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Microenvironment and Immunology

Bioactivity and Prognostic Significance of GrowthDifferentiation Factor GDF15 Secreted by Bone MarrowMesenchymal Stem Cells in Multiple Myeloma

Jill Corre1,2,3, Elodie Labat4, Nicolas Espagnolle4, Benjamin H�ebraud5, Herv�e Avet-Loiseau3,7,Murielle Roussel3,5, Anne Huynh3,5, M�elanie Gadelorge4,5, Pierre Cordelier1, Bernard Klein9,Philippe Moreau3,8, Thierry Facon3,10, Jean-Jacques Fourni�e1, Michel Attal3,5, and Philippe Bourin4,6

AbstractOverexpression of growth differentiation factor 15 (GDF15) by bone marrow mesenchymal stem cells occurs

widely in patients with multiple myeloma, but the pathophysiologic effects of GDF15 in this setting remainundefined. GDF15 has been described in numerous solid tumors but never in hematologic malignancies. In thisstudy, we report that GDF15 significantly increases survival of stroma-dependent multiple myeloma cellsincluding primary multiple myeloma cells. In particular, GDF15 conferred resistance to melphalan, bortezomib,and to a lesser extent, lenalidomide in both stroma-dependent and stroma-independent multiple myeloma cells.Akt-dependent signaling was critical to mediate the effects of GDF15, whereas Src and extracellular signal-regulated kinase 1/2 signaling pathways were not involved. Given these results, we tested the clinical significanceof plasma concentrations of GDF15 (pGDF15) in 131 patients withmultiplemyeloma and found that it correlatedwith disease prognosis. Specifically, patients with high levels of pGDF15 had lower probabilities of event-freeand overall survival 30 months after diagnosis than patients with low pGDF15 levels. Our findings suggest thattumor microenvironment-derived GDF15 is a key survival and chemoprotective factor for multiple myelomacells, which is pathophysiologically linked to both initial parameters of the disease as well as patient survival.Cancer Res; 72(6); 1395–406. �2012 AACR.

IntroductionMultiple myeloma is a clonal plasma cell malignant disease

that accounts for 13%of hematologic cancers (1). The disease isfatal and themedian survival is 5 years (2), but this survival hassignificantly increased with the introduction of novel thera-pies, (3) with some patients surviving more than 10 years (4).Prognostic factors reflecting tumor burden, tumor damage inorgans and tissues, and intrinsic characteristics includinggenetic abnormalities have been described, but so far, noneof these specifically reflect the multiple myeloma microenvi-ronment (5–12).

Nevertheless, multiple myeloma has been a prototypic dis-ease model for the study of interactions between the micro-environment and malignant cells (13) and has led to thedevelopment of novel drugs such as immunomodulatory drugsand proteasome inhibitors, which target not only multiplemyeloma cells but also their microenvironment (14). A betterunderstanding of the mechanism by which the multiple mye-loma microenvironment affects the disease is still required todefine new therapeutic targets.

Multiplemyeloma cells proliferate in close contact with cellsfrom the bone marrow microenvironment. Bone marrow-mesenchymal stem cells (BM-MSC) are the only long livedcells of the bone marrow microenvironment. They secretesurvival factors for multiple myeloma cells and cytokines thatpromote osteoclastogenesis and angiogenesis (15–18). Abnor-malities of BM-MSCs have recently been reported (19–21). Inpatients with newly diagnosed multiple myeloma, we reportedthat BM-MSCs grown without multiple myeloma cells had anabnormal gene expression profile: 145 genes were differentiallyexpressed between BM-MSCs from patients with multiplemyeloma and normal subjects. In particular, we found thatgrowth differentiation factor 15 (GDF15) was increased in BM-MSCs from patients with multiple myeloma (21).

GDF15 is a 40-kDa propeptide that is cleaved to release a25-kDa circulating protein (22). It was first described as adivergent member of the human TGF-b superfamily (23).GDF15 has a broad activity, as indicated by the diversity of

Authors' Affiliations: 1Institut National de la Sant�e et de la RechercheM�edicale (INSERM), U1037; 2Hematology Laboratory, University HospitalPurpan, Toulouse; 3Intergroupe Francophone du My�elome (IFM); 4STRO-MALab; 5Hematology Department, University Hospital Purpan; 6CSA21,Toulouse; 7Hematology Laboratory and 8Hematology Department, Uni-versity Hospital Hotel-Dieu, Nantes; 9INSERM U847, Institute of Researchin Biotherapy, Montpellier; and 10Hematology Department, University Hos-pital Huriez, Lille, France

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

Corresponding Author: Jill Corre, Hematology Laboratory, UniversityHospital Purpan, place du Docteur Baylac, Toulouse 31059, France.Phone: 33-561-779-082; Fax: 33-561-777-695; E-mail:[email protected].

doi: 10.1158/0008-5472.CAN-11-0188

�2012 American Association for Cancer Research.

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the nomenclature (MIC-1, PTGF-b, PLAB, PDF, NAG-1, andPL74). In healthy subjects, GDF15 is highly expressed in theplacenta, with serum concentration increasing during preg-nancy (24, 25). GDF15 concentration is notably increased in theserum of patients with glioma, prostate, colorectal, or pancre-atic cancer (26–29), and the factor was recently described asbelonging to a series of 20 biomarkers that best define themalignant phenotype of numerous tumors (30). Understandingthe biologic role of GDF15 in cancer growth is challenging.Some studies have documented its protumorigenic role(26, 28–32) and others have shown antitumorigenic activity(22, 33, 34). The role of GDF15 in hematologicmalignancies hasnot yet been documented.

To determine the potential impact of GDF15 overexpressionby BM-MSCs from patients with multiple myeloma, weassessed the bioactivity of GDF15 on multiple myeloma cellsunder serum-free culture conditions. We sought correlates ofplasma concentration of GDF15 (pGDF15) in patients withmultiple myeloma with biologic and clinical parameters of thedisease. We found that GDF15 is a survival and chemoprotec-tive factor for multiple myeloma cells and show correlates ofpGDF15 to initial parameters of the disease and to patientsurvival.

Materials and MethodsCell samples

The MOLP-6 stroma-dependent multiple myeloma cellline was a generous gift from Dr. Harashima (ref. 35; FujisakiCell Center, Hayashibara Biochemical Laboratories) in 2007.Cryopreserved MOLP-6 cells were defrosted and grown onBM-MSCs in RPMI-1640 medium with 10% fetal calf serumand 10 mg/mL ciprofloxacin. BM-MSCs were obtained fromhealthy bone marrow by culturing cells harvested fromfilters used to remove ossicles and aggregates from theallograft and prepared as previously described (21). TheMM1.S stroma-independent multiple myeloma cell line waspurchased from American Type Culture Collection in 2009.Cryopreserved MM1.S cells were defrosted and grown at37�C and 5% CO2 in RPMI-1640 medium with 10% fetal calfserum and 10 mg/mL ciprofloxacin. These 2 multiple mye-loma cell lines were tested and authenticated before startingand during the study (morphologic analysis, immunophe-notype, caryotype, stroma dependence, mycoplasma, andEBV detection). Bone marrow from 4 patients with newlydiagnosed multiple myeloma was obtained by sternal punc-ture after patients gave their written informed consent.Primary bone marrow myeloma cells were purified with useof magnetic anti-human CD138 microbeads (Miltenyi Bio-tec), and multiple myeloma BM-MSCs were obtained fromthe CD138-negative fraction and prepared as previouslydescribed (21).

Cell culturesAll culture experiments were carried out in SynH serum-free

culturemedium, an Iscove-based fully defined culturemediumcontaining human albumin without insulin (ABCell-Bio;ref. 12).

Cell survival. MOLP-6,MM1.S, and primarymultiplemye-loma cells were plated at 50� 103 cells permLwith 0, 10, 100, or200 ng/mL recombinant human GDF15 (R&D systems). Cellswere grown at 37�C and 5%CO2. At day 1 of culture, viable cellswere counted by Trypan blue exclusion assay. In a separate setof experiments, MOLP-6 cells were pretreated overnight withan Akt-1/2 inhibitor (124018) at 1 mmol/L (Merck) beforeadding 200 ng/mL GDF15 for 24 hours.

Chemoprotection assay. MOLP-6 and MM1.S cells wereplated at 50 � 103 cells per mL with or without 200 ng/mLGDF15 for 24 hours. Cells were grown at 37�C and 5% CO2. Thefollowing drugs were added to the cell cultures for 1 day: 8mmol/Lmelphalan (Sigma), 8 nmol/L bortezomib (MillenniumPharmaceuticals), or 100 mmol/L lenalidomide (Celgene).Appropriate controls (cells treated with dimethyl sulfoxide orbuffer alone) were included. In a separate set of experiments,both multiple myeloma cell lines were pretreated overnightwith an Akt-1/2 inhibitor (124018) at 1 mmol/L before applyingthe above mentioned procedure. Viable cells were counted byTrypan blue exclusion assay. Percentage cell survival in cellspretreated with GDF15 � Akti versus nonpretreated cells wasobtained, and percentage of control cell survival (without Aktior GDF15 pretreatment and without any drugs) was thencalculated.

Akt phosphorylationMOLP-6, MM1.S, and primary multiple myeloma cells were

treated with 20 ng/mL GDF15 (R&D systems) or 50 ng/mLrecombinant human interleukin 6 (IL-6; R&D systems) inserum-free medium for 15 minutes (a set of experiments wascarried out in 10% calf serum conditions). Cells were fixed for15 minutes, washed, permeabilized for 30 minutes with use ofan intracellular stain kit (BD Pharmingen) and stained with aphycoerythrin (PE)-conjugated anti-pT308 or S473 Akt mAb(BD Biosciences) for 30 minutes. Akt phosphorylation wasdetected by flow cytometry (EPICS XL-MCL; Beckman Coul-ter). Live cells, which could be clearly distinguished in forwardversus sideways scatter plots, were gated, and the medianfluorescence intensity (MFI) was determined. Fold changes ofMFI for each condition were defined as the fraction (MFI ofmeasured sample)/(MFI of isotype control). A set of experi-ments with anAkt-1/2 inhibitor (124018, 1mmol/L)was carriedout to verify inhibition of Akt phosphorylation.

Src and ERK1/2 phosphorylationMOLP-6 andMM1.S cells were treatedwith 20 ng/mLGDF15

(R&D systems) in serum-free medium for 15 minutes. Cellswere fixed for 15 minutes, washed, permeabilized for 30minutes with use of an intracellular stain kit (BD Pharmingen)and stained with an Alexa Fluor 488–conjugated anti-pY416Src (Millipore) for 60 minutes or PE-conjugated anti-pT202/pY204 ERK1/2 mAb (BD Biosciences) for 30 minutes. Src orextracellular signal-regulated kinase 1/2 (ERK1/2) phosphor-ylation was detected by flow cytometry (EPICS XL-MCL).

Patient and healthy individual plasma samplesBone marrow plasma. Bone marrow of 24 patients with

newly diagnosed multiple myeloma was obtained by sternal

Corre et al.

Cancer Res; 72(6) March 15, 2012 Cancer Research1396




puncture after patients gave their written informed consent.Bonemarrow from5healthy individuals was obtained from theremaining sample used for quality control during the prepa-ration of allogeneic hematopoietic grafts. Bonemarrowplasmawas obtained after centrifugation of bonemarrow. Themedianage of patients with multiple myeloma and healthy subjectswas 57� 10 and 45� 13 years, and the sex ratio (M:F) was 1.5and 1.3, respectively.Blood plasma. Peripheral blood plasma from 131

patients with newly diagnosed multiple myeloma wasobtained after patients gave their written informed consent.Patients' characteristics are in Table 1. The median age ofpatients was 59 � 10 years, and the sex ratio (M:F) was 1.4.These 131 patients were enrolled in different IntergroupeFrancophone du My�elome treatment trials: 53 receivedconventional chemotherapy, including melphalan and pred-nisone, and 78 high-dose chemotherapy. Patients with DurieSalmon disease stage I were symptomatic and thus treated.Blood plasma from 13 healthy volunteers was also collected

after written agreement; the median age was 52 � 3 yearsand the sex ratio (M:F) was 1.2.

GDF15 measurementQuantification of GDF15 in supernatants and plasma

involved use ofDuoSet ELISA for humanGDF15 (R&Dsystems)following the manufacturer's protocol. All samples wereassayed in duplicate. Intra-assay reproducibility was evaluatedin 3 independent experiments. Each assay involved 6 replicatesof 3 plasma samples containing low,median, and high values ofGDF15. The calculated overall intra-assay coefficient of vari-ation was 11%, 7%, and 7%, respectively. Interassay reproduc-ibility was evaluated in 5 independent experiments. Each assayinvolved a duplicate of the same 3 plasma samples containinglow, median, and high values of GDF15. The calculated overallinterassay coefficient of variation was 15%, 4%, and 4%,respectively.

Statistical analysisStatistical analysis of categorical variables was tested with

the c2 test or the Fisher exact test and that of continuousvariables with the Student t test, then Mann–Whitney U test.All tests were 2 tailed. Correlation between pGDF15 level andinitial biologic parameterswas tested by Spearman correlation.A P value less than 0.05 was considered statistically significant.

Survival was evaluated for the 131 patients with multiplemyelomabypGDF15 level. The threshold value of pGDF15 (0.50ng/mL), defining low and high levels, was defined as meanþ 3SD of pGDF15 of healthy subjects. The duration of event-freesurvival (EFS) was calculated for all patients from the date ofdiagnosis to the time of disease progression, relapse, or death.Kaplan–Meier curves for EFS and overall survival (OS) wereplotted and compared by the log-rank test. Prognostic factorsfor survival were determined by the Cox proportional hazardsmodel for univariate and multivariate analysis. As possibleprognostic factors, age, International Staging System (ISS;ref. 7), b2-microglobulin level, hemoglobin level, lactate dehy-drogenase (LDH) activity, C-reactive protein (CRP) level, andpresence or absence of deletion of chromosome 13 (del13)wereincluded in the regression model. For continuous variables,classical cut-off values were selected.

ResultsGDF15 increases survival of a stroma-dependentmultiple myeloma cell line and primary multiplemyeloma cells

We first measured the survival of a stroma-dependent(MOLP-6) and a stroma-independent (MM1.S) multiple mye-loma cell lines and primary multiple myeloma cells underserum-free conditions supplemented with titrated concentra-tions of GDF15. Without stromal cells, only 43.9% � 3.1% ofMOLP-6 cells and 51.2% � 5.8% of primary multiple myelomacells survived at day 1 of culture whereas 77.4% � 10.2% ofMM1.S cells survived. GDF15 increased the MOLP-6 cell andthe primary multiple myeloma cells survival: 200 ng/mLof GDF15 yielded up to 70.3% � 5.6% viable MOLP-6cells (P < 0.005) and 74.0% � 5.9% viable primary multiple

Table 1. Characteristics of the 131 patientswithnewly diagnosed multiple myeloma

Age, y 59 � 10Sex (M/F) 76/55Durie Salmon stage [no. of patients (%)]I 31 (24)II 21 (16)III 79 (60)

Serum heavy chains [no. of patients (%)]IgG 79 (60)IgA 30 (23)Other 22 (17)

Serum light chains [no. of patients (%)]Kappa 81 (62)Lambda 47 (36)Nonsecretory 3 (2)

ISS [no. of patients (%)]I 58 (44)II 47 (36)III 26 (20)

Albumin level, g/L 40 � 6b2-microglobulin level, mg/L 4.2 � 5.9Hemoglobin level, g/dL 11 � 2Bone marrow plasma cells (% of cells) 32 � 22Calcemia, mmol/L 2.4 � 0.3Creatininemia, pmol/L 97 � 36Serum LDH activity, IU/L 353 � 89del13 [no. of patients (%)]No 58 (44)Yes 73 (56)

Lytic bone lesions [no. of patients (%)]No 46 (35)Yes 85 (65)

Treatment [no. patients (%)]Melphalan/prednisone 53 (40)High-dose chemotherapy 78 (60)

Growth Differentiation Factor 15 in Multiple Myeloma

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myeloma cells (P < 0.05). In contrast, GDF15 did not signifi-cantly increase MM1.S cell survival: 89.7% � 10.2% of MM1.Scells survived with 200 ng/mL of GDF15 (P ¼ 0.2; Fig. 1).

GDF15 triggers Akt phosphorylation in a stroma-dependent multiple myeloma cell line and primarymultiple myeloma cells

Multiple myeloma cell survival involves intracellular signal-ing cascades such as the Akt pathway (36). We thus wonderedwhetherGDF15 triggered theAkt phosphorylation onT308 and

S473 residues fromMOLP-6 andMM1.S cells and from purifiedprimary multiple myeloma cells from 4 patients in serum-freeculture conditions. Intracellular immunostaining followed byflow cytometry showed that GDF15 could trigger T308 andS473 Akt phosphorylation in MOLP-6 cells (P < 0.0005 andP < 0.05, respectively) whereas treatment with an IL-6 controldid not (Fig. 2A). GDF15 was still effective on Akt phosphor-ylation in serum conditions (Supplementary Fig. S1). In con-trast, neither GDF15 nor IL-6 was able to induce p-Akt T308and S473 in MM1.S cells (Fig. 2C), reflecting their constitutive

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Figure 1. Effect of GDF15 onsurvival of MOLP-6 stroma-dependent cells (A), primarymultiple myeloma (MM) cells (B),and MM1.S stroma-independentcells (C). Cells were cultured for 1day with GDF15 (1, 10, 100, and200 ng/mL). Experiments werecarried out under serum-freeconditions. Percentage cellsurvival in control versus GDF15-treated cells was measured byTrypan blue exclusion assay. Datarepresent mean � SD of 4independent experiments carriedout in duplicate. NT, no treatment.�, P < 0.05; ��, P < 0.005.

Corre et al.





activation of Akt (Supplementary Fig. S2). In primary multiplemyeloma cells, GDF15 induced T308 and, although to a lowerextent, S473 Akt phosphorylation (P < 0.05 and nonsignificant,respectively), whereas IL-6 induced only T308 phosphorylation(Fig. 2B). Therefore, GDF15 enhances Akt phosphorylation andactivity inMOLP-6 and primarymultiplemyeloma cells but notMM1.S cells. Overnight pretreatment of MOLP-6 cells with anAkt-1/2 inhibitor (124018, 1 mmol/L) inhibited GDF15-inducedp-Akt (Fig. 3A) and abrogated the GDF15-induced survivalincrease (P < 0.05; Fig. 3B).Of note, GDF15 did not induce phosphorylation of Src and

ERK1/2 in both multiple myeloma cell lines (SupplementaryFigs. S3 and S4).

GDF15 confers drug resistance to melphalan,bortezomib, and lenalidomide in a stroma-dependentand stroma-independent multiple myeloma cell lineUsing the same culture conditions as above, we asked

whether GDF15 was chemoprotective against drugs classi-

cally used in multiple myeloma treatment. Dimethyl sulfox-ide alone did not affect multiple myeloma cell survival (datanot shown). In drug-treated cultures, the proportion ofcontrol MOLP-6 cell survival was increased when the cellswere pretreated with GDF15 (melphalan: 70.0% � 10.1% vs.48.8% � 6.6%, P < 0.01; bortezomib: 91.0% � 10.6% vs. 67.6%� 7.5%, P < 0.01; lenalidomide: 43.0% � 7.6% vs. 31.1% �3.8%, nonsignificant; Fig. 4A). Similar results were obtainedwith MM.1S cells (melphalan: 80.0% � 24.3% vs. 30.0% �6.7%, P < 0.01; bortezomib: 81.5% � 17.5% vs. 54.0% � 8.0%,P < 0.05; lenalidomide: 20.7% � 7.8% vs. 5.6% � 1.4%,nonsignificant; Fig. 4B). Thus, GDF15 decreases chemother-apy-induced cytotoxicity of the 3 drugs in both multiplemyeloma cell lines. Overnight pretreatment of MOLP-6 cellswith an Akt-1/2 inhibitor (124018, 1 mmol/L) tended toabrogate the GDF15-induced drug resistance (melphalan:52.4% � 5.7% vs. 70.0% � 10.1%, P < 0.05; bortezomib: 69.4%� 1.4% vs. 91.0% � 10.6%, P < 0.05; lenalidomide: 32.7% �6.3% vs. 43.0% � 7.6%, nonsignificant; Fig. 4A). On the

Figure 2. Effect of GDF15 on T308and S473 Akt phosphorylation inMOLP-6 stroma-dependent cells (A),primary multiple myeloma cells (B),and MM1.S stroma-independentcells (C). Cells were pretreated or notwith GDF15 (20 ng/mL) or IL-6(50 ng/mL) for 15 minutes andstained for pT308 or pS473 Akt andisotype control for flow cytometry.Experiments were carried out underserum-free conditions. Live cells,which could be clearly distinguishedin forward versus sideways scatterplots, were gated, and the MFI wasdetermined. Fold changes of MFI foreach condition were defined as thefraction (MFI of measured sample)/(MFI of isotype control). Datarepresent mean � SD of 4independent experiments.�, P < 0.05; ��, P < 0.0005.

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contrary, Akt-1/2 inhibitor had no significant effect on theGDF15-induced drug resistance for MM1.S cells (Fig. 4B).

GDF15 is not produced by multiple myeloma cellsthemselves

Because GDF15 has been described to be produced bytumors cells themselves in solid cancer, we measured simul-taneously the concentration of GDF15 in supernatants ofprimary BM-MSCs andmultiple myeloma cells from 3 patientswith newly diagnosed myeloma. Whereas the concentration ofGDF15 ranged from 4 � 10�3 to 8 � 10�3 pg per cell for theirBM-MSCs, we did not detect any GDF15 in the correspondingmultiple myeloma cells supernatants. We found similar resultswith both multiple myeloma cell lines, MOLP-6 and MM1.Scells (Supplementary Fig. S5). Hence, GDF15 is a specific factorof microenvironment in myeloma.

pGDF15 increases with multiple myeloma disease stageBecause GDF15 is oversecreted by BM-MSCs from patients

with multiple myeloma relative to healthy subjects (21) andconfers in vitro survival and chemoresistance to multiplemyeloma cells, we next wondered whether the concentration

of GDF15 was also higher in bone marrow plasma frompatients with multiple myeloma than from healthy subjects.We measured the concentration of GDF15 in bone marrowplasma from patients with multiple myeloma (n ¼ 16) andhealthy subjects (n ¼ 5) and found it significantly higher inpatients with multiple myeloma (0.95 � 0.92 ng/mL vs. 0.41 �0.19 ng/mL, P< 0.001). Amongpatientswithmultiplemyeloma,the mean concentration of GDF15 was significantly higherfor the 9 patients with ISS stage II and III disease (1.26 �0.16 ng/mL) than for the 7 patients with stage I disease (0.57�0.20 ng/mL, P < 0.001; Fig. 5A).

To study the concentration of GDF15 in a larger cohort ofsubjects, and because blood samples are easier to collect thanbone marrow samples, we next measured the concentrationsof GDF15 in bonemarrow and blood plasma in 24 patients withmultiple myeloma. We found a correlation coefficient of 0.98(Supplementary Fig. S6), so for the rest of the study, we testedonly blood plasma.

The pGDF15 was measured in blood from 131 additionalpatients with newly diagnosed multiple myeloma and 13healthy subjects. Demographic and clinical characteristics forthe patients with multiple myeloma are in Table 1. Mean

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Figure 3. Effect of Akt-1/2 inhibitor(Akti-1/2) pretreatment onGDF15-induced MOLP-6 Aktphosphorylation (A) and survival(B). A, representative experimentwhere cells were pretreated or notwith Akti-1/2 (1 mmol/L), incubatedwith GDF15, and stained for pT308Akt for flow cytometry. Isotypecontrol staining (black line) andpT308 Akt (gray line) are shownon histograms. B, cells werepretreated or not overnight withAkti-1/2 (1mmol/L) and then treatedor notwithGDF15 (200 ng/mL) for 1day. Experiments were carried outunder serum-free conditions.Percentage cell survival in controlversus Akti-1/2 pretreated cellswas obtained by Trypan blueexclusion assay. Results areexpressed according topercentage of control cell survival(without Akti-1/2 pretreatment andwithout GDF15). Data representmean � SD of 3 independentexperiments carried out induplicate. �, P < 0.05.

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pGDF15 was significantly higher for patients with multiplemyeloma (0.90� 1.10 ng/mL) than for healthy subjects (0.25�0.08 ng/mL, P < 0.001). In addition, it was significantly higherfor patients with ISS stage III disease (2.10� 2.04 ng/mL) thanstage II disease (0.81 � 0.47 ng/mL, P < 0.003) and for patientswith stage II disease than stage I disease (0.49 � 0.24 ng/mL,P < 0.001; Fig. 5B).

pGDF15 level is correlated with initial multiplemyelomaparameters

For the 131 patients withmultiplemyeloma, we searched fora correlation between pGDF15 and initial biologic factors ofthe disease. We found a significant positive correlationbetween pGDF15 level and b2-microglobulin level, creatine-mia, calcemia, and serum monoclonal protein level and a

Figure 4. Effect of GDF15 onchemoprotection in MOLP-6 (A) andMM1.S (B) cell lines. Cells werepretreated or not with GDF15 (200ng/mL) and then treated withmelphalan (8 mmol/L), bortezomib (8nmol/L), or lenalidomide (100 mmol/L)for 1 day. In a separate set ofexperiments, both multiple myelomacell lines were pretreated overnightwith an Akti-1/2 at 1 mmol/L beforeapplying the above mentionedprocedure. Experimentswere carriedout under serum-free conditions.Percentage of cell survival in cellspretreated with GDF15� Akti versusnonpretreated cells was measuredby Trypan blue exclusion assay.Results are expressed according topercentage of control cell survival(without Akti or GDF15 pretreatmentand without any drugs). Datarepresent mean � SD of 5 (MOLP-6;A) and 3 (MM.1S; B) independentexperiments carried out at least induplicate. NS, nonsignificant.�, P < 0.05; ��, P < 0.01.

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significant inverse correlation with levels of hemoglobin andalbumin. pGDF15 level was not correlated with bone marrowplasma cell frequency, LDH activity, or CRP level (Supplemen-tary Fig. S7).

Mean pGDF15 was calculated according to Durie Salmondisease stage, presence or absence of del13, and bone status.Mean pGDF15 was significantly higher in patients withDurie Salmon stage III than Durie Salmon stage II or I(1.31 � 1.55 vs. 0.68 � 0.38 or 0.53 � 0.37 ng/mL, P < 0.02).Mean pGDF15 was also significantly higher in patientswith than without del13 (1.13 � 1.41 vs. 0.64 � 0.38 ng/mL,P < 0.004). Finally, pGDF15 was significantly higher inpatients with than without osteolytic lesions (1.05 � 1.26vs. 0.66 � 0.46 ng/mL, P < 0.02).

pGDF15 level is correlated with survivalSurvival was evaluated for the 131 patients with multiple

myeloma by pGDF15. Among the 131 patients, 50 had

pGDF15 less than 0.50 ng/mL (mean 0.33 � 0.10; "pGDF15low

patients") and 81 had pGDF15 0.50 ng/mL or more (mean1.25 � 1.26; "pGDF15high patients"). For the 131 patients withmultiple myeloma, the median follow-up from time ofdiagnosis was 27 months (range, 1–60 months). The medianduration of EFS was 33 months (range, 1–60 months), andthe median duration of OS was not reached. For the 50pGDF15low patients, the median duration of EFS was 39months (range, 5–60 months), and the probability of EFS andOS 30 months after diagnosis was 80% and 97%, respectively.Among the 81 pGDF15high patients, the median durationof EFS was 29 months (range, 1–60 months) and the prob-ability of EFS and OS 30 months after diagnosis was50% and 75%, respectively. EFS and OS were significantlylonger for pGDF15low patients (P < 0.0045 and P < 0.013,respectively; Fig. 6). None of the 26 patients with pGDF15less than 0.34 ng/mL died, and median duration of EFS forthese patients was more than 60 months; among these

0.00

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A

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Figure 5. GDF15 concentrationmeasured by ELISA in bonemarrow (BM) plasma (A) and bloodplasma (B) in healthy subjects andin patients with multiple myeloma(MM). Data represent mean � SD.�, P < 0.003; ��, P < 0.001.

Corre et al.





patients were 18 with ISS disease stage I and 8 stage II, aswell as 11 with Durie Salmon stage I, 5 stage II, and 10 stageIII (data not shown).

Prognostic factors of EFSWe analyzed the impact of prognostic factors (age, ISS

stage, b2-microglobulin level, hemoglobin level, LDH activ-ity, CRP level, and presence or absence of del13) on EFSfor the 131 patients with multiple myeloma. On univariateanalysis, EFS was significantly related to pGDF15 (P¼ 0.003),age (P ¼ 0.003), and b2-microglobulin level (P ¼ 0.02).On multivariate analysis, EFS was related to only pGDF15(P ¼ 0.04) and age (P ¼ 0.001).

DiscussionOur study shows that functionally GDF15 is a survival and

chemoprotective factor for multiple myeloma cells and thatclinically pGDF15 is related to initial parameters of thedisease and survival. This study provides new insights intothe mechanism by which the abnormal multiple myelomamicroenvironment affects the pathophysiology and theprognosis of multiple myeloma. Here, we investigated thesignificance of one of multiple myeloma microenvironmentdysfunctions, the overexpression of GDF15 by BM-MSCs(21). Although the overexpression of GDF15 has beendescribed in numerous solid malignancies (26, 29, 32, 37),

its precise implication in tumors remains unknown. GDF15may have different functions in cancer, such as being atumor suppressor during early oncogenesis but a promoterat later stages (22, 38). GDF15 has never been studied inhematologic malignancies.

Here, we first determined the role of GDF15 on multiplemyeloma cell survival in vitro. This involved experiments underserum-free conditions to avoid components present in serum,in particular insulin-like growth factor 1 (IGF-1), which mightconfound interpretation of the results (12). GDF15 increasedcell survival in the MOLP-6 stroma-dependent multiple mye-loma cell line, confirming our previous work (21) but in morestringent culture conditions. Moreover, GDF15 was able tosignificantly increase cell survival in primary multiple myelo-ma cells. Although these results contrast with GDF15-inducedapoptosis of prostate cancer cells (33), they are notably con-sistent with those of GDF15 supporting malignant glioma cellproliferation (26). We previously reported that BM-MSCs frompatients with multiple myeloma induced an overgrowth ofMOLP-6 cells when compared with BM-MSCs from normalsubjects (21). To determine whether the overproduction ofGDF15 by multiple myeloma BM-MSCs is involved in thisovergrowth, we carried out MOLP-6 cells and multiple mye-loma BM-MSCs coculture experiments involving MSCs from 4patients with multiple myeloma that were transfected eitherwith a control plasmid or with a short hairpin RNA (shRNA)-targeting GDF15. The number of MOLP-6 cells after 7 days ofcoculture was significantly decreased (P < 0.05) when MSCswere transfected with shRNA GDF15 (Supplementary Fig. S8).Although this decrease is moderate, it suggests that the over-production of GDF15 participates to the MOLP-6 overgrowthwe previously observed with multiple myeloma BM-MSCs. Wedid not find any significant survival advantage with GDF15 inthe MM1.S stroma-independent multiple myeloma cell line. Ofnote, no overgrowth of stroma-independent cell line had beenobserved with multiple myeloma BM-MSCs in our previouswork (21). Interestingly, GDF15 induced Akt phosphorylationin MOLP-6 and primary multiple myeloma cells but not inMM1.S cells. In addition, pretreating MOLP-6 cells with anAkti-1/2 inhibitor abrogated GDF15-induced survival increase.These results support the presence of an Akt-dependentsurvival mechanism, which is consistent with the observationfrom Kim and colleagues (31), who found that GDF15 activatesAkt signaling in human breast and gastric cancer cells. Thecontribution of the Akt pathway to the growth of multiplemyeloma cells has been extensively studied (36, 39–41). Func-tionally, the Akt pathway is implicated in cell-cycle and apo-ptosis regulation in multiple myeloma cells. Akt is phosphor-ylated in bone marrow biopsies from patients with multiplemyeloma, notably because of bone marrow microenviron-ment-derived cytokines such as IL-6 or IGF-1 (42). Our dataindicate that GDF15 also contribute to triggering the Aktpathway in multiple myeloma cells. The absence of IL-6 effecton Akt signaling in MOLP-6 cells was expected as IL-6 has nobioactivity on their survival (35). Its marginal effect in primarymultiple myeloma cells in our serum-free conditions is due tothe fact that the bioactivity of this cytokine is highly dependenton IGF-1 presence (12). The lack of significant effect of IL-6 and

Figure 6. Kaplan–Meier plot of survival to 30 months in 131 patients withmultiple myeloma. Fifty patients with pGDF15 level less than 0.5 ng/mLand 81 patients with pGDF15 level 0.5 ng/mL or more. A, event-freesurvival. B, overall survival.






GDF15 on MM1.S stroma-independent cells survival reflectsconstitutive activation of Akt (41). Apart from Akt, GDF15 hasbeen described to activate ERK1/2 in a SNU-216 gastric cancercells (31) and Src in SK-BR-3 breast cancer cells (43). In ourculture conditions, GDF15 could not induce phosphorylationof ERK1/2 in both multiple myeloma cell lines, suggesting thatthe Ras/Raf/MEK/MAPK pathway would not be involved. Inaddition, GDF15 had no bioactivity on Src in MOLP-6 andMM1.S cells.

GDF15 conferred drug resistance in MOLP-6 and MM1.Scells to 3 drugs classically used in multiple myeloma treat-ment (melphalan, bortezomib and, to a lower extent, lena-lidomide). These data are consistent with previous observa-tions (44) showing that GDF15 protects prostate cancer cellsagainst the cytotoxic effect of docetaxel and mitoxantrone.Because the GDF15-mediated chemoprotection of MOLP-6cells was abrogated when the cells were pretreated with anAkti-1/2 inhibitor, we suggest the presence of an Akt-depen-dent chemoprotection mechanism. For MM1.S cell line, thiseffect might be Akt independent. Other signaling cascadesactivated by cytokines or growth factors in multiple mye-loma cells may be involved, including Janus-activated kinase(JAK)/Stat3, Wnt, NF-kB pathways (1, 14), and underlyingother cellular processes that might influence chemoresis-tance (drug efflux, cell death, genotoxic sensing, DNA repair,proteasome activity). The differences of biologic profilebetween MOLP-6 and MM1.S cells and the similaritiesbetween MOLP-6 and primary multiple myeloma cells pin-point the importance of the stroma dependence of multiplemyeloma cell lines chosen as in vitro models to study theimpact of microenvironment on tumor.

We next showed that GDF15 is increased in bone marrowplasma from patients with multiple myeloma. Although ourprevious studies indicated that this increase reflected GDF15overproduction by multiple myeloma BM-MSCs, macro-phages might also contribute to total GDF15 level. Macro-phages are able to secrete GDF15 (23) and constitute abun-dant components of multiple myeloma microenvironmentand able to protect multiple myeloma cells against drug-induced apoptosis (45). However, as compared with itsproduction in solid tumors, GDF15 is not produced by themalignant multiple myeloma cells themselves but specifi-cally by their microenvironment.

Although GDF15 has been described in many solidtumors, a lot remains to be uncovered on its biology; inparticular GDF15 receptor is still unknown today. There issome evidence for SMAD pathway activation, suggestingGDF15 may act through a TGF-b superfamily (46). A recentstudy identified GDF15 as an acute-phase modifier of CCR2/TGF-bRII–dependent inflammatory responses to vascularinjury (47). On the other side, Kim and colleagues elegantlyshowed that GDF15 induces the transactivation of ErbB2tyrosine kinase in SK-BR-3 breast and SNU-216 gastriccancer cells (31). We did not find any expression of TGF-bRII or ErbB2 on both multiple myeloma cell lines andprimary multiple myeloma cells (data not shown), suggest-ing that GDF15 receptor also remains to be discovered inmultiple myeloma.

To determine whether the GDF15 concentration levelincrease was indicative of the severity of the disease inpatients with multiple myeloma, and because we found thatthe concentrations of GDF15 in bone marrow and bloodplasma in 24 patients with multiple myeloma were corre-lated, we next measured the pGDF15 in 131 patients withnewly diagnosed multiple myeloma. The pGDF15 levelincrease was correlated with prognosis, as was reported forpatients with prostate, colorectal, and endometrial cancers(27, 28, 48). Finally, we found a strong relation betweenpGDF15 level and survival to 30 months in patients withmultiple myeloma.

This study allows to gain a better understanding into themechanism by which the abnormal microenvironmentaffects the pathophysiology and the prognosis of multiplemyeloma. Microenvironment has become a therapeutic tar-get that cannot be ignored in multiple myeloma. However,the identification of specific targets into this tumoral micro-environment is urgently needed for the development of next-generation therapies. Although further work need to be doneto characterize GDF15 biology, we suggest that GDF15participates in the control of minimal residual disease,possibly by maintaining in a chemoprotective niche anundetectable pool of multiple myeloma cells causing therelapse. Because of the moderately minor phenotype dis-played by GDF15-knockout mice (49, 50), therapeutic strat-egy specifically targeting GDF15 might be conceivable. Inthis regard, future studies from our laboratory will assessGDF15 as a novel target for therapeutic strategies in mul-tiple myeloma.

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

Authors' ContributionsConception and design: J. Corre, M. Attal, P. BourinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Corre, E. Labat, N. Espagnolle, B. Hébraud, H. Avet-Loiseau, M. Roussel, A. Huynh, P. Cordelier, P. Moreau, T. FaconAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Corre, E. Labat, N. Espagnolle, M. Attal, P. BourinWriting, review, and/or revision of the manuscript: J. Corre, B. Klein, J.J.Fournié, M. Attal, P. BourinAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Corre, E. Labat, N. Espagnolle, B.Hébraud, H. Avet-Loiseau, M. Roussel, A. Huynh, M. Gadelorge, P. Cordelier, P.Moreau, T. FaconSupervision of study: J. Corre, P. BourinDevelopment of methodology: E. Labat, M. Gadelorge, N. Espagnolle, P.CordelierAdministrative, technical, or material support (i.e., reporting ororganizing data, constructing databases): J. Corre, M. Gadelorge, N.Espagnolle

AcknowledgmentsThe authors thank Laura Smales for correction of the manuscript.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received January 18, 2011; revised January 20, 2012; accepted January 25, 2012;published OnlineFirst February 2, 2012.

Corre et al.





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2012;72:1395-1406. Published OnlineFirst February 2, 2012.Cancer Res Jill Corre, Elodie Labat, Nicolas Espagnolle, et al. in Multiple MyelomaFactor GDF15 Secreted by Bone Marrow Mesenchymal Stem Cells Bioactivity and Prognostic Significance of Growth Differentiation

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