scholarly journals Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sarah Gooding ◽  
Sam W. Z. Olechnowicz ◽  
Emma V. Morris ◽  
Andrew E. Armitage ◽  
Joao Arezes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Disease ◽  
Bone Destruction ◽  
Bone Homeostasis ◽  
Pain Mechanisms ◽  
Transcriptomic Profiling ◽  
Myeloma Bone Disease ◽  
Bmp Pathway ◽  
Bmp Signalling

Abstract Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

scholarly journals Development of an in vivo model of human multiple myeloma bone disease

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
M Alsina ◽  
B Boyce ◽  
RD Devlin ◽  
JL Anderson ◽  
F Craig ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Bone Destruction ◽  
Long Bone ◽  
X Rays ◽  
Myeloma Bone Disease ◽  
Human Multiple Myeloma

Osteolytic bone destruction and its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due to increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent to myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgG kappa, as a model for human multiple myeloma, SCID mice were irradiated with 400 rads and mice were injected either with 10(6) ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation. Development of bone disease was assessed by blood ionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 to 35 days after injection and developed hypercalcemia (1.35 to 1.46 mmol/L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all the hypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones were obtained, concentrated, and assayed for bone-resorbing activity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with controls (percentage of total 45Ca released = 35% +/- 4% v 11% +/- 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-forming unit-granulocyte- macrophage [CFU-GM]) showed a threefold increase in CFU-GM from ARH-77 marrow versus controls (185 +/- 32 v 40 +/- 3 per 2 x 10(5) cell plated). Bone-resorbing human and murine cytokines such as interleukin- 6 (IL-6), IL-1 alpha or beta, TGF-alpha, lymphotoxin, and TNF alpha were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat long bone resorption assay when compared with untreated media (percentage of total 45Ca released = 22% +/- 2% v 11% +/- 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total 45Ca released = 19% +/- 1% and 22% +/- 1%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful to dissect the pathogenesis of the bone destruction in multiple myeloma.

Brain-Derived Neurotrophic Factor Enhances Osteoclastogenesis in Multiple Myeloma via Upregulation of RANKL/OPG Ratio Both in Vitro and in a Murine Model of Myeloma Bone Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 568-568
Author(s):  
Li-Sha Ai ◽  
Chun-Yan Sun ◽  
Tao Guo ◽  
Ya-Dan Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Neurotrophic Factor ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Myeloma Bone Disease ◽  
Trap Staining

Abstract Abstract 568 Osteolytic bone disease is a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption uncoupled with osteoblastic bone formation. Myeloma-induced osteoclastogenesis is largely depending on the increase of receptor activator of NF-κB ligand (RANKL) and decrease of osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as an MM-derived factor correlated with increased RANKL level and contributed to myeloma bone destruction. On the other hand, tyrosine receptor kinase B (TrkB), the receptor of BDNF, was found to be abundantly expressed by osteoblasts (OBs). Since OBs are the main source of RANKL and OPG in bone, here we sought to evaluate the involvement of BDNF/TrkB in the crosstalk between myeloma cells and OBs, as well as the effects of BDNF on RANKL/OPG ratio and myeloma bone disease. Co-cultures of OBs with pre-osteoclasts were performed in a non-contacted transwell system and treated with various concentration of BDNF. Osteoclast formation was detected with a tartrate-resistant acid phosphatase (TRAP) staining kit. Then, RANKL and OPG levels were measured when OBs cultures were exposed to BDNF or co-cultured with three human myeloma cell lines (RPMI8226, ARH-77 and U266). K252a (an inhibitor of TrkB) was present or absent in these systems to assess the effects of BDNF on RANKL/OPG expression in OBs. The involvement of downstream signaling molecules activated by BDNF in OBs was also investigated in this study, with the use of U0126 and a specific small interfering RNA (siRNA) for TrkB. For in vivo study, ARH-77 cells were stably transfected with an antisense short-hairpin RNA construct to BDNF (AS-ARH) or empty vector (EV-ARH). These cells were then intravenously injected to severe combined immunodeficiency (SCID) mice, to test their capacity to induce MM bone disease. Radiographs of mice tibiae and vertebrae were taken weekly by X ray. Changes in total body bone mineral density (BMD) of mice skeleton were recorded. At the end of the experiment, bone sections were stained with hematoxylin and eosin staining or TRAP staining. Secretion levels of RANKL and OPG in mice bone marrow were measured by ELISA. We showed that BDNF increased RANKL and decreased OPG production in OBs in a time- and dose-dependent manner, thus contributing to osteoclast formation in vitro. In addition, these effects were completely abolished by K252a and TrkB-siRNA (P < 0.05). BDNF regulates RANKL/OPG expression in OBs through the TrkB/ERK signaling pathway. Our in vivo results indicated that mice injected with AS-ARH cells, which expressed low levels of endogenous BDNF, were preserved and exhibited no radiologically identifiable osteolytic lesions. In addition, mice in AS-ARH group also had a lower incidence of vertebral compression deformities and paralysis in comparison with mice in EV-ARH group (P < 0.05). Further more, bones harboring AS-ARH cells showed marked reduction of RANKL/OPG ratio and osteoclast density when compared to the controls harboring EV-ARH cells (P < 0.05). Our results demonstrate that BDNF is an important contributor to osteoclastogenesis in MM. Antisense inhibition of BDNF in MM cells remarkably inhibited osteolytic bone destruction in SCID-ARH mice model. BDNF-induced bone destruction is partially mediated by MM-OB interactions via upregulation of RANKL/OPG ratio in the bone marrow milieu. These findings suggest targeting BDNF may become a new therapeutic strategy to improve patient outcome in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Increasing Wnt signaling in the bone marrow microenvironment inhibits the development of myeloma bone disease and reduces tumor burden in bone in vivo

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2833-2842 ◽  
Author(s):  
Claire M. Edwards ◽  
James R. Edwards ◽  
Seint T. Lwin ◽  
Javier Esparza ◽  
Babatunde O. Oyajobi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Bone Disease ◽  
Critical Role ◽  
Tumor Burden ◽  
Bone Marrow Microenvironment ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of β-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

Bone Marrow Monocyte / Macrophage Derived Activin A Mediates the Osteoclastogenic Effects of IL-3 in Myeloma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3933-3933 ◽  
Author(s):  
Rebecca Silbermann ◽  
Marina Bolzoni ◽  
Paola Storti ◽  
Benedetta Dalla Palma ◽  
Sabrina Bonomini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Disease ◽  
Healthy Subjects ◽  
Research Funding ◽  
Bone Destruction ◽  
Activin A ◽  
Negative Regulator ◽  
Normal Donor ◽  
Myeloma Bone Disease

Abstract Abstract 3933 Multiple myeloma (MM) is characterized by bone destruction with suppressed new bone formation that is mediated by multiple factors including MIP-1α (CCL3), RANKL, IL-3, DKK1 and recently, Activin A (ActA). IL-3 is a bifunctional cytokine that indirectly increases osteoclastogenesis and suppresses osteoblastogenesis via CD14+ bone marrow monocytes (BMM). Additionally, IL-3 levels are elevated in the BM plasma of MM patients compared with normals, however the mediators of IL-3's effects on myeloma bone disease are unknown. To investigate this we performed gene expression profiling using Affymetrix GeneChip® analysis of IL-3 treated BMM from MM patients and found ActA gene expression was increased 180-fold and confirmed this finding at the protein level by ELISA. ActA is a negative regulator of bone mass that promotes osteoclastogenesis and is overproduced in MM patients. Interestingly, treatment of MM cells or bone marrow stromal cells (BMSC) with IL-3 did not induce secretion of ActA. We found that ActA was produced by MM and MGUS patient CD14+ cells treated with IL-3 to a significantly higher degree compared to healthy subjects. (Median ActA levels for MM and MGUS ActA levels were increased 66.57 and 51.6 fold respectively over untreated cells, while IL-3 treatment of normal cells increased ActA 8.5 fold.) ActA levels were also increased in freshly isolated marrow plasma of a cohort of patients with active MM as compared to patients with smoldering MM (SMM), MGUS, or healthy subjects (median ActA levels: active MM 453 pg/ml, SMM 328 pg/ml, MGUS 332 pg/ml, normal 286 pg/ml). ActA levels in MM patients with and without bone disease were not significantly different. (Median value with bone disease 463pg/ml vs. 407 pg/ml without bone disease.) ActA has also been reported to have a role in the differentiation and polarization of CD14+ tumor associated macrophages (TAMs), which are osteoclast precursors and can block MM cell apoptosis. Therefore, we examined the potential role of ActA in IL-3 mediated osteoclast (OCL) formation. Culture of normal BMM with ActA or IL-3 significantly enhanced osteoclastogenesis compared with control (mean number of OCL / 1×105 normal marrow non-adherent cells plated in IL-3 (100pg/ml) treated cultures 73; ActA (1ng/ml) treated cultures 123; cultures with vehicle alone, 8), and ActA enhanced RANKL-induced osteoclastogenesis. Osteoprotegerin treatment of normal donor BMM stimulated with ActA failed to block the osteoclastogenic effects of ActA, demonstrating that ActA's osteoclastogenic effects were RANKL independent. Importantly, the osteoclastogenic effect of IL-3 was dose-dependently inhibited by anti-ActA, and IL-3 induced ActA expression by BMM decreased during OCL differentiation. In support of early OCL precursors as the source of IL-3 induced ActA, we did not identify IL-3 receptors on mature OCL by flow cytometry. These results demonstrate that IL-3 induction of osteoclastogenesis is mediated by ActA produced by CD14+ BMM and is RANKL independent. Thus, we hypothesize that therapies targeting the ActA receptor, such as the recently developed ActA receptor antagonist, should block both IL-3 and ActA, and thereby significantly impact MM bone disease via their effects on TAMs. Disclosures: Bolzoni: Celgene Italy: Research Funding. Roodman:Millennium: Consultancy; Amgen: Consultancy. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

scholarly journals Non-Coding RNAs in Multiple Myeloma Bone Disease Pathophysiology

2020 ◽  
Vol 6 (3) ◽  
pp. 37 ◽  
Author(s):  
Lavinia Raimondi ◽  
Angela De Luca ◽  
Gianluca Giavaresi ◽  
Stefania Raimondo ◽  
Alessia Gallo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Osteoclast Formation ◽  
Bone Marrow Niche ◽  
Bone Homeostasis ◽  
Myeloma Bone Disease ◽  
Non Coding Rna ◽  
Cellular Components ◽  
Recent Attention

Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhanced osteoclastogenesis responsible of MM-related bone disease (MMBD). Several studies have disclosed the mechanisms underlying increased osteoclast formation and activity triggered by the various cellular components of the MM bone marrow microenvironment, leading to the identification of novel targets for therapeutic intervention. In this regard, recent attention has been given to non-coding RNA (ncRNA) molecules, that finely tune gene expression programs involved in bone homeostasis both in physiological and pathological settings. In this review, we will analyze major signaling pathways involved in MMBD pathophysiology, and report emerging evidence of their regulation by different classes of ncRNAs.

Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 812-812
Author(s):  
Claire M. Edwards ◽  
James R. Edwards ◽  
Seint T. Lwin ◽  
Gregory R. Mundy
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Disease ◽  
Tumor Burden ◽  
Bone Microenvironment ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.

Myeloma bone disease and proteasome inhibition therapies

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1098-1104 ◽  
Author(s):  
Evangelos Terpos ◽  
Orhan Sezer ◽  
Peter Croucher ◽  
Meletios-Athanassios Dimopoulos
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Bone Disease ◽  
Proteasome Inhibitors ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Bone Specific Alkaline Phosphatase ◽  
Myeloma Bone Disease

AbstractBone disease is one of the most debilitating manifestations of multiple myeloma. A complex interdependence exists between myeloma bone disease and tumor growth, creating a vicious circle of extensive bone destruction and myeloma progression. Proteasome inhibitors have recently been shown to promote bone formation in vitro and in vivo. Preclinical studies have demonstrated that proteasome inhibitors, including bortezomib, which is the first-in-class such agent, stimulate osteoblast differentiation while inhibiting osteoclast formation and bone resorption. Clinical studies are confirming these observations. Bortezomib counteracts the abnormal balance of osteoclast regulators (receptor activator of nuclear factor-κB ligand and osteoprotegerin), leading to osteoclast inhibition and decreased bone destruction, as measured by a reduction in markers of bone resorption. In addition, bortezomib stimulates osteoblast function, possibly through the reduction of dickkopf-1, leading to increased bone formation, as indicated by the elevation in bone-specific alkaline phosphatase and osteocalcin. The effect of bortezomib on bone disease is thought to be direct and not only a consequence of the agent's antimyeloma properties, making it an attractive agent for further investigation, as it may combine potent antimyeloma activity with beneficial effects on bone. However, the clinical implication of these effects requires prospective studies with specific clinical end points.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 567-567
Author(s):  
Li Nan ◽  
Haiyan Chen ◽  
Jian Ruan ◽  
Rebecca S Sollie ◽  
Cara Schafer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Resorption ◽  
Bone Disease ◽  
Bone Destruction ◽  
Chip Assay ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
D Cells ◽  
High Level

Abstract 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.

IL3 Induces Osteoclastogenesis In Vivo and Is Modulated By Bone Marrow Monocyte / Macrophage Derived Activin A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3101-3101
Author(s):  
Rebecca Silbermann ◽  
Marina Bolzoni ◽  
Paola Storti ◽  
Daniela Guasco ◽  
Judith Anderson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Disease ◽  
Research Funding ◽  
Bone Destruction ◽  
Neutralizing Antibody ◽  
Myeloma Cell ◽  
Receptor Expression ◽  
Saline Control ◽  
Downstream Effects

Abstract Multiple myeloma (MM) bone disease is characterized by bone destruction with suppressed bone formation resulting in enhanced tumor growth. We previously showed that IL3 is a multifunctional cytokine that is elevated in the marrow plasma of MM patients as compared with healthy subjects and that IL3 increases myeloma cell and osteoclast (OCL) precursor proliferation and suppresses osteoblastogenesis via an indirect mechanism mediated by bone marrow monocyte / macrophages (BMM). In addition, IL3 has widespread proliferative and differentiation effects on multiple hematopoietic cell types. Thus attempts at targeting IL3 to date have been unsuccessful due to IL3's impact on normal hematopoiesis. Therefore, identification of IL3's downstream effects in MM bone disease is critical for effective targeting of IL3 in MM. We demonstrated that IL3 induces human OCL formation and that an IL3 neutralizing antibody inhibits OCL formation stimulated by bone marrow (BM) plasma containing high levels of IL3 from MM patients. We recently reported that treatment of MM patient CD14+ BMM with IL3 induces high levels of Actvin A (ActA), a TGF-β superfamily member that, like IL3, modulates MM bone disease by enhancing osteoclastogenesis and inhibiting osteoblasts. As IL3 induces ActA secretion from CD14+ BMM, and has a very similar effect to IL3 on OCL formation, we treated OCL precursors with IL3 in the presence of a neutralizing antibody to ActA (anti-ActA) and demonstrated that anti-ActA dose-dependently inhibited the osteoclastogenic effect of IL-3 on OCL formation. We also showed that ActA stimulates osteoclastogenesis via a RANKL-independent mechanism, and that, in support of early OCL precursors as the source of ActA induced by IL3, IL3 receptor expression is highest on early OCL precursors and decreases during OCL differentiation. We now report in vivoconfirmation of IL3's osteoclastogenic effects. Female C57BL mice were injected intraperitoneally with saline or anti-ActA for 7 days. Beginning on day 3, mice were injected subcutaneously over the calvaria with IL3 or saline daily for 5 days. Mice were sacrificed after 7 days and calvaria were sectioned and stained for TRAP. A blinded observer performed quantitative histologic analyses, and TRAP+ OCL were counted on the endosteal bone surfaces. TRAP+ OCL numbers on the endosteal bone surfaces (N.Oc), corrected for bone surface area (BS), were dramatically increased by IL3 treatment compared to saline treated controls (mean N.Oc/BS supracalvarial IL3 treatment 18.51, SD 2.14 vs saline control 13.42, SD 1.18, p<0.05.) Further, treatment of mice with anti-ActA prior to and during supracalvarial IL3 treatment significantly reduced N.Oc/BS induced by IL3 (mean N.Oc/BS supracalvarial IL-3 with intraperitoneal anti-ActA 11.01, SD 1.2, p<0.01). These findings are the first demonstration that IL3 induces osteoclastogenesis in vivo and that IL3 induced osteoclastogenesis is completely blocked in vivo by a neutralizing antibody to ActA. These results suggest that therapies targeting ActA activity should block IL3's effects in MM bone disease, and supports investigation of IL3 receptor blockade for the modulation of IL3's downstream effects in MM bone disease. Disclosures: Roodman: Amgen: Consultancy; Eli Lilly and Co.: Research Funding. Giuliani:Celgene Italy: Research Funding.

scholarly journals Wnt3a signaling within bone inhibits multiple myeloma bone disease and tumor growth

Blood ◽  
2008 ◽  
Vol 112 (2) ◽  
pp. 374-382 ◽  
Author(s):  
Ya-Wei Qiang ◽  
John D. Shaughnessy ◽  
Shmuel Yaccoby
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Bone Disease ◽  
Tumor Burden ◽  
Bone Homeostasis ◽  
Mineral Density ◽  
Myeloma Bone Disease ◽  
Empty Vector

Abstract Canonical Wnt signaling is central to normal bone homeostasis, and secretion of Wnt signaling inhibitors by multiple myeloma (MM) cells contributes to MM-related bone resorption and disease progression. The aim of this study was to test the effect of Wnt3a on bone disease and growth of MM cells in vitro and in vivo. Although Wnt3a activated canonical signaling in the majority of MM cell lines and primary cells tested, Wnt3a had no effect on MM cell growth in vitro. Moreover, forced expression of Wnt3a in H929 MM cells conferred no growth advantage over empty vector-transfected cells in vitro or importantly when grown subcutaneously in severe combined immunodeficient (SCID) mice. Importantly, although H929 cells stably expressing an empty vector injected into human bone grew rapidly and induced a marked reduction in bone mineral density, bones engrafted with Wnt3a-expressing H929 cells were preserved, exhibited increased osteoblast-to-osteoclast ratios, and reduced tumor burden. Likewise, treatment of myelomatous SCID-hu mice, carrying primary disease, with recombinant Wnt3a stimulated bone formation and attenuated MM growth. These results provide further support of the potential anabolic and anti-MM effects of enhancing Wnt signaling in the bone.

Sign in / Sign up

Export Citation Format

Share Document