MM Bone Disease Research Articles

Mechanisms of Altered Bone Remodeling in Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy of plasma cells proliferating in the bone marrow and it is associated with the osteolytic bone disease. About 70% of myeloma patients show the bone disease at diagnosis and 15–20% have pathologic fractures. The osteolytic disease is due to the altered bone remodeling with increased activity of osteoclasts, the bone resorbing cells, and the reduced activity of osteoblasts, the bone forming cells. In the bone marrow microenvironment, the deregulated activity of bone cells is due to the malignant plasma cells through the release of soluble molecules or cell-cell contact. Recently, the key role of osteocytes, the orchestrators of bone remodeling, and the adipocytes has been highlighted in MM-bone disease. In this review, we overview the mechanisms leading to increased bone resorption as well as suppressed osteoblast formation and activity in MM-bone disease, thus highlighting cytokines that will represent new therapeutic targets to improve bone health in MM-bone disease.

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

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

IgM Multiple Myeloma Presenting As Hyperviscosity Syndrome with Isolated Skin Manifestation

Background:Immunoglobulin M-producing multiple myeloma (IgM MM) accounts for less than 1% of all myeloma cases. Because the disorder is rare,its clinical characteristics and long-term prognosis remain unknown. Hyperviscosity occurs from pathologic changes of either cellular or protein fractions of the blood such as are found in MM (particularly IgA and IgG3) and other diseases.Case PresentationA 65-year-old male smoker presented to emergency room (ER) with headache, dizziness, shortness of breath, symmetrical finger pain, nose bleeds, and blood in stool. On lab work hemoglobin was 7.9 gm/dl. Review of the peripheral blood smear showed atypical lymphocytes. CT chest showed left axillary and supraclavicular lymphdenopathy. Serum protein was 13 gm/dl, Serum protein electrophoresis showed a monoclonal IgM kappa gammopathy with UIEP confirming the bence jones proteinuria. A bone marrow biopsy showed about 80% cellularity with diffuse and solid neoplastic plasma cell infiltrates that were CD138+, CD20- and CD3-. Flow cytometric analysis of the bone marrow aspirate demonstrated a small fraction of clonal plasma cell population that were CD38+, CD138+, CD45-, and CD56-, Fluorescent in Situ Hybridization confirmed neoplastic plasma cells with kappa light chain restriction, and cyclinD1-. On examination, the patient had a erythematous skin changes and edematous bilateral symmetrical finger findings.Discussion:MM with IgM gammaglobulin is a rather distinct subtype of MM displaying clinical and pathologic features of both MM and WM. The clinical presentation of WM is similar to that of MM except that organomegaly is common in WM but is uncommon in MM and lytic bone disease and renal disease are uncommon in WM but are common in MM.The criteria for diagnosis of IgM myeloma (and its differentiation from WM) requires the presence of either lytic bone lesions and/or evidence of the t(11;14) translocation. It is also characterized by negative CD20, D56 and CD117 phenotype. All the reported cases of IgM myeloma had been treated with melphalan and prednisolone with suboptimal results. Some patients were treated with VAD (vincristine, adriamycin and dexamethasone) regimen, others with CTD protocol. The results were similar to the MP regimen. Radiotherapy was mainly used as palliative measure.Conclusion:Our case suggests that IgM multiple myeloma can presents as hyperviscosity syndrome with isolated skin manifestation and may have unique clinical characteristics. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry for Minimal Residual Disease

60% clonal plasma cells in the marrow, serum free light chain (FLC) ratio 100 (provided involved FLC level 100), and more than 1 focal lesion ( 5mm in size) on magnetic resonance imaging (MRI) or whole body or spine/pelvis (Table1). Each of these biomarkers has been validated in 2 or more independent studies to have a very high risk (w80%) of progression to symptomatic disease within 2 years. In addition, the update allows modern imaging methods including computed tomography (CT) and positron emission tomography-CT to diagnose MM bone disease. The new diagnostic criteria provide 3 important clarifications on requirements for bone and renal disease in the diagnosis of MM. First, the presence of osteoporosis, vertebral compression fractures, or bone densitometric changes in the absence of lytic lesions is not considered sufficient evidence of myeloma bone disease. Second, only suspected or proven light chain cast nephropathy is considered as meeting the renal component of the CRAB criteria. Renal disorders associated with M proteins such as light chain deposition disease, membranoproliferative glomerulonephritis, and AL amyloidosis, are considered unique diseases and not MM. Third, an estimated GFR less than 40 ml/ minute is preferred to the serum creatinine concentration for purposes of fulfilling the CRAB criteria. Table 1 International Myeloma Working Group Diagnostic Criteria for Smoldering Multiple Myeloma and Multiple Myeloma Disorder Disease Definition Smoldering multiple myeloma Both criteria must be met: Serum monoclonal protein (IgG or IgA) 3gm/dL, or urinary monoclonal protein 500 mg per 24h and/or clonal bone marrow plasma cells 10-60% Absence of myeloma defining events or amyloidosis Multiple Myeloma Both criteria must be met: Clonal bone marrow plasma cells 10% or biopsy-proven bony or extramedullary plasmacytoma Any one or more of the following myeloma defining events (MDE): BCRAB features felt attributable to the underlying plasma cell proliferative disorder, specifically:

LIGHT/TNFSF14 increases osteoclastogenesis and decreases osteoblastogenesis in multiple myeloma-bone disease.

LIGHT, a TNF superfamily member, is involved in T-cell homeostasis and erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT has a role in Multiple Myeloma (MM)-bone disease. We found that LIGHT was overproduced by CD14+ monocytes, CD8+ T-cells and neutrophils of peripheral blood and bone marrow (BM) from MM-bone disease patients. We also found that LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis. In cultures from healthy-donors, LIGHT induced osteoclastogenesis in RANKL-dependent and -independent manners. In the presence of a sub-optimal RANKL concentration, LIGHT and RANKL synergically stimulated osteoclast formation, through the phosphorylation of Akt, NFκB and JNK pathways. In cultures of BM samples from patients with bone disease, LIGHT inhibited the formation of CFU-F and CFU-OB as well as the expression of osteoblastic markers including collagen-I, osteocalcin and bone sialoprotein-II. LIGHT indirectly inhibited osteoblastogenesis in part through sclerostin expressed by monocytes. In conclusion, our findings for the first time provide evidence for a role of LIGHT in MM-bone disease development.

Notch- and TNFα-Activated Signaling Pathways Mediate Osteocyte Apoptosis Triggered By Multiple Myeloma Cells

Osteocytes comprise 95% of all bone cells and are central regulators of bone homeostasis and skeletal integrity. However the role of osteocytes in MM bone disease is unknown. We have previously shown that interactions with multiple myeloma cells have a profound effect on osteocytic gene expression, increasing Sost and RANKL transcripts and decreasing OPG. More recently, we and others have shown that myeloma cells increase the prevalence of osteocyte apoptosis, which might contribute to MM induced bone disease by increasing resorption in specific areas of bone. However, the mechanisms responsible for and the potential for targeting osteocyte apoptosis in myeloma are unknown. Osteocytes extensively communicate with each other and with cells on the bone surface and in the marrow, through cytoplasmic connections that run within canaliculi, which allows direct cell-to-cell contact and the distribution of secreted molecules among all bone and marrow cells including myeloma cells. Therefore, to explore the mechanism underlying osteocyte apoptosis induced by myeloma cells we used a co-culture system that allows both cell-to-cell contact and exchange of soluble factors between osteocytic MLO-A5 cells and the human JJN3 MM cell line or primary CD138+ cells isolated from MM patients. Osteocyte apoptosis was quantified by trypan blue uptake and chromatin condensation/nuclear fragmentation in the absence or presence of the caspase3 specific inhibitor DEVD. We found that osteocyte apoptosis was increased 2-3 fold when osteocytes were co-cultured with JJN3 cells compared to osteocytes cultured alone. Apoptosis occurred within 8h of co-culture and gradually increased for up to 48h. Osteocyte apoptosis was completely inhibited by DEVD. Importantly, co-culture with primary CD138+ MM cells from 5 different patients also increased osteocyte apoptosis, which was also blocked by DEVD. We next determined if direct cell-to-cell contact was required for MM cells to induce osteocyte apoptosis and whether Notch signaling, a signaling pathway with profound effects on the skeleton activated by such interactions, was involved. We found that osteocytes co-cultured with JJN3 cells exhibited 3-7 fold higher levels of expression of the Notch target genes Hes1 and Hey1, detected as early as 4h and maintained up to 48h of co-culture. The pharmacological specific Notch inhibitor GSIXX completely blocked osteocyte apoptosis induced by either JJN3 cells or primary CD138+ MM cells measured at 8h and 24h. Moreover, cultures of osteocytes grown on plates coated with the Notch ligand Delta 1 fused to IgG2 exhibited 2-5 times higher levels of apoptosis compared to osteocytes cultured on IgG2 control, and this effect was inhibited by GSIXX. In addition, overexpression of the Notch intracellular domains 1 or 2, known to activate Notch signaling, increased osteocyte apoptosis by 2 fold. These findings demonstrate that rapid activation Notch signaling in osteocytes triggered by direct cell-to-cell contact with myeloma cells induces osteocyte apoptosis. Interestingly, Notch inhibition by GSIXX only partially prevented osteocyte apoptosis induced by JJN3 cells measured at 48h, suggesting the involvement of other mechanisms. To further investigate this finding, we measured soluble levels of TNFα in JJN3 cultures since TNFα is a recognized inducer of osteocyte apoptosis. The levels of TNFα secreted by JJN3 cells increased 5 fold (from 2 to 9 pg/ml) during 4 to 48h of culture. Conditioned medium (CM) from JJN3 cells cultured alone for 48h increased osteocyte apoptosis, and this effect was blocked by DEVD and by a neutralizing anti-human TNFα antibody, but not by GSIXX. Moreover, combination of GSIXX and anti-TNFα antibody completely inhibited osteocyte apoptosis induced by co-culture with JJN3 cells, while each agent added separately only partially inhibited osteocyte apoptosis measured at 48h. These results demonstrate that direct interactions with MM cells induces caspase3-dependent osteocyte apoptosis, triggered by rapid activation of Notch signaling through cell-cell contact and is maintained by accumulation of MM-derived TNFα. Our findings suggest both Notch and TNFα signaling pathways are potential targets to reverse or prevent myeloma induced osteocyte apoptosis. DisclosuresNo relevant conflicts of interest to declare.

The Role of LIGHT in Multiple Myeloma-Bone Disease

Background. The TNF superfamily member LIGHT, known to play a major role in T-cell homeostasis, has been reported in erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT is implicated in the mechanisms leading to Multiple Myeloma (MM)-bone disease.Methods. Peripheral blood (PB) and bone marrow (BM) aspirates were obtained from 40 patients (23M/17F, median age: 64 years), newly diagnosed as having symptomatic MM with or without bone disease, smoldering MM (sMM) or M.G.U.S. Bone disease assessment was performed by skeleton x-Ray, and spine and pelvis NMR or CT. The control group included PB and BM aspirates from 15 patients with non-neoplastic disease without any skeletal involvement, and PB from 25 healthy-donors matching for age and sex with the MM group. Patients and controls gave their written informed consent to the study, approved by Ethical Committee of University Hospital of Bari, and performed according to Declaration of Helsinki. By means of flow cytometry, western blotting, and real-time PCR, LIGHT expression was assessed in freshly purified CD14+ monocytes, CD2+ T-cells and neutrophils from PB and BM aspirates of patients and controls. Osteoclasts (OCs) were obtained from unfractionated PBMC cultures treated or not with an anti-LIGHT neutralizing monoclonal antibody (mAb). Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. In cultures from BM mononuclear cells (BMNCs), the formation of CFU-F and CFU-OB was evaluated in the presence or absence of anti-LIGHT neutralizing mAb. CFU-F and CFU-OB were identified with alkaline phosphatase (ALP) or Von Kossa staining, respectively. Further, in CFU-F and CFU-OB cultures, the expression of OB differentiation markers was analyzed by real-time PCR.Results. We found overexpression of LIGHT on CD14+ monocytes, CD8+ T-cells and neutrophils of PB and BM from MM-bone disease patients, in whom LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis, as we demonstrated by culture treatment with an anti-LIGHT antibody. Moreover, in cultures from healthy-donors, we found that LIGHT induced osteoclastogenesis in RANKL-dependent and –independent manners. In particular, in the presence of a sub-optimal RANKL concentration, LIGHT and RANKL showed synergic effects on osteoclast formation, associated to early and sustained activation of Akt, NFκB and JNK pathways. Otherwise in cultures of BM samples from patients without bone disease, LIGHT treatment inhibited the formation of CFU-F and CFU-OB, and the expression of osteoblastic markers such as collagen-I, osteocalcin and bone sialoprotein-II, supporting a LIGHT indirect inhibition of osteoblastogenesis, possibly and to some extent, through sclerostin expression by monocytes.Conclusions. Our findings, for the first time, provide evidence that LIGHT plays a role in MM-bone disease development by increasing osteoclastogenesis and decreasing osteoblastogenesis. DisclosuresNo relevant conflicts of interest to declare.

Zoledronic Acid Improves Overall Survival Compared To Pamidronate In Multiple Myeloma

BackgroundRecent practice guideline changes suggest all patients with MM should receive bisphosphonates, regardless of the presence of bone disease. Nitrogen-containing bisphosphonates, ZA and PAM, inhibit protein prenylation, a process crucial for osteoclast survival. This inhibition also interferes with tumor cell adhesion, angiogenesis, and proliferation suggesting potential for anti-tumor activity of bisphosphonates. Morgan et al showed increased OS with use of ZA versus clodronate in MM. This increase was independent of the effect on bone disease, supporting anti-tumor activity of ZA. ZA and PAM are the two approved bisphosphonates for MM in the United States. In in-vitro studies, ZA has increased potency compared to PAM, a finding that may result in increased apoptosis of tumor cells. A meta-analysis by Mhaskar et al demonstrated an association between higher bisphosphonate potency and improved OS in MM. In an effort to clarify the effect of ZA versus PAM on OS in MM, we evaluated outcomes in a large cohort of United States Veterans with MM. MethodsWe identified 1,018 patients with newly diagnosed MM in the Veterans Administration Cancer Registry between 2002 and 2009, who were treated with either PAM or ZA, but not both. Data was collected on age, co-morbidities, date of diagnosis and death, myeloma specific and supportive medications, and baseline lab data. Cox proportional-hazards was used to assess association between bisphosphonate use and OS while controlling for other prognostic factors. Propensity score analyses were performed to reduce confounding by indication, using the inverse probability weighting (IPW) approach of Cole and Hernan and propensity score matching. The covariates for each propensity model were the same as those in the main analysis. ResultsOf the 1,018 patients in the cohort, 383 received ZA and 635 received PAM. The median follow-up was 26.9 months. After adjustment using propensity score groups, baseline characteristics were well balanced between the groups (Table 1). Kaplan-Meier curves showing OS of patients receiving ZA versus PAM are shown in Figure 1. After controlling for age, weight, comorbidity score, era of diagnosis (before or after 2006), baseline lab characteristics, and treatment, OS was significantly improved with ZA compare to PAM (HR 0.84; 95% CI, 0.72-0.98). In both the IPW and propensity score matching analyses, ZA significantly improved OS in MM (HR 0.84; 95% CI, 0.72-0.97) and (HR 0.83; 95% CI, 0.69-0.99) respectively.Table 1Baseline CharacteristicsUnadjusted DataAdjusted with IPWPropensity Score MatchingPAMZAP-valuePAMZAP-valuePAMZAP-valueAge (mean)Male Sex (%)9896.10.077397.397.10.830496.696.61.0000Race (%)White and other74.57072.87370.571.6Black25.5300.116927.2270.938129.528.40.7397Mean Charlson score2.62.40.18512.62.50.37212.62.40.3503Transplant (%)15.6110.038513.713.80.959111.411.41.0000Melphalan_use (%)35.336.80.619936.235.50.811238.436.70.6404Lenalidamide_use(%)21.727.40.039423.423.90.853224.727.60.3910Thalidomide_use(%)51.352.70.664351.551.30.939651.152.80.6508Steroid Alone (%)22.716.20.012520.520.80.913319.017.10.4926Bortezomib_use(%)34.332.90.639733.732.90.783634.133.20.8109Hemoglobin (mean)10.710.70.744210.810.60.149310.910.70.2788Creatinine (mean)1.91.80.00121.81.90.53611.51.60.7944Albumin (mean)3.33.30.88033.33.20.65173.33.20.6348Calcium (mean)9.79.50.05819.79.60.56309.79.50.0962 [Display omitted] ConclusionIn this large, multicenter cohort study, ZA improved OS compared to PAM in patients with MM. The benefit persisted even after controlling for known patient and treatment related prognostic factors, as well as controlling for differences in baseline patient characteristics. Bisphosphonates play a key role in the treatment of MM for the prevention of MM bone disease. Evidence suggests that bisphosphonates may also have a direct anti-tumor effect in MM. Our study adds to this growing body of evidence and provides rationale for selecting ZA over PAM in most patients with MM. Disclosures:No relevant conflicts of interest to declare.

In Vitro Osteoclastogenesis and T-Cell RANKL Expression In Multiple Myeloma-Bone Disease At Diagnosis and In The Setting Of Frontline Treatment

IntroductionMultiple myeloma (MM)-bone disease occurs in 70% to 80% of patients at MM diagnosis, and up to 90% at relapse; skeletal related events cause high morbidity and mortality. MM-bone disease is mostly consisting of lytic lesions arising as a consequence of an unbalanced bone remodelling due to activation of osteoclasts (OCs), and inactivation of osteoblasts. Osteoclastogenesis may be under immunoregulation by T-cells through the production of receptor activator of NF-kB ligand (RANKL), that is the OC differentiating molecule. AimBy means of an in vitro osteoclastogenesis model derived from peripheral blood mononuclear cells (PBMCs) of patients with MM-lytic bone disease, we purposed to evaluate OC formation and T-cell RANKL expression at diagnosis, and in the setting of frontline therapy. MethodsPB was taken by venipuncture from 18 patients (7 men and 11 women), aged from 63 to 84, diagnosed as having symptomatic MM by International Myeloma Working Group (IMWG) criteria. In all of them, lytic lesions were demonstrated by skeleton standard radiography, and in some cases by nuclear magnetic resonance (NMR) too. On the basis of patients' age and fitness, initial standard therapy consisting of proteasome inhibitor (Bortezomib) associated with Melphalan and Prednisone (VMP) was administered for nine cycles. The therapy response was assessed according to IMWG criteria. The controls included PB collected from 20 healthy donors, matched for age and sex with the patients. Controls and patients gave their written informed consent to the study, that was approved by the local Ethical Committee and performed according to the Declaration of Helsinki. PB was taken from the patients at diagnosis, and following the fourth and the ninth VMP cycles, respectively. OCs were obtained from unfractionated PBMC cultures either stimulated or not with the exogenous pro-osteoclastogenic cytokines [macrophage-colony stimulating factor (M-CSF) and RANKL]. Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. Freshly isolated T-cells from patients' and controls' PBMCs were lysed and analyzed by Western Blotting to evaluate RANKL expression. ResultsSpontaneous osteoclastogenesis was demonstrated in the cultures derived from PBMCs isolated at diagnosis. With particular regard to the patients achieving a deep treatment response, at the end of the culture period we found that PBMCs formed few small-sized OCs, when cultured in the absence of M-CSF and RANKL. Moreover, fresh T-cells purified after four and nine VMP cycles expressed progressively lower RANKL levels than fresh T-cells purified at diagnosis. Otherwise the cultures derived from non-responsive patients' PBMCs, collected during and after treatment respectively, showed in vitro spontaneous formation of numerous and large OCs, overlapping the results at diagnosis. Fresh T-cells purified during or after treatment expressed comparable or even higher RANKL levels respect to diagnosis. ConclusionsThese findings show that in vitro spontaneous osteoclastogenesis and T-cell RANKL expression may be correlated with the response to treatment in patients with MM-lytic bone disease. However, further studies will be necessary to confirm these data, and to better translate them from a biologic to a clinical point of view. Disclosures:No relevant conflicts of interest to declare.

CK2 Kinase Inhibitors Display Anti-Myeloma Effects and Antagonize Osteoclast Activity in Models of Multiple Myeloma Bone Marrow Microenvironment

AbstractAbstract 444 Background.Multiple myeloma (MM) plasma cell growth in the bone marrow (BM) microenvironment is fueled by survival signals delivered by the surrounding non-malignant cells (stromal and other types) and through contacts with the extracelllar matrix. Interactions of MM cells with osteoclasts and osteoblasts generate a milieu, in which bone resorption and bone loss occur more rapidly than bone deposition. Novel agents, such as bortezomib and lenalidomide, which target the MM BM microenvironment, have shown unprecedented anti-myeloma efficacy in part due to their ability to somewhat revert these microenvironmental alterations. However, often resistance occurs also to novel drugs and the disease progresses. We have described that targeting protein kinase CK2 with chemical inhibitors or RNA interference causes MM cell death, increases the sensitivity to chemotherapeutics and compromises the NF-κB and STAT3 activity (Piazza FA et al. 2006, Blood; 108: 1698). We also found that CK2 inhibitors synergize with Hsp90 inhibitors (Manni S et al. 2012, Clinical Cancer Res; 18: 1888) and bortezomib (Manni S et al., Blood (2011 ASH Annual Meeting Abstracts); 118; 1849) in inducing MM cell death. Moreover, a phase I clinical trial is ongoing in USA (ID: NCT01199718) testing the oral CK2 inhibitor CX4945 (Cylene Pharmaceuticals, CA, USA) in MM patients. Purpose.We investigated whether and how CK2 inhibition with ATP-competitive CX4945 and tTBB inhibitors could affect the growth of MM cells and of osteoprogenitors in models of BM microenvironemnt. The aim of the study was to provide further insights into the mechanism of action of CK2 inhibitors also in the MM microenvironment, in particular on the stromal cell-mediated MM cell survival and on the unbalanced bone metabolism. We ultimately aimed at generating original data useful for the design of novel rational combination therapies incorporating CK2 inhibitors in the therapy of MM and of MM-bone disease. Methods.MM plasma cells from patients and MM cell lines were cultured in the presence of BM stromal cells obtained from MM patients or BM stromal cell lines or in the presence of osteoclasts. ATP-competitive CK2 inhibitors were added to the co-cultures or to cultures of osteoblast cell lines or progenitors. Cell growth was evaluated with different means and signaling pathways were studied in MM plasma cells and in the stromal cells. NF-κB target gene expression and DNA binding was tested with microplate arrays. For osteoclast generation, CD14+ peripheral blood monocytes were stimulated in alpha-MEM medium with 10% FBS plus RANKL (60ng/ml) plus M-CSF (25ng/ml) for 28 days; early-osteoblasts colonies were obtained from BM cells stimulated under appropriate conditions. Results.CK2 inhibition with CX4945 or tTBB caused apoptosis of MM cells (either freshly isolated from patients or cell lines) cultured on patient-derived mesenchymal stromal cells (MSC) or on the BM stromal cell line HS-5. The inhibitors did not significantly affect MSC viability. A reduction of NF-κB activity evaluated in MM cells was found upon CK2 inhibition, with a parallel reduction of the production of NF-κB-dependent cytokines. When assayed on osteoprogenitors, CX4945 displayed an inhibitory effect on osteoclast formation from CD14+ monocytes even at low concentrations (1 μM up to 7 μM, comparable with the effects of zolendronate 1 μM), whereas it inhibited the formation of osteoblasts from BM colonies at day 14 at fairly higher concentrations (>5 μM). Moreover, CX4945 inhibited osteoblast proliferation at even higher concentration (>7.5 μM). The anti-myeloma effect of CK2 inhibitors was present also when MM cells (INA-6 cell line) were cultured in the presence of osteoclasts generated from CD14+ monocytes. Conclusions.Our study shows that inhibition of CK2 could profoundly affect the growth of MM cells in models of BM microenvironment while substantially sparing the normal cellular stromal counterparts and osteoblasts and suggests that CK2 inhibitors could be exploited to target the hyperactivity of osteoclast seen in MM bone disease. Disclosures:Giuliani:Celgene: Research Funding.

Matrix Metalloproteinase 13 (MMP13) Upregulation Is Essential for Multiple Myeloma Related Bone Lytic Lesion

AbstractAbstract 4025 Background:MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo. Methods and Results:Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p<0.01) induced OCL fusion, while this effect was largely impaired in the co-culture group with 5TGM1-MMP13 KD MM cells. Treatment with IL-6 further enhanced the OCL fusion activity in both groups. However, in 5TGM1-MMP13 KD group, IL-6 did not compensate for impaired OCL formation due to MMP13 silencing. This indicates that MMP13 secreted from MM cells is one of the key factors facilitating OCL formation/activity in MM. We further investigated the effects of MMP13 silencing on MM tumor progression and bone disease in vivo using the 5TGM1 intratibial tumor model. Recombination activating genes 2 (RAG2) knockout mice were intratibially injected with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells, and 3 weeks later, mice were sacrificed and tumor growth and bone lytic lesion were monitored by serum IgG2b level (by Elisa) and micro-QCT respectively. Preliminary results show that MMP13 KD in MM cells inhibited tumor growth and lytic lesions in trabecular bone. Conclusion:Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions. Disclosures:Lentzsch:Celgene: Consultancy, Research Funding.

Molecular Sequaele of Activin A-Dependent Osteoblast Inhibition in Myeloma.

Abstract 1789Poster Board I-815Understanding the pathogenesis of cancer-related bone disease is critical to the discovery of new therapies. The development of osteolytic lesions in multiple myeloma (MM) results from unopposed osteoclast activity due to decreased osteoblast (OB) function. We recently demonstrated that activin A contributes to MM-mediated OB inhibition. The availability of a clinical-grade activin A inhibitor that reduces tumor burden by restoring the bone structure in vivo, underscores the relevance of this pathway in the development of MM-bone disease. Here, we characterize the signaling pathway mediating OB inhibition by activin A. Activin A secretion by bone marrow stromal cells (BMSC) is enhanced in the presence of MM cells which was completely abrogated and using a transwell system. To identify the receptors and pathways involved in activin A secretion, we used neutralizing antibody against several integrins, CD40 and osteopontin. Only VLA4 neutralizing antibodies partially inhibited activin secretion by about 20% (range 10-30%, p<0.05). Since activin A promoter contains a cJUN-binding sequence, we explored the relations between JNK and activin. We show that upon MM cell binding to BMSC, stromal-derived JNK is phosphorylated and SP600125 (20 μM), a specific JNK inhibitor, impairs activin A secretion by BMSC both at basal conditions and in the presence of MM (40% and 54%, respectively; p<0.01). These results suggest that activin secretion requires JNK activation induced by cell-to-cell contact. We next investigated the mechanism underlying activin-mediated OB inhibition by assessing the activity of several signaling pathways critical to OB differentiation. First, we showed that activin A induces SMAD2 phosphorylation, while no effects were noted on SMAD1, β-catenin, pP65 and pERK. Then, using a doxycycline-inducible β-catenin system, we demonstrate that β-catenin activation overcomes activin A inhibition of OB differentiation. These results suggest that activin/SMAD2 and β-catenin modulate OB differentiation by affecting a common downstream target. We next evaluated the expression of two candidates, the transcription factors RUNX2 and DLX5. Only DLX5 expression was downregulated by exogenous activin A (5 fold decrease at 96 hours, p< 0.01). Specific inhibition of SMAD2 via shRNA-mediated knock-down upregulated basal DLX5 and ALP mRNA expression (1.5 and 4.7 fold increase respectively, p<0.05), and partially overcame activin A inhibitory effects. In turn, DLX5 knock-down abrogated OB differentiation without additive effects by exogenous activin A. The clinical relevance of DLX5 was confirmed by the strong correlation between its expression levels in BM biopsies from MM patients and activin A levels in BM serum. Finally, we demonstrate by in vitro and in-vivo studies that MM-mediated DLX5 inhibition is restored by treatment with RAP-011, the specific activin inhibitor. In conclusion, we show that MM cell engagement of BMSC enhances activin A secretion via adhesion-mediated JNK activation and activin A, in turn, inhibits osteoblast differentiation via SMAD2-dependent DLX5 downregulation. This study identifies a novel pathway relevant to OB differentiation and amenable to drug targeting. DisclosuresChauhan:Nereus Pharmaceuticals, Inc: Consultancy. Seehra:Acceleron Pharma: Employment. Anderson:Celgene : Research Funding; Novartis: Research Funding; Millennium: Research Funding. Scadden:Fate Therapeutics: Consultancy. Raje:Astrazeneca, Novartis, Celgene: Research Funding.

The Novel, Orally Available Multi-Kinase Inhibitor BAY 73-4506 in Multiple Myeloma

Agents targeting not only myeloma cells directly but also bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) that cause enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for multiple myeloma. Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506 (BAY), currently in phase II clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1–3, PDGFR-β), as well as oncogenic, (cKIT, RET, FGFR, Raf) kinases We first tested the ability of BAY to suppress proliferation and survival in a wide array of MM cell lines, including those resistant to conventional chemotherapeutics. Our data show that BAY, in a low micromolar range that is well below concentrations achieved in patient plasma during the first clinical trial in solid tumors, induces apoptosis by caspase-9 and caspase-3 activation in all cell lines tested. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, co-culture system. BAY treatment abrogates growth factor-stimulated MEK, ERK and AKT phosphorylation at sub-micromolar concentrations. Since the VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1–3, we examined its anti-angiogenic properties. BAY inhibits endothelial cell growth and endothelial cell tubule formation in vitro at concentrations less than 1μM; moreover, it also markedly inhibited VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; and importantly, BAY also inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts. Finally, BAY significantly delays tumor growth and abrogates blood vessel formation in vivo in a xenograft mouse model of human MM. These in vitro and in vivo results provide the basis for further clinical evaluation of BAY to improve patient outcome in MM.

Evaluation of NCAM and DKK1 Expression in Multiple Myeloma and MGUS by Gene and Tissue Microarrays: Expression Accompanies Progression.

Introduction: Expression NCAM1, a cell adhesion molecule involved in neuron-neuron adhesion, is also expressed by multiple myeloma (MM PC). Osteolytic bone lesions are a hallmark of MM and elevated expression of NCAM in MM PC has been correlated with this process (Ely and Knowles, 2003). We have previously reported that MM PC express DKK1 and MM blocks osteoblast differentiation in a DKK1-specific manner, suggesting that secretion of the Wnt signaling inhibitor plays a role in MM bone disease. Herein we used gene expression microarrays and tissue microarrays (TMAs) to investigate the simultaneous expression of DKK1 and NCAM in MM and MGUS.Methods: The study population consisted of 198 newly diagnosed MM and 44 MGUS. RNA from CD138-selected plasma cells was hybridized to Affymetrix U133Plus microarrays and data processed with Affymetrix Microarray Suite GCOS1.1 software. TMAs were constructed from formalin-fixed, paraffin embedded bone marrow biopsies. Serial sections of TMA were immunostained for CD138, NCAM and DKK1. TMAs were scanned using ScanScope using 20x lens, assessed using TMA lab software (Aperio Technologies) and scored as an average number of cells in the context of CD138 staining.Results: When put in context of a recently described molecular classification (Zhan et al., 2006), NCAM and DKK1 were found significantly co-over-expressed (DKK1+/NCAM+) in HYPERDIPLOID MM (P < 0.01); NCAM+/DKK1− was typical for MMSET-spike MM (P<0.001); NCAM−/DKK1+ DKK1 was characteristic of MM in MAF-spike disease (P < 0.001), CCND1 spikes with co-expression of CD20 (P<0.01), and the so-called MYELOID subgroup (P<0.001). In contrast, virtually all cases of MGUS were DKK1−/NCAM-. On TMAs, DKK1 expression varied in CD138-positive cells in MM, and in osteocytes and megakaryocytes in MM and MGUS, but was clearly negative in osteoblasts/lining cells. NCAM expression, was also variably expressed in PC of MM cases and in virtually 100% of osteoblasts/lining cells in both MM and MGUS. Osteocytes were distinctly negative for NCAM in both diseases. Of 195 analyzable MM biopsies, PC were DKK1+ in 1% to > 90% of PC in 191 (98%); of these, 94 were also NCAM+ in 5% to > 90% of PC; 1 case was positive for NCAM+/DKK− and 3 were NCAM−/DKK−. There was no significant difference in clinical parameters and survival in a comparison of NCAM+/DKK+ and NCAM−/DKK+ groups. DKK1 gene expression was higher in DKK1+/NCAM+ than in DKK1+/NCAM− MM (P=0.006); NCAM gene expression was higher in DKK1+/NCAM+ than in DKK+/NCAM− MM (P<0.0001) and NCAM was the number one SAM-defined gene over-expressed in DKK1+/NCAM+ relative to DKK1+/NCAM− disease. Consistent with GEP data, DKK1+/NCAM+ MM was over-represented in HYPERDIPLOID MM (32% v. 10%; P<0.001), while DKK1+/NCAM− disease was overrepresented in MAF-spike (15% v. 2%; P=0.003) and MYELOID subgroups (32% v. 18%; P=0.03). DKK1+/NCAM+ MM was completely absent in CCND1-spike /CD20-negative disease, with only 4% of DKK1+/NCAM− cases in this subgroup. No difference was observed for the other subtypes.Conclusion: DKK1 is expressed in all cases of MM with half also expressing NCAM. Neither gene is expressed in MGUS PC. These data suggests that expression of these genes in plasma cells accompanies disease progression.

Serum Levels of OPG and MIP-1α in Untreated Multiple Myeloma Patients. Correlations with Staging, Survival and Bone Disease.

Cytokines, such as MIP-1a (macrophage inhibiting factor) and OPG (osteoprotegerin) are assumed to play a role in MM disease biology and bone disease, by mechanisms that are still under investigation. MIP-1a is constitutively secreted by myeloma cells, plays a possible role in the development of MM bone lesions, enhance MM cell adhesion to stromal cells and its serum levels have been recently related to survival in MM. OPG is a soluble decoy receptor which acts as a soluble receptor antagonist that prevents osteoclasts activation and the development of bone disease. Reported findings on serum OPG levels in MM patients are controversial as well as its possible role in the biology of the disease. In order to investigate the possible relationship of MIP-1a and OPG levels in MM patients with prognosis and bone disease, we determined by ELISA serum MIP-1a and OPG levels in 20 MGUS, 82 MM patients and 27 healthy individuals (HI). Both cytokines were determined by ELISA (R&D, Quantikine, USA) in frozen sera collected at dignosis, before treatment. The median age of MM patients was 69 years (44–84) and 50% were males. MM patients' stage was as follows: 23 stage I, 28 stage II, 31 stage III according to Durie-Salmon staging system and 27 stage I, 17 stage II, 35 stage III according to the International Scoring System (ISS). In HI median MIP-1a was 28 pg/ml (15–54) and median OPG 1600 pg/ml (450–4600). In subjects with MGUS, median MIP-1a was 34 pg/ml (17–58) and median OPG 2300 pg/ml (820–6200). In MM patients, median pretreatment serum MIP-1a was 32 pg/ml (16–100) and OPG 3000 pg/ml (820–25000). No statistical significant difference was observed between HI, MGUS and MM patients with regard to MIP-1a levels but for OPG levels differences between HI and MM patients and between MGUS and MM patients were both significant (0.01 and 0.05 respectively). No relationship was found between MIP-1a or OPG levels and bone disease. However, there was a trend for longer survival in patients with MIP-1a or OPG levels lower than median (5-year overall survival 60 ± 12 vs 38 ± 14, p=0.08 and 66 ± 13 vs 29 ± 13, p=0.07 respectively). In addition MIP-1a levels were correlated with ISS stage: MIP-1a levels were 28.3±11.3 in ISS stage 1, 29.8±11.1 in ISS stage 2, 39±19.2 in ISS stage 3 (p=0.02). In conclusion, in our experience serum OPG levels are higher in MM patients than in MGUS or HI, MIP-1a levels are correlated with the ISS stage and both high serum MIP-1a and OPG levels at diagnosis are related with a shorter survival.

The OPG/TRAIL Interaction in an In Vitro Osteoclastogenesis Model Derived from Human Multiple Myeloma-Bone Disease.

The development of multiple myeloma (MM)-bone disease is mediated by increased recruitment and activity of osteoclasts (OCs). Osteoclastogenesis is regulated by a complex signaling system, that involves receptor activator of nuclear factor-kappa B (RANK), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), all belonging to the Tumor Necrosis Factor (TNF) family. The aims of our study were to investigate: the MM T cell involvement in osteoclastogenesis, and the expression of the major osteoclastogenic mediators. Unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) isolated from 32 MM patients with or without osteolytic bone lesions, and parallel T cell-depleted cultures were used as in vitro osteoclastogenesis models. In addition, unstimulated and unfractionated PBMC cultures from 32 controls with nonneoplastic disease without any skeletal involvement were also established. Our results showed that the OCs derived from MM-bone disease PBMCs spontaneously developed and displayed a longer survival in a T cell-dependent way. Differently in T cell-depleted MM PBMC cultures, the addition of macrophage-colony stimulating factor (M-CSF) and RANKL was necessary to promote the formation of OCs, that however did not exibit a longer survival. MM-bone disease T cells overexpressed RANKL, OPG and TNF-related apoptosis inducing ligand (TRAIL), also detected in large amounts in the culture media. Despite high OPG levels, the persistence of osteoclastogenesis in our system can be related to the interaction between OPG and TRAIL, that were coimmunoprecipitated by a monoclonal antibody (mAb) against TRAIL. The evidence that TRAIL binds to OPG blocking OPG anti-osteoclastogenic effect is also supported by the addition of different concentrations of functional anti-TRAIL mAb, significantly decreasing the OC formation. The OCs developed from MM-bone disease PBMCs expressed a T cell-modulated balance of death and decoy TRAIL receptors. In particular, we found these OCs overexpressed TRAIL decoy receptor DcR2 in the presence of T cells, and death receptor DR4 in the T cell-depleted cultures. In conclusion, our results highlight that MM-bone disease T cells support the spontaneous OC formation with longer survival, involving the OPG/TRAIL interaction and the unbalanced OC expression of TRAIL death and decoy receptors.