple myeloma (14). PCs (r ¼ 0.56; P ¼ 0.070).

5460 Cancer Res; 77(20) October 15, 2017 Cancer Research

CCR1, CXCL12, and Hypoxia in MM Dissemination

Here, we demonstrate that increased HIF-2a–mediated expres- hematopoietic progenitor cells from the bone marrow. In addi- sion of CXCL12 in PCs desensitizes cells to CXCL12 in vitro, tion, CCL3/CCR1 signaling may lead to heterologous desensiti- decreasing migration and adhesion to stroma. This is consistent zation of CXCR4, as has been shown for other cell types (35, 36). with previous findings demonstrating that high endogenous Our data show that CCL3 stimulation does not affect cell CXCL12 expression could lead to homologous desensitization surface CXCR4 expression, suggesting that the effects of CCL3 of CXCR4, downregulating cell surface CXCR4 expression and are downstream of CXCR4. It is possible that CXCR4 and CCR1 decreasing response to CXCL12 (11). In addition, we found that may heterodimerize, as has been shown for CXCR4 and CCR5 cell surface CXCR4 is strongly downregulated in response to (46). However, as CCL3 downregulates cell surface CCR1 (data HIF-2a or sustained hypoxic culture in human multiple myeloma not shown) without affecting cell surface CXCR4 levels, this cell lines, suggesting that chronic hypoxia may be sufficient to would suggest that stable CCR1–CXCR4 heterodimers are not downregulate response to CXCL12. While short-term hypoxia present. In addition, CCL3 signaling through CCR1 may activate may increase CXCR4 expression (as shown previously in ref. 15 kinases that modulate activation of CXCR4 downstream targets, at 24 hours and less), sustained hypoxia, as shown here, can thereby altering downstream signaling (47). In support of this, we downregulate CXCR4 protein expression, at least in part through found that the abrogation of CXCL12-mediated migration by increased expression of CXCL12. CCL3 was associated with decreased F-actin remodeling in Our in vitro data suggest that, in response to hypoxia, CXCL12 response to CXCL12. overexpression and subsequent CXCR4 downregulation in mul- In addition to CCR1, CCL3 is also the ligand for the chemokine tiple myeloma PCs may decrease responsiveness to CXCL12, receptor CCR5. However, the effects of CCL3 observed here are facilitating egress from the bone marrow. However, while we due to CCR1 and not CCR5 as the cell lines used do not express found that increased CXCL12 expression was associated with early detectable CCR5 (45, 48, 49). Furthermore, we demonstrated that poor survival outcomes for patients, we found no association either knockout of CCR1 or treatment with an inhibitor specific between expression of CXCR4 on peripheral blood or bone for CCR1, but not CCR5, restored the migratory response of U266 marrow multiple myeloma PCs and the number of circulating cells to CXCL12, further supporting the role of CCR1 in this multiple myeloma cells. This strongly suggests that mechanisms process. other than regulation of cell surface CXCR4 levels may be more We also demonstrated that CCR1 gene expression is associated important in regulating multiple myeloma PC dissemination in with poor prognosis in multiple myeloma patients and there is a patients. Using microarray, we identified that the most highly significant positive association between peripheral blood multi- upregulated gene following HIF-2a overexpression in LP-1 cells ple myeloma PC numbers and the expression of CCR1 on either was the chemokine receptor CCR1. CCR1 has previously been peripheral blood or bone marrow multiple myeloma PCs. In suggested to play an important role in multiple myeloma with the contrast, cell surface CXCR4 expression was not associated with levels of the CCR1 ligand CCL3 being elevated in the peripheral circulating multiple myeloma tumor cell numbers, suggesting blood of multiple myeloma patients with advanced disease that CCR1 may be more important than CXCR4 in determining (43, 44). Notably, CCL3 acts as a potent stimulus for the migra- the numbers of peripheral blood multiple myeloma PCs. As the tion of multiple myeloma cells in vitro (45). numbers of patients included in flow cytometry studies were Here, we show that HIF-2a increases CCR1 expression, increas- insufficient to conduct multivariate analysis, it is unclear whether ing migration toward CCL3 and abrogating the chemotactic the adverse survival effect of CCR1 expression is dependent on the response to CXCL12 in HMCL. This provides a novel means increase in peripheral blood multiple myeloma PCs; future stud- whereby hypoxia may increase PC dissemination in patients with ies in this area are warranted. multiple myeloma. While the mechanisms responsible for this CXCR4 plays a key role in the homing of multiple myeloma PCs remain unclear, CCL3/CCR1 signaling has been shown to have from the peripheral blood to the bone marrow (11, 12, 45) and CXCR4-dependent and -independent roles in mobilization of reactivation of CXCR4 when in the peripheral blood is required to Normoxic MM PC Hypoxic MM PC ++++ CXCR4 + CXCR4 +CCR1 ++++CCR1 Figure 7. Proposed mechanism of hypoxic regulation of CXCR4 CCR1 CXCR4 and CCR1 and subsequent plasma cell Tumor growth dissemination in multiple myeloma. Increased BMSC CXCR4 CCR1 expression in multiple myeloma (MM) PCs Hypoxia HIF-2 in response to bone marrow hypoxia and HIF-2a activation overrides the BMSC-derived CXCL12 retention signal and triggers egress of multiple CCR1 myeloma PCs to the peripheral circulation. A Bone marrow subsequent increase in CXCR4 receptor Homing expression and resensitization to CXCL12 in Egress multiple myeloma PCs in the peripheral circulation allows homing to other bone marrow Blood vessel sites. MM PC

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Vandyke et al.

enable subsequent homing of the cells in the circulation to Authors' Contributions secondary sites in the bone marrow. Our data show that CCR1 Conception and design: K. Vandyke, D.R. Hewett, L.B. To, D.J. Peet, expression is decreased, and CXCR4 expression increased, in A.C.W. Zannettino multiple myeloma tumor cells in the peripheral blood when Development of methodology: K. Vandyke, P. Diamond, D.J. Peet Acquisition of data (provided animals, acquired and managed patients, compared with the bone marrow. While the mechanisms respon- provided facilities, etc.): K. Vandyke, M.N. Zeissig, S.K. Martin, K.M. Mrozik, sible for this remain to be fully elucidated, we hypothesize that the C.M. Cheong, P. Diamond increased oxygen tension in the peripheral blood compared with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, the bone marrow (17, 50, 51) may be sufficient to decrease CCR1 computational analysis): K. Vandyke, K.M. Mrozik, L.B. To, S. Gronthos expression, allowing resensitization to CXCL12. In addition, our Writing, review, and/or revision of the manuscript: K. Vandyke, M.N. Zeissig, data suggest that CXCL12 levels are lower in the peripheral blood D.R. Hewett, S.K. Martin, C.M. Cheong, S. Gronthos, D.J. Peet, P.I. Croucher, A.C.W. Zannettino than in the bone marrow in patients with high CXCL12, support- fi Administrative, technical, or material support (i.e., reporting or organizing ing previous ndings (11, 18). Decreased local CXCL12 concen- data, constructing databases): L.B. To trations in the peripheral blood may therefore lead to increased Study supervision: K. Vandyke, D.R. Hewett, A.C.W. Zannettino CXCR4 levels and allow multiple myeloma PCs to subsequently rehome to the bone marrow. Acknowledgments Taken together, our studies point to a potential role for hypoxi- Biospecimens were provided by the South Australian Cancer Research cally mediated changes in the CCR1/CXCR4 axis that drive egress Biobank (SACRB), which is supported by the Cancer Council SA Beat Cancer Project, Medvet Laboratories Pvt. Ltd., and the Government of South Australia. of multiple myeloma PCs from the bone marrow (Fig. 7). We The authors would like to thank Jo Gardiner for providing clinical follow up data suggest that increased CCR1 expression in multiple myeloma PCs from the Royal Adelaide Hospital Paraproteinaemia Database, which is sup- in response to hypoxia overrides the stroma-derived CXCL12 ported by funding from Celgene and the Royal Adelaide Hospital Haematology retention signal and triggers egress to the peripheral bood. Future Private Practise Fund. studies are required to determine whether the desensitization of CXCR4 in response to CXCL12 and CCL3 is involved in the Grant Support mobilization and dissemination of multiple myeloma tumor This research was supported in part by a Young Investigator Project Grant cells in vivo. Moreover, identification of the factor(s) involved in from the Cancer Australia Priority-driven Collaborative Cancer Research Scheme, funded by Cure Cancer Australia (APP1100358 to K. Vandyke), the recirculation and dissemination process in multiple myeloma a National Health & Medical Research Council Project Grant (APP626911 to is key in the development of therapeutic strategies that limit overt A.C.W. Zannettino, S. Gronthos, D.J. Peet, L.B. To), and a grant from the Royal relapse. We suggest that further studies are warranted to determine Adelaide Hospital Contributing Haematologists' Committee to K. Vandyke, whether therapeutic targeting of CCR1 is a potential strategy for L.B. To, A.C.W. Zannettino, D.R. Hewett. preventing multiple myeloma cell dissemination to limit disease The costs of publication of this article were defrayed in part by the payment of advertisement progression and relapse. page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest Received January 18, 2017; revised May 11, 2017; accepted August 18, 2017; No potential conﬂicts of interest were disclosed. published OnlineFirst August 30, 2017.

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HIF-2α Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1

Kate Vandyke, Mara N. Zeissig, Duncan R. Hewett, et al.

Cancer Res 2017;77:5452-5463. Published OnlineFirst August 30, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0115

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