Osteolytic Bone Disease Research Articles

Bone Marrow Stromal Mediated Pro-Oncogenic Wnt/β-Catenin Antagonize sDKK1 and Promote Osteoclast Activity In Multiple Myeloma

IntroductionBone marrow stromal cells (BMSC) provide a suitable microenvironment for multiple myeloma (MM) cell survival, resistance and related osteolytic bone disease. Wnt signaling enhances osteoblast proliferation and mineralization, whereas it blocks osteoblast apoptosis and osteoclast differentiation by increasing the OPG/RANKL ratio. The Wnt inhibitor Dickkopf 1 (Dkk1) negatively regulates canonical Wnt signaling by binding to and antagonizing the Wnt co-receptors Lrp5/6 (He et al., 2004). Serum levels of DKK1 have been reported to be elevated in patients with MM, while osteoprotegrin levels are decreased (Tian et al 2003). Crosstalk between Wnt/β-catenin and other signaling pathways within the bone marrow microenvironment that regulate the osteogenic differentiation of mesenchymal stem cells (MSCs) has been reported previously (Cawthorne et al. 2012 and Krishnan et al. 2006). However the question of whether the pro-oncogenic effect of Wnt-β-catenin signal from the stroma antagonizes the elevated serum DKK1 and thus actually enhance the osteolytic actvities in MM relapse remains to be answered. MethodsBone marrow-derived stromal fibroblasts from myeloma patients were maintained in culture for 21 days. Adherent stromal fibroblast (confirmed with staining for Vimentin) and non-adherent cells were examined for DKK1 by ELISA and for Wnt β-catenin by performing (a) immunofuoresence detection, (b) luciferase reporter assays and (c) Western blot analysis. ResultsAdherent stromal fibroblasts showed β-catenin expression after 21 days in culture (Fig 1), the signal for β-catenin expression was 68% compared to that in non-adherent BM cells (27%). Consistently, luciferase reporter assays of adherent stromal fibroblasts transfected with Wnt/β-cat responsive reporter plasmids (STF16) showed >4 fold increase in the reporter activity compared to that in the MM cells. As expected, the level of soluble DKK1 was significantly lower in patients with early MM (ranges from 5167 ± 379 pg/ml) vs. advanced stage MM (53967±1323 pg/ml) among patients who are relapsed (n=16), and the difference in the DKK1 levels between these two groups of samples (early vs. advanced stage MM, n=8 each) are statistically significant (p<0.05). The increase in serum DKK1 is also directly correlated with increase in the serum VEGF and vascular tube formation suggesting the stromal influence of Wnt/β-catenin in mediating angiogenesis in MM relapse patients. Transfection of stromal fibroblasts with siRNA for NF-κBp65 down regulated both β-catenin and DKK1 at the mRNA and protein levels, suggesting a close interaction between Wnt/β catenin and inflammatory signaling within the BM stromal microenvironment.To further determine the effect of potential drugs on the regulation of β-catenin and DKK1, we measured soluble DKK1 activity in the cell culture medium of growing BMSC cells stimulated with human M-CSF and RANKL to generate osteoclasts and osteoblasts, followed by treatment with different concentrations of Bortezomib, or Dipyridamole (an agent which increases exracellular adenosine levels) at a concentration of 2.5, 5.0 and 10ug/ml respectively) for 24h. As previously reported, dipyridamole increased obsteoblast differentiation, surprisingly, diminished sDKK1 levels, as well as bortezomib (11667±1528 pg/ml) at 10 ug/ml. SummaryOur preliminary findings suggest that Wnt/β-catenin and sDKK1are co-produced in BM stromal cells and are both diminished by drugs that modulate both Wnt and RANKL pathways. Another interesting message from this study is that the stromal derived β-catenin may influence bone metastasis of myeloma, as seen in other cancer types, via transcriptionally regulating DKK1 within the BM niche. Since the bone marrow stromal cells from MM relapse patients still express an elevated level of β-catenin, in spite of the higher serum DKK1 level in MM relapse patients, we believe that stromal contribution of β-catenin is critical in patients with multiple myeloma.▪ Disclosures:No relevant conflicts of interest to declare.

Relationship Between Presence Of Osteolytic Lesions, Cytogenetic Features and Bone Marrow Levels Of Cytokines and Chemokines In Multiple Myeloma Patients: Role Of Chemokine (C-C motif) Ligand 20

Osteolytic lesions are the main feature of multiple myeloma (MM) patients. In the last years the pathophysiological role of several cytokines and chemokines such as RANKL, osteoprotegerin (OPG), DKK-1, Interleukin- (IL)-3, IL-7, Activin A, chemokine (C-C motif) ligand (CCL)-3 and CCL20 has been highlighted in MM bone disease although the relationships between their bone marrow (BM) levels, cytogenetic features and the skeletal involvement are not yet defined.A total cohort of 365 consecutive patients with monoclonal gammopathy has been evaluated in this study including 250 patients with symptomatic MM, 62 patients with smoldering MM (SMM) and 53 patients with MGUS. A group of 29 healthy subjects was also included. X-ray survey, magnetic resonance imaging (MRI) and positron emission tomography (PET)/computerized tomography (CT) scan were performed in MM patients The presence of the main cytogenetic abnormalities (hyperdiploid karyotype, del13, t(4;14), t(11;14), 17p-, amp1) has been investigated in purified CD138+ MM cells by FISH analysis and the BM plasma levels of soluble (s) and total RANKL, OPG, IL-3, IL-7, DKK-1, Activin A, CCL3 and CCL20 were measured by ELISA assay. Quantitative variables were compared by non-parametric Kruskal-Wallis and Mann-Whitney tests as appropriate and categorical variables were analyzed by Chi-square test.MM patients showed higher BM median levels of Activin A, sRANKL, DKK-1, CCL20 and IL-7 as compared to N (p<0.05) and those of Activin A, DKK-1 and CCL20 as compared to MGUS and to SMM (p<0.01). BM plasma levels of Activin A, sRANKL and sRANKL/OPG ratio were significant higher in high-risk cytogenetic MM patients as compared to standard risk ones (p<0.05) as well as Activin A, CCL3 and CCL20 BM levels correlated with ISS staging being higher in stage III as compared to stage I and II (p<0.005).Regarding the bone status we found that 60%, 86% and 66% of MM patients were positive at X-ray survey, MRI and PET/CT scan respectively. Any significant relationship was not observed between X-rays or PET/CT scan positivity and the presence of cytogenetic abnormalities (p=NS) whereas MM patients with high-risk cytogenetic showed higher positivity to the MRI scan than standard risk (100% vs. 77%, p=0.03). Significant higher BM levels of DKK-1 and CCL20 (p<0.005) were found in all MM patients with almost one osteolytic lesion as compared to those negative at the X-ray survey whereas Activin A (p=0.05), DKK-1 (p=0.009) CCL3 (p=0.05), and CCL20 (p<0.001) were significantly higher into the BM of patients with high bone disease (more than three lesions) as compared to those with low bone disease. MM patients with a positive MRI scan have higher BM levels of RANKL, DKK-1, CCL3 and CCL20 as compared to those negative (p<0.05). On the other hand, MM patients positive at the PET/CT scan with more of three lesions have significant higher BM levels of RANKL and CCL3 as compared to those negative or with less of three lesions (p<0.05). Finally by a logistic multivariate analysis we found that among the cytokines and chemokines analyzed and the clinical and cytogenetic features CCL20 levels were the only significant predictor of the presence of skeletal involvement (OD ratio: 1.03; p=0.014).In conclusion our study defines the relationship between skeletal involvement, cytogenetic feature and the profiles of BM cytokines and chemokines showing that CCL20 is a critical marker related to the presence of osteolytic bone disease in MM patients. Disclosures:Giuliani:Celgene Italy: Research Funding.

Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging.GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28days exposure in rats. After 7days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT.GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption.

T Cells Induce Pre-Metastatic Osteolytic Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.

Critical role for inflammasome-independent IL-1β production in osteomyelitis (P6211)

Abstract The immune system plays an important role in the pathophysiology of many acute and chronic bone disorders, but the specific inflammatory networks that regulate individual bone disorders remain to be elucidated. Here, we characterized the osteoimmunological underpinnings of osteolytic bone disease in Pstpip2cmo mice. These mice carry a homozygous L98P missense mutation in the Pombe Cdc15 homology (PCH) family phosphatase PSTPIP2 that is responsible for the development of a persistent autoinflammatory disease resembling chronic recurrent multifocal osteomyelitis (CRMO) in humans. We found that improper regulation of IL-1β production resulted in secondary induction of inflammatory cytokines, inflammatory cell infiltration in the bone and unremitting bone inflammation. Aberrant Il1β expression precedes the development of osteolytic damage in young Pstpip2cmo mice, and genetic deletion of Il1β, but not Il1α, rescued osteolytic bone disease in mutant mice. Intriguingly, caspase-1 activation in the inflammasome complex was dispensable for Pstpip2cmo-mediated bone disease. Thus, our findings establish a critical role for inflammasome-independent production of IL-1β in osteolytic bone disease.

Abstract 2143: Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models.

Abstract Anthracyclines (doxorubicin) are one of the most effective and widely used chemotherapeutic agent for the treatment of several cancer types, including early stage and metastatic breast cancer. However, doxorubicin has been known to cause long-term bone damage and growth defects in patients with childhood cancers, which was also confirmed by several preclinical studies. This raises concern regarding the usage of doxorubicin for the treatment of patients with metastatic breast cancer who are already at increased risk of osteolytic bone damage leading to severe bone loss due to. A number of clinical and preclinical studies have indicated that doxorubicin causes both long term and short term bone damages by lowering bone mineral density, increasing bone loss and may disrupt bone homeostasis by directly affecting bone marrow stem cells, however, the underlying mechanism is poorly understood. We have previously reported that treatment with doxorubicin increases circulating levels of TGFβ, a cytokine which has been implicated in the increase in osteoclast differentiation and survival, suppression of osteoblast differentiation and has been associated with advancement of breast cancer to bone metastasis. We hypothesize, breast cancer patients undergoing doxorubicin treatment will experience accelerated bone loss due to an increase in serum TGFβ levels and blocking TGFβ will rescue this bone loss, at least partially. We have tested the efficacy of anti-TGFβ antibody 1D11, in blocking doxorubicin-mediated bone damage in cardiac injection and orthotopic models of breast cancer to bone metastasis. X-ray image analysis revealed that doxorubicin treatment resulted in an eightfold increase in osteolytic lesion areas (P=0.002) and microCT analysis has shown almost 50% decrease in BV/TV (P=0.0005), in a preclinical murine model metastatic breast cancer. Doxorubicin treatment has also decreased BV/TV in non-tumor bearing animals, suggesting a direct damaging effect of this agent to bone at the sites which are not affected by cancer infestation. As anticipated, treatment with anti-TGFβ antibody 1D11 was able to rescue doxorubicin-mediated bone loss in all three preclinical models tested. At the cellular level, direct effect of doxorubicin treatment on the bone marrow microenvironment included decreased osteoblast survival and reduced mineralized matrix formation and increased osteoclast formation, thereby affecting both bone formation and bone resorption. Taken together, we conclude that, doxorubicin therapy decreases bone formation and increases bone resorption causing additional bone loss in patients with osteolytic bone disease and an anti-TGFβ therapy may be affective in controlling that bone loss. Citation Format: Tapasi Rana, Anwesa Chakrabarti, Michael Freeman, Swati Biswas. Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2013-2143

Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Lenalidomide in combination with an activin A-neutralizing antibody: preclinical rationale for a novel anti-myeloma strategy

Given the prevalence of osteolytic bone disease in multiple myeloma (MM), novel therapies targeting bone microenvironment are essential. Previous studies have identified activin A to be of critical importance in MM-induced osteolysis. Lenalidomide is a known and approved treatment strategy for relapsed MM. Our findings demonstrate that lenalidomide acts directly on bone marrow stromal cells via an Akt-mediated increase in Jun N-terminal kinase-dependent signaling resulting in activin A secretion, with consequent inhibition of osteoblastogenesis. Here, we attempted to augment the antitumor benefits of lenalidomide while overcoming its effects on osteoblastogenesis by combining it with a neutralizing antibody to activin A. Increased activin A secretion induced by lenalidomide was abrogated by the addition of activin A-neutralizing antibody, which effectively restored osteoblast function and inhibited MM-induced osteolysis without negating the cytotoxic effects of lenalidomide on malignant cells. This provides the rationale for an ongoing clinical trial (NCT01562405) combining lenalidomide with an anti-activin A strategy.

A mixed exercise training programme is feasible and safe and may improve quality of life and muscle strength in multiple myeloma survivors

BackgroundExercise programmes are beneficial for cancer patients however evidence is limited in patients with multiple myeloma (MM), a cancer that is characterised by osteolytic bone disease, giving rise to high levels of bone morbidity including fractures and bone pain.MethodsWe conducted a single arm phase 2 study of an exercise programme (EP) as rehabilitation for treated MM patients, to evaluate feasibility, effects on QOL and physiological parameters. Patients were given individualised programmes, comprising stretching, aerobic and resistance exercises, carried out under supervision for 3 months then at home for a further 3 months.ResultsStudy uptake was high, 60 of 75 (80%) patients approached consented to the study. Screen failures (11, due to fracture risk and disease relapse) and patient withdrawals (12) resulted in a final 37 patients enrolling on the programme. These 37 patients demonstrated high attendance rates in the supervised classes (87%), and high levels of adherence in home exercising (73%). Patients reported better QOL following the EP, with improvement in FACT-G and Fatigue scores over time from baseline (p<0.01 for both, one-way repeated measures ANOVA) to 6 months. Upper and lower limb strength also improved on the EP, from baseline to 6 months (p<0.01 for both). There were no adverse reactions.ConclusionsAn EP in MM patients is feasible and safe, with high attendance and adherence. Benefits in QOL, fatigue and muscle strength await confirmation in randomized studies, prompting urgent evaluation of the benefits of EP in the rehabilitation of MM patients.

Potential Use of Bisphosphonates in Invasive Extramammary Paget’s Disease: An Immunohistochemical Investigation

Invasive extramammary Paget's disease (EMPD) is relatively rare and is reported to be highly metastatic to lymph nodes or even other organs, including bone. Histologically, EMPD shows significant numbers of lymphocytes around the tumor mass, suggesting the possible development of novel immunomodulatory therapy for EMPD by targeting these infiltrating lymphocytes. Previously, bisphosphonates (BPs) were administered for the treatment of malignancy, especially osteolytic bone disease. Recent reports also suggested that BPs might have a direct antitumor effect through several pathways beyond their beneficial effect on bone metastasis. Among them, the abrogation of immunosuppressive cells, myeloid derived suppressor cells (MDSC), by BPs might be one of the optimal methods to induce an antitumor immune response both locally and at sites remote from the tumor. In this study, we employed immunohistochemical staining for immunosuppressive macrophages and cytotoxic T cells in the lesional skin of patients with noninvasive EMPD and those with invasive EMPD.

Rapamycin increases neuroblastoma xenograft and host stromal derived osteoprotegerin inhibiting osteolytic bone disease in a bone metastasis model

PurposeOsteoprotegerin (OPG) is a decoy receptor for the Receptor of NF-κB (RANK) ligand that can inhibit osteoclastogenesis. Previous studies have suggested that Mammalian Target of Rapamycin (mTOR) inhibition upregulates OPG production. We tested the hypothesis that the mTOR inhibitor rapamycin could inhibit neuroblastoma bone metastases through its action on OPG. Experimental DesignAn orthotopic model of bone metastasis was established. Mice with established disease were subsequently treated with rapamycin (5mg/kg IP daily) or vehicle control (DMSO 1:1000). X-rays were obtained twice a week to detect pathologic fractures. Serum OPG levels were measured by ELISA after two weeks of treatment. ResultsMice with bone disease receiving rapamycin had increased serum levels of OPG in the CHLA-20 mice compared to controls (36.89pg/mL±3.90 vs 18.4pg/mL±1.67, p=0.004) and NB1691 tumor-bearing groups (46.03±2.67pg/mL vs 17.96±1.84pg/mL, p=0.001), and a significantly longer median time to pathologic fractures with CHLA-20 (103days vs 74.5days, p=0.014) and NB1691 xenografts. ConclusionIn a xenograft model, increased OPG expression correlated with a delay to pathologic fracture suggesting a potential role for mTOR inhibitors in the treatment of neuroblastoma bone metastases.

Cell Fusion in Myeloma Marrow Microenvironment: Role in Tumor Progression

Multiple myeloma (MM) is a B cell malignancy characterized by uncontrolled expansion of malignant plasma cells within the bone marrow that contribute to formation of multiple osteolytic bone disease and severe skeletal devastation. Recently, direct and indirect observations suggest that fusion events between cells housed within the MM marrow microenvironment often occur and may play a role in tumor progression, including myeloma bone disease (MBD). A number of cells resident in the marrow, such as myeloid progenitors and dendritic cells, have inherited fusogenicity and osteoclastogenic potential due to the expression of a number of fusogenic proteins as well as a high sensitivity to fusogenic factors produced within the MM marrow milieu. Similarly, osteoclasts (OC), as bone-resorbing multinucleated cells resulting from the fusion of marrow monocyte/ macrophages, have been reported to improperly fuse with malignant plasma cells and drive transition of these cells into OC-like cells exerting bone-resorbing capacity. Further, based on indirect cytogenetic and molecular evidence, it has been proposed that MM cells may generate a hybrid progeny with high metastatic potential and drug resistance, ultimately pointing to uncontrolled homotypic fusions that accelerate MBD progression.

Selective tyrosine kinase inhibition of insulin-like growth factor-1 receptor inhibits human and mouse breast cancer–induced bone cell activity, bone remodeling, and osteolysis

Insulin-like growth factor 1 (IGF-1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF-1 receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA-MB-231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF-1 and conditioned medium-induced osteoclast formation by preventing phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M-CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF-1 and conditioned medium and totally prevented conditioned medium-induced osteoclast formation in osteoblast-bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast-osteoclast coupling. PQIP also inhibited IGF-1-induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA-MB-231 conditioned medium-induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer-associated bone turnover. We conclude that inhibition of IGF-1 receptor tyrosine kinase activity by PQIP suppresses breast cancer-induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer.

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that invivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

The promise and dilemma of cannabinoid therapy: lessons from animal studies of bone disease

The endocannabinoid system plays an important role in numerous physiological processes and represents a potential drug target for diseases ranging from brain disorders to cancer. Recent preclinical studies implicated endocannabinoids and their receptors in the regulation of bone cell activity and in the pathogenesis of bone loss. Cells and intervening nerves in the skeleton express cannabinoid receptors and the machinery for the synthesis and breakdown of endocannabinoids. In healthy adult mice, pharmacological and genetic inactivation of the cannabinoid type 1 receptor (CB1) and putative cannabinoid receptor GPR55 (G protein-coupled receptor 55) inhibit osteoclastic bone resorption and increase bone mass, suggesting that both receptors have a negative role in early bone development. Although no distinct abnormalities in bone development were observed in healthy adult mice deficient in cannabinoid type 2 receptors (CB2), pharmacological blockage of this receptor was effective in suppressing bone loss associated with increased bone turnover, particularly in mouse models of osteoporosis, arthritis and osteolytic bone disease. In the aging skeleton, CB1 deficiency causes accelerated osteoporosis characterized mainly by a significant reduction in bone formation coupled to enhanced adipocyte accumulation in the bone marrow. A similar acceleration of bone loss was also reported in aging CB2-deficient mice but found to be associated with enhanced bone turnover. This perspective describes the role of cannabinoid ligands and their receptors in bone metabolism and highlights the promise and dilemma of therapeutic exploitation of the endocannabinoid system for treatment of bone disorders.

Bortezomib-Dexamethasone As Induction Therapy for Light Chain Deposition Disease (LCDD): A Single Center Experience

AbstractAbstract 5027 Background:LCDD is a rare plasma cell dyscrasia characterized by deposition of immunoglobulin light chain in kidneys and, occasionally, in other organs such as liver and heart. Most patients present with rapidly deteriorating renal function and nephrotic proteinuria.There is no standard treatment for LCDD. High dose dexametasone (HDD) with or without alkylating agents and high dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT) have been used, the latter with better results. Recently the combination therapy with Bortezomib-Dexametasone (BD) has been used in small series of patients and has shown promising results. Here we report on four patients with LCDD treated in our center from September 2010 to September 2011 with BD as induction therapy. Patients and Methods:The characteristics of the patients are shown in Table 1. Three patients were male; the median age was 44. 5 years (range 37–64 years). Two patients had more than 20% bone marrow plasma cell count with no evidence of active multiple myeloma (MM) defined by osteolytic bone disease, hypercalcemia or myeloma cast nephropaty. All patients had renal biopsy with histologic and immunofluorescence studies. In all patients except one, the diagnosis was confirmed by electron microscopy examination. One patient was therapy naive and three patients were refractory to HDD. All patients but one presented with impaired renal function and all of them showed nephrotic albuminuria. Serum free light chains values (sFLC) were high in all patients, with abnormal kappa to lambda ratio (R k/λ). Noteworthy, in three patients serum immunofixation electrophoresis did not succeed in detecting the circulating monoclonal light chain. Patients were given Bortezomib (1. 3 mg/m2days 1, 4, 8 and 11) and Dexamethasone (40 mg days 1–4) every 21 days, for three to six cycles. Results:Two patients achieved normalization of R k/λ. One patient achieved reduction of more than 50% of involved sFLC and reduction of more than 50% of the M protein after three cycles. One patient had progressive disease. None of the responding patients with renal impairment achieved improvement of the renal function, but all responding patients showed reduction of more than 50% of initial albuminuria. After BD one patient achieved hematological CR, one VGPR and one PR (Gertz MA et al., Amyloid 2010). All responding patients were eligible for ASCT. Two patients underwent stem cell mobilization with cyclophosphamide 4 g/m2; one patient was mobilized with G-CSF alone. Melphalan dose was reduced to 140 mg/m2in the only patient undergoing hemodyalisis. There were no complications related to stem cell harvest and engraftment (only one patient showed a late platelet engraftment). After ASCT two patients achieved at least VGPR; one patient achieved a PR and he underwent second ASCT achieving again a PR. Dose reduction of Bortezomib was required in two patients because of grade 2 neuropathy. Conclusions:BD is feasible and effective in LCDD patients, and it can be used as an induction regimen before ASCT.Table n°1:Patients characteristicsAge, sex64, M39, M50, M37, FImmunofluorescence on renal biopsyYesYesYesYesElectron microscopy study on renal biopsyYesNoYesYesPrior therapyHDDHDDNoneHDDBone Marrow plasma cells (%)15256610Type of light chain involvedkkkλSerum immunofixation pre/post BDNeg./Neg.k/kIgGk/IgGkIgGλ/Neg.Urine immunofixation pre/post BDk/kk/Neg.Neg./Neg.λ/Neg.sFLC (mg/L) pre/post BD718/10601580/19.1804/18135/53.9sFLC R k/λ pre/post BD27.5/36.983.6/0.5152/1.10.57/1.09Serum Creatinine (mmol/L) pre/post BD318/792158/170100/73443/56024h Urine Protein (gr) pre/post BD4.5/2.35.4/1.148.28/0.86.66/1.58Serum Albumin (gr/L) pre/post BD34/3631/3531/4036/40Serum MC& (g/L) pre/post BD01.08/<0.635.7/101.04/Neg.Hematologic response to BD (Gertz MA et al., Amyloid 2010)PDVGPRPRCRMonths without progression*NR#6310ASCTNoYesYesYesHematologic response to ASCT (Gertz MA et al., Amyloid 2010)—VGPRPRCRConditioning regimen = Melphalan (mg/m2)—200200/140140#NR: non responder.*from the end of BD cycles to HDM and ASCT.&MC= Monoclonal Component. Disclosures:Off Label Use: Bortezomib for light chain deposition disease.

High CCN1 Levels Are Associated with Longer Progression-Free and Overall Survival in GEP-Defined High-Risk Myeloma

Abstract 3940 Background:An imbalance between osteoblasts and osteoclasts results in osteolytic bone disease, a major complication in multiple myeloma (MM). Using gene expression profiling (GEP), we identified CYR61 as the most significantly overexpressed gene in bone biopsies in subsets of MGUS and MM patients compared with healthy controls. CYR61 is induced during osteoblast differentiation by endothelin-1, (Clines et al., 2007) and by Wnt3a (Si et al., 2006). CYR61 also inhibits the formation of multinucleate osteoclasts in vitro (Crockett et al., 2007). GEP revealed that elevated levels of CYR61 in 90/264 (34%) of newly diagnosed total therapy 3 (TT3) patients is associated with superior overall survival (p=0.006, HR-0.78). The CYR61 protein, CCN1, is significantly elevated in 57/177 (32%) TT3 patients at diagnosis and is associated with superior overall survival (p=0.024, HR-0.1). Recombinant CCN1 reduces MM cell growth and survival in vitro, and ectopic CYR61 produced by MM cells implanted into the SCID-hu model show that CCN1 inhibits osteolysis and reduces MM tumor growth in vivo. Taken together these data suggest that elevated CCN1 may be indicative of a dysfunctional microenvironment (ME) and could serve as a biomarker to monitor the MM ME. Methods:To test whether CCN1 could serve as an MM ME biomarker, CCN1 protein was quantitated by ELISA (DRG International). In BM sera from patients with MGUS/AMM (n=84), newly diagnosed myeloma (n=239), and in paired samples at diagnosis and 48h after a test dose of velcade (n=168 pairs), in nCR/CR (n=156 pairs) or at relapse (n=41 pairs). Differences between groups were analyzed using the t-test and survival using the log-rank test. Results and Discussion:CCN1 levels in myeloma patients at diagnosis (464.5 ± 30.4, p=0.0007), near complete remission (nCR, 466.1 ± 51.9 pg/ml, p=0.0055), complete remission (CR, 510.4 ± 36.5 pg/ml, p< 0.0001) and at relapse (692.5 ± 166.2 pg/ml, p=0.0185) were significantly elevated compared with healthy donors (274.4 ± 40.1 pg/ml). In paired samples, CCN1 levels at 48h post test-dose of velcade were significantly higher than at diagnosis (p=0.005). This suggests that elevated levels of CCN1 post-velcade may reflect recovery of the MM ME. To determine whether CCN1 may be associated with CR duration, CCN1 levels at nCR/CR were compared in patients that remained in CR for < 5 years and ≥ 5 years. CCN1 levels were significantly elevated in patients in CR ≥ 5 years (563.2 ± 53.2 pg/ml, p=0.01) compared to those in CR < 5 years (411.4 ± 22.8). CCN1 levels at nCR/CR and molecularly-defined risk were examined to determine whether they were independently associated with survival. Four groups were compared: CCN1 quartiles 1–3 levels [Q1-Q3] with low- or high-risk by GEP 70 or CCN1 [Q4] with low- or high-risk. While CCN1 levels were not prognostic for low-risk patients, Q4 high-risk patients had significantly longer PFS (Figure 1a, p=0.0120) and OS (Figure 1b, p<0.0001) than those with Q1-Q3 levels (median OS 6.9 and 3.8 years, respectively).To determine whether baseline CCN1 levels are associated with time to CR, CCN1 levels in patients who had not achieved CR (368.0 ± 38.0 pg/ml n=25) were compared to those that achieved CR within 6 months (525.6 ± 60.0 pg/ml, p=0.0284) and within 1 year (548.2 ± 74.5 pg/ml n=96, p=0.033). Patients who achieve CR had significantly higher CCN1 levels than those who did not reach CR. At diagnosis, MM CCN1 levels were lower than those of MGUS/AMM (608.3 ± 71.5 pg/ml, p=0.0324). We thus examined CCN1 levels in untreated MGUS and AMM patients to determine whether they were associated with time to progression to overt MM. Patients who progressed in less than 3 years had CCN1 levels significantly lower (441.6 ± 28.5 n=38) than those who progressed later (726.7 ± 121.7 pg/ml n=46, p=0.0399). CCN1 levels at the upper quartile were associated with significantly longer time to progress to overt MM (p<0.0056).Taken together, these data suggest that elevated CCN1 levels indicate a recovery effort by the ME and due to its positive association with survival of myeloma patients may be useful in the monitoring of disease course and thus personalized therapeutic management. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HMOX1 Is a Targeted Therapy for Myeloma-Induced Bone Disease.

Abstract 2944Multiple myeloma (MM) bone disease is characterized by increased activity of osteoclasts and reduced osteoblast numbers. We recently reported that cytotherapy with mesenchymal stem cells (MSCs) promotes bone formation, inhibits bone disease and reduces MM growth (Yaccoby et al., 2006; Li et al., 2011) and that MSCs secrete bone remodeling associated factors such as decorin that directly inhibits osteoclastogenesis and promotes osteoblastogenesis (Li et al., 2012). To shed light on molecular mechanisms associated with the cytotherapeutic effects of MSCs we exploited the SCID-hu model engrafted with MM cells from various patients and cytotherapeutically treated with fetal or healthy donors MSCs. Human global gene expression profile was performed in nonmyelomatous implanted bones (n=5) and in myelomatous implanted bones injected with human MSCs (1×106 cells/bone) and analyzed immediately (control group, n=17) or 24 hours later (n=16). We indentified heme oxygenase 1 (HMOX1) as one of the top significant upregulated genes consistently induced by 24 hours cytotherapy using fetal MSCs or MSCs from 3 different donors. HMOX1 induction was confirmed by qRT-PCR and immunohistochemistry and was also found to be consistently induced by MSCs cytotherapy in myelomatous bones engrafted with MM cells from different patients (n=4). Further analysis revealed lower expression of HMOX1 in myelomatous versus nonmyelomatous bones. HMOX1, an inducible antioxidant, degrades intracellular heme into carbon monoxide, free iron and biliverdin and is involved in oxidative stress response and intracellular iron homeostasis both of which are known to regulate osteoclastogenesis. Culture of human blood mononucleated cells or committed osteoclast precursors with MSCs (non-contact co-culture) in osteoclastogenic medium supplemented with RANKL and M-CSF inhibited multinucleated osteoclast formation by 70% (p<0.0001) and 97% (p<0.0001), respectively. MSCs induced HMOX1 expression in osteoclast precursors by 3 folds (p<0.02) and HMOX1 levels remained higher in these cells during osteoclast formation. Concurrently, RANK (RANKL receptor) and NFATC1 (osteoclast main transcription factor) were constantly downregulated in osteoclast precursors by MSCs by 65±10% (p<0.01) and 42±11% (p<0.05), respectively, followed by reduced expression of the osteoclastic markers cathepsin K (p<0.0002), acid phosphatase 5 (p<0.01) and vitronectin receptor (p<0.02) at the end of the differentiation process (7 days). Similar to MSCs, overexpression of HMOX1 in osteoclast precursors using lentiviral vector markedly reduced their ability to form osteoclasts while HMOX1 inducer, hemin (50 μM), induced HMOX1 in osteoclast precursors and inhibited their differentiation into osteoclasts by 49±2% (p<0.0001). In MM-bearing SCID-rab mice (9 mice/group) bone mineral density (BMD) was reduced by 17±3% from pretreatment levels in control group whereas in hosts treated with hemin for 3 weeks BMD was reduced by 2±3% (p<0.005). Histological bone sections revealed reduced number of TRAP-expressing osteoclasts in myelomatous bones from hemin-treated hosts (p<0.01). These data suggest that HMOX1 is suppressed in myelomatous bone and that therapeutic induction of HMOX1 is a promising approach to control MM-induced osteoclastogenesis and osteolytic bone disease. Disclosures:No relevant conflicts of interest to declare.

Runx2 Transcription Factor Regulates Heparanase-Induced Bone Resorption in Multiple Myeloma

Abstract 567 Background.Despite advances in treatment strategies, myeloma remains incurable, and bone destruction is a major cause of morbidity in myeloma patients. We have documented in earlier studies that heparanase enzyme is preferentially expressed in myeloma cells and induces severe bone destruction in myeloma. We also discovered that heparanase increases the production of two major bone resorbing factors named Receptor Activator of NF-κB Ligand (RANKL) and Matrix Metalloproteinase 9 (MMP-9) by myeloma cells. Runx2, a member of the runt-related gene family, is a bone-specific transcription factor. Runx2 regulates osteoblast differentiation and is essential for bone tissue development. Interestingly, Runx2 also controls expression of RANKL and MMP-9 genes in osteoblasts. Recent evidence indicates that ectopic induction and overexpression of Runx2 in breast, uterine and prostate cancer cells is associated with bone-metastasis, and osteolytic bone disease in these cancers. However, very little is known about the function of Runx2 in myeloma cells. Here we report for the first time that heparanase engages the Runx2 pathway to promote expression of RANKL and MMP-9 in myeloma cells. Methods.Molecular, biochemical, cellular and in vivo approaches were used to assess the role of Runx2 in heparanase-induced expression of RANKL and MMP-9. These included: (1) Real-time PCR and Western blot analysis to monitor Runx2 levels in CAG myeloma cells expressing high level of heparanase (HPSE-high cells) or with knockdown of endogenous heparanase (HPSE k/d cells), and the corresponding control cells. (2) Chromatin Immunoprecipitation (ChIP) assay to determine in vivo occupancy of the RANKL and MMP-9 gene promoters in myeloma cells by Runx2. (3) Zymography to determine MMP-9 activity in both human and murine myeloma cells. (4) Real-time PCR to determine changes in RANKL and MMP-9 gene expression in Runx2 knockdown MM1.S and 5TGM1 myeloma cells. (5) Assessment of tumor growth/burden and bone resorption in the 5TGM1 syngenic model of murine myeloma. 5TGM1 cells with specific knockdown of Runx2 or non-target shRNA were injected into C57BL/KaLwRij mice through the tail vein and levels of IgG2b and TRAP5b were monitored in the sera of the mice by ELISA. Results.We find Runx2 expression is significantly increased in CAG myeloma cells expressing a high level of heparanase and dramatically reduced in HPSE k/d cells. Increased Runx2 in HPSE-high myeloma cells results in an increase in expression of RANKL and MMP-9 mRNA as well as MMP-9 activity. In sharp contrast, knockdown of Runx2 in human myeloma MM1.S and murine myeloma 5TGM1 cells results in a significant reduction of RANKL, MMP-9 gene expression and MMP-9 activity. Thus heparanase promotes RANKL and MMP-9 expression via Runx2. Analysis of initial 1-kb sequences of RANKL and MMP-9 gene promoter in human and mice, revealed presence of multiple high affinity Runx2-binding sites. ChIP assay confirmed that Runx2 mediates induction of RANKL and MMP-9 gene transcription in the both human and murine myeloma cells by direct association with the proximal promoter of these genes. Together these results demonstrate that Runx2 is a positive regulator of RANKL and MMP-9 gene expression in myeloma cells. Finally, in the syngenic model, knockdown of Runx2 in 5TGM1 murine myeloma cells remarkably inhibits the growth of these cells in vivo and bone resorption. Conclusions.Runx2 is a central regulator of heparanase induced myeloma bone disease. Our discoveries provide new insight into the mechanism of myeloma-induced bone disease and identify Runx2 as a novel target to block myeloma bone disease. Disclosures:No relevant conflicts of interest to declare.

Increased Sclerostin Secretion in Multiple Myeloma Plays a Central Role in Osteolytic Bone Disease

Abstract 3989Bone Marrow Stromal Cells (BMSC), Osteoblasts (OB) and osteoclasts (OC) are a central part of the bone microenvironment and play a crucial role in multiple myeloma (MM) growth and survival. Their imbalance results in osteolytic lesions. Understanding the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of MM.The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis.However, the role of Sclerostin in MM remains to be elucidated. Our objective was to evaluate the role of sclerostin in MM bone disease and confirm that sclerostin directed strategies are an effective approach in MM.We observed higher levels of sclerostin in MM patients’ plasma compared to leukemia patients, gastric cancer patients and healthy volunteers. Importantly, sclerostin levels were associated with an increase in tumor burden suggesting that MM cells are associated with the increase levels of sclerostin. Sclerostin concentrations similar to those detected in MM patients’ plasma inhibited OB differentiation and an anti-sclerostin neutralizing antibody (R&D Systems) reversed this effect. Furthermore, sclerostin increased TRAP positive OC numbers differentiated from MM patients’ peripheral blood mononuclear cell (PBMC) and their function as detected by pit formation assay. This was associated with stimulation of Ca2+/calmodulin-dependent protein kinases II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling in preosteoclasts reversed by specific inhibitors with consequent inhibition of osteoclastogenesis. Moreover, sclerostin stimulated JNK and CaMKII phosphorylation, stimulated mRNA expression of RANKL and inhibited mRNA expression of OPG in MM patient derived BMSC. RANKL plays a crucial role in promoting OC differentiation and OPG, the decoy receptor for RANKL, inhibits OC differentiation; therefore our results indicate that sclerostin accelerates OC differentiation by JNK and CaMKII signaling stimulation in BMSC in addition to its direct affect against OC.We next examined OB derived from MM patients' BMSC cocultured with the MM cell line INA6 by using cell culture inserts to avoid cell-cell contact. INA6 inhibited OB differentiation and sclerostin neutralizing antibody reversed the INA6 effect as assessed by qPCR and alizarin red staining. Interestingly, co-culture with MM cells stimulated sclerostin mRNA expression and sclerostin protein expression in OB well as in OB cocultures with MM cells. Moreover recombinant CCL3 protein stimulated sclerostin mRNA expression in MM cells. Because CCL3 is secreted by MM cells, these data suggest in part the mechanism by which sclerostin is increased in MM-OB cocultures.These data suggest sclerostin is secreted by MM cells and OB and inhibits osteoblastogenesis and stimulates osteoclastogenesis directly and indirectly. Neutralizing sclerostin levels reverses these effects. Taken together, our data suggest that sclerostin is a good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures:Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.