Bidirectional Notch Signaling and Osteocyte-Derived Factors in The

Published OnlineFirst February 1, 2016; DOI: 10.1158/0008-5472.CAN-15-1703 Cancer Molecular and Cellular Pathobiology Research

Bidirectional Notch Signaling and Osteocyte- Derived Factors in the Bone Marrow Microenvironment Promote Tumor Cell Proliferation and Bone Destruction in Multiple Myeloma Jesus Delgado-Calle1,2, Judith Anderson3, Meloney D. Cregor1, Masahiro Hiasa3, John M. Chirgwin2,4, Nadia Carlesso5, Toshiyuki Yoneda3, Khalid S. Mohammad1,4, Lilian I. Plotkin1,2, G. David Roodman2,3, and Teresita Bellido1,2,4

Abstract

In multiple myeloma, an overabundance of monoclonal sion, the osteocytic Rankl/Opg (TNFRSF11B) ratio, and the plasma cells in the bone marrow induces localized osteolytic ability of osteocytes to attract osteoclast precursors to induce lesions that rarely heal due to increased bone resorption and local bone resorption. Furthermore, osteocytes in contact suppressed bone formation. Matrix-embedded osteocytes with multiple myeloma cells expressed high levels of Sost/ comprise more than 95% of bone cells and are major reg- sclerostin, leading to a reduction in Wnt signaling and ulators of osteoclast and osteoblast activity, but their con- subsequent inhibition of osteoblast differentiation. Impor- tribution to multiple myeloma growth and bone disease is tantly, direct contact between osteocytes and multiple mye- unknown. Here, we report that osteocytes in a mouse model loma cells reciprocally activated Notch signaling and of human MM physically interact with multiple myeloma increased Notch receptor expression, particularly Notch3 and cells in vivo, undergo caspase-3–dependent apoptosis, and 4, stimulating multiple myeloma cell growth. These studies express higher RANKL (TNFSF11) and sclerostin levels reveal a previously unknown role for bidirectional Notch than osteocytes in control mice. Mechanistic studies reveal- signaling that enhances MM growth and bone disease, sug- ed that osteocyte apoptosis was initiated by multiple mye- gesting that targeting osteocyte-multiple myeloma cell inter- loma cell-mediated activation of Notch signaling and was actions through speciﬁc Notch receptor blockade may repre- further ampliﬁed by multiple myeloma cell-secreted TNF. sent a promising treatment strategy in multiple myeloma. The induction of apoptosis increased osteocytic Rankl expres- Cancer Res; 76(5); 1–12. 2016 AACR.

Introduction increased bone resorption and concomitant long-term suppres- sion of bone formation. The bone/BM microenvironment is a Multiple myeloma is characterized by expansion of monoclo- major contributor to tumor growth and bone destruction in nal plasma cells in the bone marrow (BM) that induce marked multiple myeloma (3). The bone remodeling compartment is bone destruction in the majority of patients (1). Multiple mye- disrupted in multiple myeloma, allowing exchange of soluble loma patients present with severe bone pain caused by osteolytic factors and direct cell-to-cell contact between multiple myelo- lesions that rarely heal (2). The osteolytic lesions result from ma cells and bone cells (4). Previous studies demonstrated that multiple cytokines are secreted or induced by the interaction of 1Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana. 2Roudebush Veterans Administra- multiple myeloma cells with the different cell types in the bone tion Medical Center, Indianapolis, Indiana. 3Division of Hematology/ microenvironment to increase bone destruction, including the Oncology, Department of Medicine, Indiana University School of Med- receptor of activator nuclear factor-kappa B ligand (RANKL), 4 icine, Indianapolis, Indiana. Division of Endocrinology, Department of the chemokine (C-C motif) ligand 3 (MIP1a), and IL3. Further, Medicine, Indiana University School of Medicine, Indianapolis, Indiana. 5Department of Pediatrics Indiana, University School of Medicine, multiple myeloma–derived IL7, dickkopf WNT signaling path- Indianapolis, Indiana. way inhibitor 1 (DKK1), and IL3, as well as direct contact by Note: Supplementary data for this article are available at Cancer Research multiple myeloma cells with osteoblasts inhibit osteoblast Online (http://cancerres.aacrjournals.org/). differentiation (1, 5). Corresponding Authors: Teresita Bellido, Indiana University School of Medicine, Although osteoclasts and osteoblasts remodel bone by 635 Barnhill Drive Medical Science Building, Room 5045A, Indianapolis, IN resorbing or forming bone respectively, osteocytes, which con- 46202. Phone: 317-274-7410; Fax: 317-278-2040; E-mail: [email protected]; stitute >95% of all bone cells, are the central regulators of these or G. David Roodman, Indiana University School of Medicine, 980 W. Walnut processes (6, 7). Osteocytes, although buried within bone Street, Suite C312, Indianapolis, IN 46202. Phone: 317-278-6255; Fax: 317-274- mineral, extensively communicate with each other and with 0396; E-mail: [email protected] cells on the bone surface and in the marrow via cytoplasmic doi: 10.1158/0008-5472.CAN-15-1703 projections that run along canaliculi and form the osteocyte 2016 American Association for Cancer Research. network. This network allows cell-to-cell communication and

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distributes osteocyte-secreted molecules that regulate osteo- Ex vivo bone organ cultures blast and osteoclast function. Osteocytes are the primary pro- These assays were performed as previously described (21). ducers of sclerostin, the product of the SOST gene, a potent Detailed description of the assay can be found in Supplementary inhibitor of bone formation, and are a major source of RANKL, Methods. the central osteoclastogenic factor (8, 9). Lack of sclerostin increases osteoblast number and activity, and deletion of Cell viability and apoptosis osteocytic RANKL inhibits osteoclast formation and bone Multiple myeloma cells were separated from adherent osteo- resorption, demonstrating that osteocytes are key regulators cyte-like cells using EDTA. Cell death was quantified by trypan – of osteoblast and osteoclast activity (10 12). Moreover, apo- blue uptake and apoptosis by chromatin condensation and ptotic osteocytes, which accumulate with skeletal disuse, glu- nuclear fragmentation of cells transfected with nuclear green fi cocorticoid excess, or estrogen de ciency, increase local bone fluorescent protein (nGFP; refs. 22, 23). At least 100 cells in five resorption by attracting osteoclast precursors to particular different fields selected by systematic random sampling were areas of bone (6, 13). Although knowledge of the role of examined for each experimental condition. Representative photo- osteocytes and the osteocytic network in bone homeostasis micrographs were taken with an EVOS FLCell Imaging System and common skeletal diseases has greatly increased, the con- (Life Technologies). tribution of osteocytes to the development and progression of cancer involving bone is just beginning to be defined. In the current study, we determined if reciprocal communica- Cell proliferation analysis tion between multiple myeloma cells and osteocytes occurs and Viable cells were enumerated by trypan blue exclusion. Fluo- explored the mechanisms involved and the consequences of these rescence emitted by 5TGM1-GFP cells was measured in a Spec- interactions for the progression of multiple myeloma bone traMaxi3 microplate reader (Molecular Devices), set at 485 nm/ disease. 520 nm. 5TGM1-GFP cell numbers linearly correlated with relative fluorescent units (RFU; Supplementary Fig. S1A). Materials and Methods Transfections Reagents MLO-A5 cells were transiently transfected using Lipofectamine- Reagents used in this study can be found in Supplementary Plus (Invitrogen; refs. 23, 24). Methods. Gene expression analysis Cells and culture conditions mRNA expression was quantified as previously described (25). L. Bonewald (University of Missouri–Kansas City, Kansas Murine soluble RANKL, OPG, or human TNFa were quantified by City, MO) provided the murine MLO-A5 in 1997 and MLO-Y4 ELISA (R&D Systems) of culture supernatants. in 2001 osteocyte-like cells (14, 15). JJN3, 5TGM1, and MM1.S multiple myeloma cell lines were provided by N. Giuliani (University of Parma, Parma, Italy) in 2006, B. Oyajobi (Uni- Western blot analysis versity of Texas at San Antonio, San Antonio, TX) in 2007, and The assays were performed as described previously (25). S. Rosen (Northwestern University, Evanston, IL) in 2003 (16– Detailed descriptions of antibodies can be found in Supplemen- 18). R. Jilka (University of Arkansas for Medical Sciences, Little tary Methods. Rock, AR) provided the OB-6 osteoblast–like cells in 1997 (19). Nonadherent osteoclast precursors were collected as described þ Osteoclast precursor migration before (20). After informed consent, CD138 cells from multiple Forty-eight–hour CM from JJN3, MLO-A5, or from JJN3 and myeloma patients were prepared as previously detailed (16). MLO-A5 cocultured in direct contact was placed in the bottom of Studies were approved by the Indiana University School of 8-mm pore chambers to attract murine nonadherent BM cells (26). Medicine Institutional Review Board. Cell lines were authenti- Detailed description of the assay can be found in Supplementary fi cated by morphology, gene expression pro le, and tumorigenic Methods. capacity (multiple myeloma cells). Cocultures were established by (i) adding multiple myeloma cells on top of osteocyte-like cells, (ii) adding multiple myeloma cells in transwell chambers Mouse model of human multiple myeloma Six-week-old female SCID mice B6.CB17-Prkdcscid/SzJ (The in a 1:5 ratio (osteocytic:multiple myeloma), or (iii) adding 50% bg nu xid conditioned media (CM) from 48-hour culture of multiple Jackson laboratories) and NIH-Lyst Foxn1 Btk (Charles Riv- myeloma cells to osteocytes. DEVD (50 nmol/L), anti-TNFa er Wilmington) were injected intratibially with JJN3 cells or saline fi (0.3 mg/mL), or GSIXX (2.5–10 mmol/L) were added 1 hour and sacri ced 4 weeks later (16). Detailed information regarding before addition of multiple myeloma cells or CM. MLO-A5 cells imaging and micro-CT analysis can be found in Supplementary were treated with 0.01 ng/mL TNFa, 5 ng/mL TGFb, or 10 ng/mL Methods. Studies were approved by the Institutional Animal Care IL6 for 4 to 24 hours. For Notch activation, MLO-A5 cells were and Use Committee of the Indiana University School of Medicine. cultured on delta-like 1 ligand (DLL1)-IgG2 or control IgG2- The sample size was calculated based on a previous study (16). coated plates for 24 hours. For OB-6 osteoblast–like cell differentiation, cells were cultured with osteogenic media (OM; Immunohistochemistry 0.2 mmol/L ascorbic acid, 10 mmol/L b-glycerophosphate) or Antigen detection was performed on paraffin-embedded tibiae OM containing 50% of CM from MLO-A5 or JJN3 cells cultured as described before (25). Detailed descriptions of antibodies can alone, or from JJN3 directly cocultured with MLO-A5 cells. be found in Supplementary Methods.

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Acid etching scanning electron microscopy A5 alone vs. MLO-A5 cells cocultured with JJN3 cells; Fig. 2A). Cell The assays were performed as described previously (27). death was detected at 8 hours and progressively increased at 24 Detailed description of the assay can be found in Supplementary and 48 hours. To elucidate the effect of factors released by Methods. multiple myeloma cells on osteocyte apoptosis, the cell types were separated by a porous membrane that prevents physical Statistical analysis interactions and only allows exchange of soluble factors. Cocul- Data were analyzed using SigmaStat (SPSS Science). Differ- ture of JJN3 and MLO-A5 cells separated by transwell chambers ences between mean were evaluated using unpaired t test, one- did not increase cell death at 8 or 24 hours, but it did at 48 hours – – way, or two-way ANOVA, followed by pairwise multiple compar- (1% 3% vs. 8% 13%; Fig. 2B). These results suggest that different isons using the Student–Newman–Keuls method. Mean SD are mechanisms mediate the early and late increases in osteocyte cell reported. P values <0.05 were considered significant. death induced by multiple myeloma cells. Coculture of MLO-A5 cells with JJN3 cells or JJN3-CM increased the percentage of MLO- Results A5 cells exhibiting chromatin condensation and nuclear fragmentation, sine qua non features of apoptosis (Fig. 2A and B). More- Multiple myeloma cells increase osteocyte apoptosis and over, osteocyte death was inhibited by the CASP3 inhibitor, in vivo osteocytic RANKL and sclerostin production in an model DEVD, at all time points confirming that it was due to apoptosis of multiple myeloma bone disease (Fig. 2A and B; Supplementary Fig. S1C). Coculture of MLO-A5 þ Our murine model of human multiple myeloma is well cells with CD138 cells from 6 different multiple myeloma þ established and reproduces all features of human multiple mye- patients (Fig. 2A) or CM from CD138 cells from a multiple – fi loma induced bone disease (16). Osteolytic lesions were rst myeloma patient (Fig. 2B) also increased MLO-A5 cell apoptosis. detected radiographically 2 weeks after JJN3 cell injection (Sup- Similar results were obtained when MLO-A5 were cocultured with plementary Fig. S1B), and their size progressively increased up to human MM.1s or murine 5TGM1 multiple myeloma cells, or 4 weeks (Fig. 1A). Micro-CT analysis of JJN3-involved tibia when MLO-Y4 cells, another murine osteocytic cell line, were fi performed at 4 weeks con rmed the presence of osteolytic lesions cocultured with JJN3 cells (Supplementary Fig. S1D). and demonstrated decreased trabecular bone volume (BV/TV), Notch signaling is activated by cell-to-cell contact and reg- trabecular number (Tb.N.), trabecular thickness (Tb.Th.), and ulates cell proliferation and apoptosis in multiple cell types increased trabecular separation (Tb.Sp.) compared with controls (28). Further, Notch signaling is aberrantly activated in mul- (Fig. 1B). The percentage of apoptotic osteocytes, stained with an tiple myeloma cells due to the overexpression of Notch recep- antiactive caspase-3 (CASP3) antibody, was increased 2-fold in tors and ligands (29, 30), demonstrating that multiple myelo- bones injected with JJN3 cells compared with controls (Fig. 1C). ma cells are potential signalers as well as targets of Notch. We Moreover, the percentage of osteocytes expressing RANKL and therefore assessed if direct interactions between multiple mye- fi sclerostin was signi cantly higher in JJN3-injected mice com- loma cells and osteocytes activate Notch in these cells and the fi pared with controls. These ndings suggest that in addition to potential biologic consequences. Direct contact of MLO-A5 survival, osteocyte gene expression is altered in multiple myelo- cells with JJN3 multiple myeloma cells activated Notch signal- – – ma bearing bones. Histologic analysis of multiple myeloma ing in MLO-A5 cells, as shown by the increased expression of bearing bones showed that multiple myeloma cells were adjacent the Notch target gene hairy/enhancer-of-split related with to the bone surface, and acid etching-scanning electron micros- YRPW motif 1 (Hey1; Fig. 2C) and hairy and enhancer of split copy (SEM) images revealed that osteocytic dendritic processes 1 (Hes1), which persisted up to 48 hours (Supplementary Fig. were in direct contact with multiple myeloma cells in the marrow S1E). JJN3 cell activation of osteocytic Notch signaling was (Fig. 1D and E). Thus, osteocytes are in close contact with suppressed by the Notch inhibitor GSIXX, confirming the – multiple myeloma cells, suggesting that multiple myeloma specificity of the effect (Fig. 2C). Hey1 expression was also þ induced changes in osteocytes may result from cell-to-cell contact increased in MLO-A5 cells cocultured with CD138 cells from 3 and/or exchange of soluble factors between osteocytes and mul- of 5 multiple myeloma patients (Fig. 2C). As expected, Notch tiple myeloma cells. activation (Hey1 and Hes1) in osteocytes did not occur when MLO-A5 cells were cocultured with multiple myeloma cells in Osteocyte apoptosis is initiated by multiple myeloma cell– transwell chambers (Supplementary Fig. S1F). MM1.s or mediated activation of Notch signaling in osteocytes and 5TGM1 multiple myeloma cells also increased the Notch target amplified by multiple myeloma cell–derived TNFa gene expression in osteocyte-like MLO-A5 or MLO-Y4 cells In vitro cocultures between osteocyte-like MLO-A5 cells and (Supplementary Fig. S1F). In addition, interactions with JJN3 multiple myeloma cells were performed to determine the cells rapidly upregulated the expression of receptors Notch3 and mechanisms responsible for the increased osteocyte apoptosis Notch4 in MLO-A5 cells, whereas Notch1 or Notch2 receptor and upregulation of RANKL and sclerostin observed in osteocytes expression was unchanged (Fig. 2D). Moreover, the levels of in multiple myeloma–bearing bones. Communication between NOTCH3 and of Notch intracelular domain 3 (NICD3), the multiple myeloma cells and bone cells, including osteocytes, can active form and downstream effector of the receptor NOTCH3, occur in different manners: (i) via secretion of soluble factors, (ii) were elevated in tumor-bearing bones compared with control via direct cell-to-cell contact, or (iii) a combination of both. First, bones, demonstrating that Notch signaling activation also direct cell-to-cell cultures were established to determine the effect occurs in vivo (Fig. 2E and F). of physical interactions with multiple myeloma cells on osteocyte To evaluate the potential impact of gain-of-function of Notch apoptosis. Coculture of MLO-A5 cells in direct contact with signaling on osteocyte life span, MLO-A5 cells were transfected human JJN3 multiple myeloma cells induced a 2- to 3-fold with nGFP and the Notch intracellular domains (NICD) 1 or 2, increase in MLO-A5 cell death (1%–4% vs. 6%–15% in MLO- which translocate to the nucleus and activate Notch target gene

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Figure 1. Osteocytes interact with multiple myeloma tumors in the BM to increase apoptosis, RANKL, and sclerostin production in osteocytes in a murine model of human multiple myeloma. A, osteolytic lesions were detected by radiographs, micro-CT, and histology 4 weeks after injection. Representative images of tibiae are shown. H&E, hematoxylin and eosin. B, micro-CT analysis of cancellous bone in tumor- bearing tibias (n ¼ 5/group). C, percentage of cortical osteocytes stained for active CASP3, RANKL, or sclerostin (n ¼ 5–7/group). , P < 0.05 vs. saline. D, multiple myeloma cells adjacent to osteocytes in bone detected by hematoxylin and eosin (H&E). Black arrows, osteocytes; yellow arrow, osteoclasts on the bone surface. E, osteocytes in direct contact with multiple myeloma tumors detected by acid etching-SEM. Areas indicated by boxes a and b are magniﬁed on the right. Arrows, osteocytic cytoplasmic projections in contact with the BM compartment.

transcription. Cells expressing either NICD1 or NICD2 exhibited contribute to osteocyte apoptosis at later time points. In support increased apoptosis compared with vector controls (Fig. 3A). of this notion, levels of TNFa, a recognized inducer of osteoblast Moreover, MLO-A5 cells cultured on plates coated with the Notch and osteocyte apoptosis (31), increased 4-fold from 4 to 48 hours ligand DLL1 expressed higher levels of Hes1, confirming Notch in CM from JJN3 cell cultures (Fig. 3D), and addition of TNFa activation, and had increased cell death (2- to 5-fold) compared increased MLO-A5 cell death (Supplementary Fig. S1G). Consis- with control cells cultured on IgG2 (1%–2% vs. 5%–9% in control tent with a role for multiple myeloma–secreted TNFa in multiple vs. DLL1-treated cells). DLL1-induced death was completely myeloma–induced osteocyte apoptosis, the decrease in osteocyte inhibited by the Notch inhibitor GSIXX or by DEVD (Fig. 3B), viability induced by JJN3-CM was blocked by DEVD or by a indicating that Notch activation is sufficient to trigger osteocyte neutralizing anti-human TNFa antibody, but not by GSIXX (Fig. apoptosis. Consistent with this observation, GSIXX completely 3D). Moreover, GSIXX or anti-TNFa only partially inhibited cell blocked osteocytic cell death induced by direct coculture with death, whereas the combination of both completely blocked the þ JJN3 cells or CD138 cells from a multiple myeloma patient at 8 increase in osteocytic cell death induced by direct coculture of or 24 hours (Fig. 3C). In contrast, GSIXX only partially prevented MLO-A5 cells with JJN3 cells for 48 hours (Fig. 3E). These findings MLO-A5 cell death measured at 48 hours, suggesting that factors suggest that multiple myeloma cells induce osteocyte apoptosis secreted by multiple myeloma cells accumulated during culture by activating Notch and TNFa signaling pathways in osteocytes.

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Figure 2. Multiple myeloma–induced osteocyte apoptosis is triggered by activation of Notch signaling in osteocytes and is sustained by multiple myeloma–derived TNFa. A, cell death, measured by trypan blue uptake, and apoptosis (24 hours), measured by nuclear morphology, were quantiﬁed in MLO-A5 cells cocultured in þ direct contact with JJN3 multiple myeloma cells or CD138 cells isolated from six multiple myeloma patients, with or without the caspase inhibitor DEVD for 24 hours. B, MLO-A5 cell death or apoptosis (24 hours) was quantiﬁed in cocultures in indirect contact with JJN3 cells, cultured with 48-hour CM collected from JJN3 cultured alone, or 48-hour CM from primary CD138þ cells from a multiple myeloma patient with or without the caspase inhibitor DEVD. Representative experiments out of three (n ¼ 3–8) are shown. C and D, Hey1, Notch1, 2, 3, and 4 expression in MLO-A5 cells cocultured for 4 hours in direct contact with JJN3 multiple myeloma cells or CD138þ cells from multiple myeloma patients. Data from one-well per condition are reported for patient #1; 3 wells per condition are reported for the other patients. E, percentage of cortical osteocytes stained for NOTCH3 (n ¼ 3–7/group). , P < 0.05 vs. saline. F, protein levels of NICD3, the activated form of the NOTCH3 receptor, in bone lysates obtained from saline- and JJN3-injected tibias.

Osteocyte apoptosis increases the osteoclastogenic potential of myeloma cells, suggesting that soluble factors secreted by osteocytes and stimulates osteoclast precursor recruitment multiple myeloma cells were responsible. Similar results were We then investigated the consequences of osteocyte apo- obtained with other multiple myeloma and osteocyte-like cell ptosis on the osteoclastogenic potential of osteocytes. MLO- lines (Supplementary Fig. S2A). Among several cytokines A5 cells cocultured with JJN3 multiple myeloma cells exhib- secreted by multiple myeloma cells known to upregulate ited increased Rankl expression at the mRNA and protein Rankl (32), only TNFa increased Rankl mRNA in MLO-A5 þ levels (Fig. 4A). Further, CD138 cells from 3 of 4 multiple cells after 4 hours (Supplementary Fig. S2B) or 24 hours. myeloma patients also increased Rankl transcripts in MLO-A5 Moreover, anti-TNFa signiﬁcantly blunted the increased Rankl cells. Like apoptosis, Rankl expression in osteocytes was mRNA levels in MLO-A5 cells treated with CM from JJN3 increased by either direct or indirect contact with multiple cells (Fig. 4B). Inhibition of osteocyte apoptosis by DEVD

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Figure 3. Activation of Notch signaling induces osteocyte apoptosis. A, apoptosis was measured by nuclear morphology in MLO-A5 cells cotransfected with nGFP and vectors overexpressing NICD1 or NICD2 and quantified after 24 hours. B, cell death measured by trypan blue uptake and Hes1 expression (qPCR) were quantified in MLO-A5 after 24-hour culture on DLL1-coated plates with or without GSIXX or DEVD. C, cell death of MLO-A5 cells cocultured in direct contact with JJN3 cells or CD138þ from a multiple myeloma patient with or without the Notch inhibitor GSIXX or the caspase inhibitor DEVD. D, TNFa protein levels secreted to the media by JJN3 multiple myeloma cells. MLO-A5 cell death quantified in cocultures in indirect contact with JJN3 cells, with or without DEVD, anti-TNFa (aTNFa), or GSIXX, as indicated. E, cell death of MLO-A5 quantified in cocultures in direct contact with JJN3 cells, with or without DEVD, GSIXX, anti-TNFa, or a combination of GSIXX and anti-TNFa. Representative experiments out of three (n ¼ 3–8) are shown. , P < 0.05 vs. MLO-A5 cultured alone (veh), vs. vector-transfected cells (A), or vs. cells plated on IgG2-coated plates (B).

similarly reduced Rankl levels to those observed with anti- Multiple myeloma cells upregulate Sost expression in TNFa. osteocytes, decrease Wnt signaling, and inhibit osteoblast We next determined the effect of osteocyte apoptosis on oste- differentiation oclast precursor recruitment. CM from MLO-A5 cells cultured Consistent with the increased sclerostin expression in osteo- alone signiﬁcantly increased osteoclast precursor migration com- cytes in the in vivo multiple myeloma mouse model, direct pared with control media, whereas CM from JJN3 cells did not coculture with JJN3 multiple myeloma cells upregulated Sost (Fig. 4C). CM from JJN3 and MLO-A5 cells cocultured in direct mRNA expression in MLO-A5 cells by 3-fold as early as 4 hours contact enhanced osteoclast precursor chemotaxis by 50% com- (Fig. 5A), which remained elevated up to 24 hours (Supplemen- pared with CM from MLO-A5 cultured alone. This effect was tary Fig. S2C). Simultaneously with Sost upregulation, the expres- inhibited by blocking osteocyte apoptosis with DEVD or by the sion of osteoprotegerin (Opg), a Wnt target gene, was decreased by combination of GSIXX and anti-TNFa. Taken together, these 50%, both at mRNA (Fig. 5A) and protein level (Supplementary results suggest that multiple myeloma–induced osteocyte apo- Fig. S2D). Similarly, the mRNA levels of the other Wnt target genes ptosis, even when modest, is sufﬁcient to increase osteocytic Rankl Axin2 and Smad family member 6 (Smad6; refs. 25, 33, 34) were expression and potentiate osteocyte-mediated recruitment of also decreased. In contrast, Sost/Wnt target gene expression was osteoclast precursors. not altered when multiple myeloma and MLO-A5 cells were not in

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Figure 4. Osteocyte apoptosis induced by multiple myeloma increases the osteoclastogenic potential of osteocytes. A and B, Rankl gene expression in MLO-A5 cells cocultured in direct or indirect contact with JJN3 þ cells (4 hours) or primary CD138 cells from multiple myeloma patients (24 hours), with or without anti-TNFa or DEVD (24 hours). C, osteoclast precursor migration induced by CM collected from JJN3 cells cultured alone, MLO-A5 cultured alone, or MLO-A5 cocultured with JJN3 cells in direct contact for 48 hours, with or without DEVD or a combination of GSIXX and anti-TNFa (aTNFa). Representative experiments out of two (n ¼ 3–4) are shown. , P < 0.05 vs. MLO-A5 cells cultured alone (veh), vs. osteoclast precursors cultured in the presence of CM from MLO-A5 cells cultured alone (C).

direct cell-to-cell contact. Sost upregulation induced by multiple cells cultured alone (Fig. 6A). Similar upregulation in HES1 þ myeloma cells was also recapitulated in a three-dimensional ex expression was found in CD138 cells from 3 of 4 multiple vivo bone organ model containing authentic osteocytes (Fig. 5B). myeloma patients examined (Fig. 6A and E) and in 5TGM1 cells These ﬁndings suggest that interactions between osteocytes and cocultured in direct contact with MLO-Y4 cells (Supplementary multiple myeloma cells upregulate the expression of Sost in Fig. S3A). GSIXX blocked the osteocyte-induced upregulation of osteocytes, which in turn decreases Wnt signaling. Hes1 and Hey1 in 5TGM1 cells (Fig. 6B). These results indicate that We next examined the consequences of multiple myeloma– osteocytes activate Notch signaling in multiple myeloma cells. induced upregulation of osteocytic Sost/sclerostinonosteo- Coculture of 5TGM1 cells in direct contact with MLO-A5 cells blast differentiation. Wnt/b catenin signaling is critical for markedly increased the proliferation of 5TGM1 cells in a time- osteoblast differentiation, and this pathway is tightly regulated dependent manner (Fig. 6C), whereas no changes were observed by prereceptor antagonists, including the potent osteoblast when cells were cocultured without direct contact (Supplemen- inhibitor sclerostin secreted by osteocytes (35). CM from tary Fig. S3B). Further, inhibition of Notch signaling with GSIXX MLO-A5 cells cocultured with JJN3 multiple myeloma cells in prevented in a dose-dependent manner the increased prolifera- þ direct contact markedly decreased the expression of the oste- tion of 5TGM1 cells and CD138 cell from a multiple myeloma oblast markers alkaline phosphatase (Alp), collagen 1a (Col1a), patient induced by MLO-A5 cells (Fig. 6C and E). Higher doses of runt related transcription factor 2 (Runx2), and bone gamma GSIXX also inhibited proliferation of 5TGM1 cells cultured alone carboxyglutamate protein (Bglap) in OB-6 osteoblastic cells at 48 hours, and all doses were inhibitory at 72 hours of culture cultured under osteogenic conditions (Fig. 5C). Expression of (Fig. 6C). the Wnt target genes Axin2 and Smad6 was also signiﬁcantly Osteocyte-like MLO-A5 cells did not activate Notch signaling in decreased by CM from MLO-A5-JJN3 cocultures. The expression all multiple myeloma cells tested. Thus, MLO-A5 cells did not of these genes was unchanged by treatment with CM from increase Notch target gene expression or stimulate multiple mye- þ MLO-A5 or JJN3 cells cultured separately. loma cell proliferation in 1 of 4 CD138 cell preparations from Taken together, these results demonstrate that direct interac- multiple myeloma patients (Fig. 6F), or in MM1.s or JJN3 mul- tions between osteocytes and multiple myeloma cells increase tiple myeloma (Supplementary Fig. S3C). We next examined expression of Sost/sclerostin in osteocytes, decrease Wnt signa- whether differences in proliferative responses of multiple mye- ling/bcatenin, and inhibit osteoblast differentiation. loma cells to osteocytes were due to differences in their Notch receptor repertoire. 5TGM1 cells, which readily respond to osteo- Osteocytes activate Notch signaling to increase multiple cytic cell interactions by activating Notch, express higher Notch3 myeloma cell proliferation and alter the Notch receptor levels (20-fold), and lower Notch1 and 2 compared with JJN3 cells repertoire in multiple myeloma cells in which osteocytic interactions do not activate Notch (Supple- Because Notch signaling can be bidirectional, we next exam- mentary Fig. S3D). None of the multiple myeloma cell lines ined if osteocytes modulate Notch signaling in multiple myeloma expressed detectable levels of Notch4 when cultured alone. These cells. Coculture of 5TGM1 cells in direct contact with MLO-A5 results show that different multiple myeloma cells express differ- cells for 4 hours increased Hes1 and Hey1 expression by 2- to 4- ent levels of Notch receptors and suggest that osteocytes may fold in multiple myeloma cells compared with multiple myeloma activate Notch signaling in multiple myeloma cells through

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Figure 5. Multiple myeloma increases Sost expression in osteocytes, which decreases Wnt signaling and osteoblasts differentiation. A, Sost, Opg, and Wnt target genes mRNA levels in MLO-A5 cells cocultured in direct or indirect contact with JJN3 cells (4 hours). B, Sost expression in authentic osteocytes cocultured with 5TGM1 multiple myeloma cells in ex vivo bone organ cultures (48 hours). C, osteoblast marker expression (Alp, Col1a, Runx2,andBglap) and Wnt target genes (Axin2 and Smad6) in osteoblasts cultured in OM with or without 50% CM from JJN3 cells cultured alone, MLO-A5 cultured alone, or MLO-A5 cocultured with JJN3 cells in direct contact for 48 hours. Representative experiments out of two (n ¼ 3–6) are shown. , P < 0.05 vs. MLO-A5 cells cultured alone (A), vs. authentic osteocytes (bone; B), vs. osteoblast cultured with OM and without CM (C).

Notch3, leading to increased multiple myeloma cell proliferation. osteocytes physically interact with multiple myeloma tumors in Moreover, osteocytic interactions rapidly upregulated Notch3 vivo, which increases sclerostin and RANKL production by osteo- expression in 5TGM1 cells and induced the expression of Notch4, cytes and reduces osteocyte viability. Second, the reduced viability which is undetectable in multiple myeloma cells cultured alone of osteocytes is due to apoptosis triggered by multiple myeloma (Fig. 6D). Further, culture of 5TGM1 multiple myeloma cells with cell–mediated activation of Notch signaling and sustained by authentic osteocytes in ex vivo bone organ cultures activated Notch multiple myeloma–derived TNFa. Third, elevated osteocyte apo- signaling and increased the expression of Notch3 and 4 without ptosis increases osteocytic Rankl expression and enhances the þ altering Notch1 or 2 mRNA levels (Fig. 6G). Similarly, CD138 ability of osteocytes to attract osteoclast precursors. Fourth, the cells from a multiple myeloma patient that exhibit Notch acti- increase in Sost/sclerostin decreases Wnt signaling and inhibits vation induced by osteocytes also displayed upregulation of osteoblast differentiation. Fifth, osteocytes induce reciprocal acti- NOTCH3 (Fig. 6E), whereas no changes were found in other vation of Notch signaling in multiple myeloma cells, which in Notch receptors. In contrast, no changes in the expression of turn enhances multiple myeloma cell proliferation and alters the þ NOTCH3 were observed in CD138 cells from a multiple mye- Notch receptor repertoire on multiple myeloma cells. Important- loma patient that did not show activation of Notch signaling after ly, these results were validated in in vivo and in vitro, as well as in a contact with MLO-A5 cells (Fig. 6F). MLO-A5 cells also upregu- novel ex vivo model, using several multiple myeloma cell lines and þ lated Notch1 and 2 in 5TGM1 cells at later time points (Fig. 6D). CD138 cells from multiple myeloma patients. Taken together, These results demonstrate that osteocytes change the Notch these ﬁndings demonstrate that interactions between multiple receptor repertoire expressed by multiple myeloma cells. Thus, myeloma cells and osteocytes within the bone/BM microenvi- the capacity of osteocytes to alter the Notch receptor repertoire on ronment generate a permissive niche for multiple myeloma cell multiple myeloma cells may regulate osteocyte enhancement of growth and bone destruction (Fig. 7). Thus, targeting osteocytes multiple myeloma cell growth. and their derived factors might represent a novel approach to treat multiple myeloma bone disease. Consistent with our ﬁndings showing that osteocytes induce Discussion multiple myeloma cell growth by activating Notch signaling, Osteocytes play a central role in bone homeostasis by regulat- several studies have shown that dysregulation of Notch signaling ing osteoclast and osteoblast activity, and premature death of increases multiple myeloma growth (36–38), and paracrine osteocytes leads to targeted bone resorption during skeletal disuse Notch activation mediated by BM stromal cells increases multiple and estrogen loss (6). We report that osteocytes also contribute to myeloma cell proliferation, through NOTCH1 and 2 signaling a microenvironment favorable for the progression of multiple (30, 39, 40). However, NOTCH3 and 4 appear to mediate the myeloma and its associated bone disease. First, we show that osteocytic effects, suggesting that regulation of Notch activation in

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Role of Osteocytes in Multiple Myeloma

Figure 6. Osteocytes activate Notch signaling, regulate Notch receptor expression, and increase proliferation in multiple myeloma cells. A and B, Hes1 and Hey1 gene expression in 5TGM1 multiple myeloma cells or CD138þ cells from three different multiple myeloma patients cocultured in direct contact with MLO-A5 cells, with or without GSIXX. Data from one well per condition are reported for patient #1; 3 wells per condition are reported for the other patients. C, cell growth of 5TGM1 cells measured as RFU, cultured alone or cocultured in direct contact with MLO-A5 cells, with or without GSIXX. D, time course of Notch receptors expression in 5TGM1 cells cocultured with MLO-A5 cells for 4, 24, 48, and 72 hours. For Notch1, 2,and3 expression, fold changes were calculated vs. 5TGM1 cells cultured alone. For Notch4 expression, fold change was calculated vs. Notch4 expression in 5TGM1 cells cocultured with MLO-A5 for 4 hours. Cell proliferation (number of viable cells), Hey1,andNotch1, 2, 3,and4 gene expression in CD138þ cells from a multiple myeloma patient in which MLO-A5 cells induce (E) or not (F) Notch activation. G, Hey1 and Notch1, 2, 3,and4 gene expression in 5TGM1 cells cocultured with authentic osteocytes in ex vivo bone organ cultures. A representative experiment of two (n ¼ 4) is shown. , P < 0.05 vs. multiple myeloma cells cultured alone (veh), vs. multiple myeloma cultured with MLO-A5 for 4 hours (D; Notch4). multiple myeloma is mediated by complex paracrine interac- impact both the homotypic and heterotypic cell-to-cell interactions with different cell types within the bone/BM microenviron- tions of multiple myeloma cells. Although the speciﬁc roles of ment. Further, the changes in the Notch receptor repertoire on NOTCH3 and 4 in multiple myeloma are not fully understood, multiple myeloma cells induced by osteocytic interactions could selective Notch receptor targeting may allow control of multiple

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Figure 7. Interactions between multiple myeloma (MM) cells and osteocytes generate a microenvironment conducive to increased tumor growth and bone destruction. Cell-to-cell contact activates bidirectional Notch signaling in osteocytes and multiple myeloma cells and triggers osteocyte apoptosis that is maintained by multiple myeloma–secreted TNFa. Apoptosis enhances the osteoclastogenic potential of osteocytes by increasing osteoclast precursor recruitment and osteocytic RANKL expression. Notch signaling in multiple myeloma cells increases proliferation and upregulates Notch1, 2, and 3 in patient multiple myeloma cells. Multiple myeloma cells increase Sost/sclerostin expression in osteocytes, which decreases Wnt signaling and inhibits osteoblast differentiation.

þ myeloma cell growth and its associated bone disease. Future of neural progenitor cells (42), CD34 hematopoietic stem/ studies to investigate this possibility and to identify relevant progenitor cells (43), and osteocytes (this report). Similarly, Notch ligands on osteocytes are warranted. activation of canonical Wnt signaling inhibits apoptosis in The increased prevalence of osteocyte apoptosis observed in different cells of the osteoblastic lineage (31, 44), whereas it our in vivo model is consistent with a previous report in bone increases apoptosis in hematopoietic stem/progenitor cells biopsies from multiple myeloma patients (17), although the (45) or in melanoma cells (46). These findings show that the mechanism of this phenomenon was not addressed. We show same signaling pathway can elicit different biologic responses here that osteocyte apoptosis induced by multiple myeloma depending on the ligands/signals present in a particular micro- cells is triggered by activation of Notch signaling followed by environment, the receptor repertoire exhibited by the target induction of the caspase cascade, and maintained by multiple cell, and the intracellular machinery that integrates extracellular myeloma–cell derived TNFa. However, Giuliani and colleagues cues and transduces them into biologic outcomes. were unable to block osteocyte apoptosis by neutralizing TNFa Osteocyte-derived interleukin 11 has been shown to con- (17). This discrepancy, together with their inability to detect tribute to osteoclast differentiation in multiple myeloma (17). changes in osteocytic RANKL, could be explained by the use of We show that apoptotic osteocytes attract osteoclast precursors cells lines at different stages of differentiation of the osteoblas- more potently than multiple myeloma cells in our in vitro tic lineage. We used the late osteoblast/early osteocyte-like system. Further, although relatively modest, the increased MLO-A5 cell line that expresses the recognized osteocytic genes osteocyte apoptosis was sufficient to upregulate osteocyte Sost/sclerostin and Rankl, whereas the HOB-01 preosteocytic Rankl.Thesefindings suggest that similar to other pathologic cells used in the Giuliani study do not. Moreover, we validated conditions, multiple myeloma–induced osteocyte apoptosis our in vivo observations and mechanistic studies using MLO-Y4 plays a central role in osteoclast recruitment and may initiate cells, an additional osteocytic cell line widely used as an and/or sustain bone resorption within the focal lesions osteocyte model, and confirmed our findings in authentic observed in multiple myeloma patients. Further studies are osteocytes using ex vivo bone organ cultures. Our results dem- warranted to determine whether osteocyte apoptosis leads to onstrate that murine and human multiple myeloma cell lines osteoclast differentiation in multiple myeloma and to identify þ and CD138 from multiple myeloma patients act as Notch the molecular mediators. signalers in osteocytes that concurrently with multiple myelo- Sclerostin levels are elevated in the sera of multiple myeloma ma–secreted TNFa induce osteocyte apoptosis. patients and correlate with reduced osteoblast function and poor Although our current findings demonstrating different out- patient survival (47). However, the source of sclerostin remains þ comes of Notch activation in multiple myeloma cells (prolif- unclear. CD138 cells from multiple myeloma patients were eration) versus in osteocytes (apoptosis) may appear counter- reported to express sclerostin (48), but we could not detect SOST þ intuitive, they are consistent with earlier studies showing the mRNA in any patient CD138 cells studied. In contrast, our in vitro cell type– and context-dependent nature of Notch signaling. and in vivo models demonstrate that Sost/sclerostin expression is Thus, whereas activation of Notch signaling protects several elevated in osteocytes. Further, Sost/sclerostin upregulation cancer cell types from apoptosis (36, 41), it induces apoptosis in osteocytes was associated with decreased Wnt signaling in

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Role of Osteocytes in Multiple Myeloma

osteoblasts and reduced osteoblast differentiation. In addition, Disclosure of Potential Conflicts of Interest the expression of Wnt target genes, including OPG, was reduced, No potential conflicts of interest were disclosed. thereby further increasing the Rankl/Opg ratio. These results suggest that osteocytes contribute to the generation of a multiple Authors' Contributions myeloma microenvironment with high sclerostin concentrations, Conception and design: J. Delgado-Calle, K.S. Mohammad, L.I. Plotkin, affecting both bone formation and bone resorption. G.D. Roodman, T. Bellido Physical interactions between multiple myeloma cells Development of methodology: J. Delgado-Calle, J.M. Chirgwin, L.I. Plotkin, and different cells in the bone microenvironment, including T. Bellido Acquisition of data (provided animals, acquired and managed patients, stromal cells, osteoclasts, and immune cells, were shown to be provided facilities, etc.): J. Delgado-Calle, J. Anderson, M.D. Cregor, M. Hiasa, important for tumor proliferation and survival (49). Our T. Yoneda, K.S. Mohammad, T. Bellido results suggest that osteocytes also contribute to multiple Analysis and interpretation of data (e.g., statistical analysis, biostatistics, myeloma cell growth and multiple myeloma–induced bone computational analysis): J. Delgado-Calle, J. Anderson, M. Hiasa, J.M. Chirg- disease by stimulating osteoclast recruitment and inhibiting win, L.I. Plotkin, T. Bellido bone formation via direct and indirect contact with multiple Writing, review, and/or revision of the manuscript: J. Delgado-Calle, J. Anderson, M. Hiasa, J.M. Chirgwin, N. Carlesso, K.S. Mohammad, L.I. Plotkin, myeloma cells. Because osteocytes comprise more than 95% of T. Bellido the bone cells, they should be major contributors to generating Administrative, technical, or material support (i.e., reporting or organizing a microenvironment favorable for multiple myeloma progres- data, constructing databases): J. Delgado-Calle, L.I. Plotkin, T. Bellido sion. However, the relative contribution of osteocytes versus Study supervision: G.D. Roodman, T. Bellido other cell types in the bone/BM microenvironment to multiple Other (provided advises and expertise on the experimental design of the myeloma disease remains to be determined. experiments addressing Notch signaling): N. Carlesso Our study identified pathways activated by interactions between multiple myeloma cells and osteocytes that provide Acknowledgments potential new ways to inhibit multiple myeloma tumor growth The authors thank Kevin McAndrews, Hannah M. Davis, Keith Condon, Amy Sato, Dan Zhou, Doug Tompkins, and Ning Ma for assistance in tissue collection and bone disease. Prevention of osteocyte apoptosis might sup- and Caroline Miller (Electron Microscopy Center, Indiana University School of press initiation of bone resorption by inhibiting osteoclast recruit- Medicine) for assistance with SEM. ment as well as improving the bone fragility syndrome associated with multiple myeloma and with some of the current antitumor Grant Support drugs (e.g., glucocorticoids that also increase osteocyte apopto- This work was supported by the NIH grants (Indiana-CTSI P30, sis). Pharmacologic inhibition of Notch signaling can inhibit 1R21CA179017-02, and R01AR059679 to G.D. Roodman; R01AR059357, multiple myeloma cell growth (50, 51) and could provide addi- R01 DK076007, and S10-RR023710 to T. Bellido), the Veteran's Administration tional benefits by inhibiting osteocyte apoptosis, either alone or (Merit Review to T. Bellido and G.D. Roodman), and the IBMS Gideon and Sevgi in combination with TNFa signaling blockade. Our findings Rodan Fellowship (J. Delgado-Calle), and funds from the CTSI at Indiana demonstrate that osteocytes regulate the expression and repertoire University. The costs of publication of this article were defrayed in part by the payment of of Notch receptors on multiple myeloma cells, supporting devel- fi page charges. This article must therefore be hereby marked advertisement in opment of therapeutic approaches that block speci c Notch accordance with 18 U.S.C. Section 1734 solely to indicate this fact. receptors. This approach might be an effective alternative to overcome the side effects associated with generalized Notch Received June 23, 2015; revised November 18, 2015; accepted December 14, inhibition (36). 2015; published OnlineFirst February 1, 2016.

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OF12 Cancer Res; 76(5) March 1, 2016 Cancer Research

Bidirectional Notch Signaling and Osteocyte-Derived Factors in the Bone Marrow Microenvironment Promote Tumor Cell Proliferation and Bone Destruction in Multiple Myeloma

Jesus Delgado-Calle, Judith Anderson, Meloney D. Cregor, et al.

Cancer Res Published OnlineFirst February 1, 2016.

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