Non-Coding RNAs in Multiple Myeloma Bone Disease Pathophysiology

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.

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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 ◽

10.1182/blood.v120.21.568.568 ◽

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.

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Brain-Derived Neurotrophic Factor Is a Potential Osteoclast Stimulatory Factor In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.1917.1917 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1917-1917

Author(s):

Chun-Yan Sun ◽

Yu Hu ◽

Xiao-Mei She ◽

You Qin ◽

Lu Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Resorption ◽

Bone Disease ◽

Neurotrophic Factor ◽

Brain Derived Neurotrophic Factor ◽

Osteoclast Formation ◽

Bone Lesions ◽

Bone Marrow Plasma

Abstract Abstract 1917 Background and Objective: Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was present in marrow from patients with MM and possibly involved in MM cell-induced osteolysis. Methods and Results: Levels of bone marrow plasma BDNF was measured by ELISA in a cohort of individuals with MM and controls. The concentration of BDNF was found to be significantly elevated in patients with MM (879 ± 93) pg/ml when compared with bone marrow plasma derived from normal control subjects (186 ± 52) pg/ml (p < 0.001). Moreover, bone marrow plasma levels of BDNF positively correlated with plasma cell burden and extent of bone disease in MM patients. In osteoclast formation assay, bone marrow plasma from 31 of 37 patients with MM tested significantly stimulated the formation of osteoclast when compared to controls (61.8 ± 7 [mean ± SEM for the 31 patients] versus 25.2 ± 6 TRAP+ multinucleated cells/well [mean ± SEM for the 12 controls]; p < 0.01). The effect was significantly blocked by a neutralizing antibody to BDNF (p < 0.05), suggesting a critical role for BDNF in osteoclast activation. Furthermore, BDNF was found to dose-dependently increased the formation of multinucleated, TRAP+ osteoclast. The direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. Using reverse-transcriptase polymerase chain reaction analysis and western blotting assay, we demonstrated that BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF. These data suggested that TrkB is the functional receptor mediating BDNF's effect on osteoclast formation. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1α (r = 0.45, p < 0.005) and receptor activator of nuclear factor-κB ligand (RANKL) (r = 0.68, p < 0.0001), two major osteoclast stimulatory factors in MM. Conclusion: Taken together, our results demonstrate the ability of MM cells to secret BDNF correlates with the severity of osteoclastic bone resorption, and provide evidence that BDNF play a causal role in the development of MM bone lesions through TrkB receptor. Disclosures: No relevant conflicts of interest to declare.

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Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease

Blood ◽

10.1182/blood.v126.23.5318.5318 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5318-5318 ◽

Cited By ~ 1

Author(s):

Patrik Flodr ◽

Pavla Latalova ◽

Petra Pusciznova ◽

Tomas Pika ◽

Jaroslav Bacovsky ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Conflicts Of Interest ◽

Activin A ◽

Annexin A2 ◽

Bone Marrow Microenvironment ◽

Therapeutic Approaches ◽

Variable Expression ◽

Myeloma Bone Disease

Abstract Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens. Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2. Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%). Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A). Supported by the grant NT 14393. Disclosures No relevant conflicts of interest to declare.

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Multiple Myeloma Bone Disease: Implication of MicroRNAs in Its Molecular Background

International Journal of Molecular Sciences ◽

10.3390/ijms22052375 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2375

Author(s):

Aristea-Maria Papanota ◽

Paraskevi Karousi ◽

Christos K. Kontos ◽

Ioannis Ntanasis-Stathopoulos ◽

Andreas Scorilas ◽

...

Keyword(s):

Multiple Myeloma ◽

Signaling Pathways ◽

Bone Disease ◽

Plasma Cells ◽

Scientific Data ◽

Rna Molecules ◽

Symptomatic Disease ◽

Myeloma Bone Disease ◽

Osteoblast Activity ◽

Non Coding Rna

Multiple myeloma (MM) is a common hematological malignancy arising from terminally differentiated plasma cells. In the majority of cases, symptomatic disease is characterized by the presence of bone disease. Multiple myeloma bone disease (MMBD) is a result of an imbalance in the bone-remodeling process that leads to increased osteoclast activity and decreased osteoblast activity. The molecular background of MMBD appears intriguingly complex, as several signaling pathways and cell-to-cell interactions are implicated in the pathophysiology of MMBD. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the expression of their target mRNAs. Numerous miRNAs have been witnessed to be involved in cancer and hematological malignancies and their role has been characterized either as oncogenic or oncosuppressive. Recently, scientific research turned towards miRNAs as regulators of MMBD. Scientific data support that miRNAs finely regulate the majority of the signaling pathways implicated in MMBD. In this review, we provide concise information regarding the molecular pathways with a significant role in MMBD and the miRNAs implicated in their regulation. Moreover, we discuss their utility as molecular biomarkers and highlight the putative usage of miRNAs as novel molecular targets for targeted therapy in MMBD.

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Osteoclast Gene Expression Profiling in Multiple Myeloma

Blood ◽

10.1182/blood.v112.11.2735.2735 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2735-2735

Author(s):

Jerome Moreaux ◽

Dirk Hose ◽

Thierry Rème ◽

Philippe Moine ◽

Karène Mahtouk ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Bone Resorption ◽

Bone Disease ◽

Plasma Cells ◽

Osteoclastic Bone Resorption ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Osteolytic Bone Disease

Abstract Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption. Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.

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

Blood ◽

10.1182/blood.v87.4.1495.bloodjournal8741495 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1495-1501 ◽

Cited By ~ 76

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.

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

Nature Communications ◽

10.1038/s41467-019-12296-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

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.

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Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease

Oncotarget ◽

10.18632/oncotarget.2058 ◽

2014 ◽

Vol 5 (18) ◽

pp. 8284-8305 ◽

Cited By ~ 26

Author(s):

Antonio Garcia-Gomez ◽

Javier De Las Rivas ◽

Enrique M. Ocio ◽

Elena Díaz-Rodríguez ◽

Juan C. Montero ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Bone Disease ◽

Stromal Cells ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Transcriptomic Profile

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

Blood ◽

10.1182/blood-2007-10-120253 ◽

2008 ◽

Vol 112 (2) ◽

pp. 374-382 ◽

Cited By ~ 57

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.

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Involvement of LIGHT in multiple myeloma bone disease

Bone Abstracts ◽

10.1530/boneabs.3.pp152 ◽

2014 ◽

Author(s):

Angela Oranger ◽

Giacomina Brunetti ◽

Giorgio Mori ◽

Claudia Carbone ◽

Isabella Gigante ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Disease ◽

Myeloma Bone Disease

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