The Effect of Myeloma Cells on Bone Homeostasis Is Heterogenous and Correlates with Underlying Genetic Lesions and Bone Disease In Vivo

Abstract Abstract 4052 Osteolytic bone disease is a major clinical burden in multiple myeloma. Uncoupling of normal homestatic bone remodelling by an increase in osteoclast numbers and suppression of osteoblasts is the main causative mechanism. Subgroups of patients, identified by recurrent IgH translocation partners and D-type cyclin expression, have different clinical features, including prevalence of bone disease. Molecular groups characterised by t(4:14) or t(14:16) translocations, have a lower incidence of lytic bone lesions when compared with subgroups characterised by t(11:14) and/or cyclin D1 expression. We hypothesize that while osteoclast activation is a common phenomenon, osteolysis depends upon the presence and degree of osteoblast suppression, which in turn is dictated by the underlying genetic lesion. We have recently established a medullary model of myeloma using the human KMS12BM cell line that bears t(11;14). This model exhibits lytic bone disease as evidenced by micro-computed-tomography (microCT) and histomorphometry, accompanied by increased osteoclastogenesis and suppressed osteoblast numbers. These features are typical of the clinical disease bearing t(11;14). In addition, we have established a second medullary model of an alternative molecular disease group using the MM1.s human myeloma cell line, bearing t(14;16). Non-irradiated β2M NOD/SCID mice intravenously injected with either the KMS12BM or MM1s cells develop tumours confined to the bone marrow with little extramedullary disease. Histomorphometric analysis of femora from diseased animals revealed several striking differences in bone biology between these two models. Early disease progression in the KMS12BM model is characterised by an increase in osteoblast numbers (p < 0.05) with little change in osteoclast numbers at trabecular surfaces. As homeostatic bone remodelling processes become uncoupled in advanced disease there is a loss of osteoblasts and an increase in osteoclast numbers. In contrast, in the MM1.s model, there are no significant changes in osteoblast or osteoclast numbers at the trabecular surfaces. The distinct impact of t(11:14) and t(14:16) modelled disease on bone is further illustrated by microCT analysis of femora of diseased animals. Femora of KMS12BM mice show a reduction in trabecular number and thickness (p < 0.05) with bone loss further highlighted by an increased structure model index (SMI, p < 0.05). MM1.s disease results in increased bone volume (p < 0.01) with a concordant increase in trabecular thickness (p < 0.05) and number (p < 0.01), and little change in SMI. Importantly, while bone disease in the KMS12BM model is typified by osteolytic lesions, MM1.s animals do not appear to develop osteolytic lesions, despite perturbation of bone biology. These models, representing distinct genetic subtypes of myeloma, produce different clinico-pathological effects on bone remodelling, perhaps by differential effects on osteoblast biology. They will facilitate the identification of critical molecular pathways involved in osteoblast suppression leading to bone loss. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.3989.3989 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3989-3989

Author(s):

Homare Eda ◽

Loredana Santo ◽

Diana Cirstea ◽

Andrew J. Yee ◽

Anuj Mahindra ◽

...

Keyword(s):

Multiple Myeloma ◽

Mrna Expression ◽

Bone Disease ◽

Crucial Role ◽

Research Funding ◽

Neutralizing Antibody ◽

Bone Homeostasis ◽

Osteolytic Lesions ◽

Osteolytic Bone Disease

Abstract Abstract 3989 Bone 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.

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Increased Sclerostin Secretion in Multiple Myeloma Results in Stimulation of Osteoclastogenesis and Inhibition of Osteoblastogenesis

Blood ◽

10.1182/blood.v118.21.1819.1819 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1819-1819

Author(s):

Homare Eda ◽

Loredana Santo ◽

Diana D. Cirstea ◽

Samantha Pozzi ◽

Miriam Canavese ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Plasma Concentrations ◽

Bone Homeostasis ◽

Advisory Committees ◽

Good Target ◽

Osteolytic Bone Disease

Abstract Abstract 1819 Objectives: Osteoblasts (OB) and osteoclasts (OC), are an integral part of the bone microenvironment, and play a crucial role in myeloma growth and survival. Their imbalance results in osteolytic disease and elucidating the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of multiple myeloma (MM). The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. It is regarded as a good target for osteoporosis treatment, but its role in MM remains to be determined. Our objective was to study the role of sclerostin in MM bone disease and determine if sclerostin directed strategies were a reasonable approach in MM. Methods and Results: Sclerostin concentration in patients' blood plasma and MM cell line supernatant stimulated by IL-6, FGF-2, TNFalpha, BMP7 and TGFbeta was detected by ELISA (ALPCO immunoassays). Increased level of sclerostin was detected in MM patient plasma (n=20, median: 4.73 ng/mL, range: 1.5–19.5 ng/mL). Plasma concentrations were significantly higher (p<0.01) when compared to sclerostin concentration in the plasma of leukemia patients (n=3), gastric cancer patients (n=40) and healthy volunteers (n=4). High sclerostin levels were not associated with extent of bone disease but rather correlated with tumor burden (High B2M, creatinine and LDH, and low Hb) suggesting an autocrine loop for sclerostin production. Because sclerostin is derived from mature OB or orteocytes, we measured levels during OB differentiation but we were unable to detect increased levels. We then measured sclerostin levels in RPMI-8226 MM cell line supernatant either alone or stimulated by cytokines D Systems). Conclusions: These data demonstrate that increased sclerostin levels in MM patients inhibit osteoblastogenesis and stimulate osteoclastogenesis. Taken together, sclerostin may be good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures: Raje: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

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Inhibitory Effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside on RANKL-Induced Osteoclast Differentiation and ROS Generation in Macrophages

International Journal of Molecular Sciences ◽

10.3390/ijms22010222 ◽

2020 ◽

Vol 22 (1) ◽

pp. 222

Author(s):

Eun-Nam Kim ◽

Ga-Ram Kim ◽

Jae Sik Yu ◽

Ki Hyun Kim ◽

Gil-Saeng Jeong

Keyword(s):

Bone Loss ◽

Bone Formation ◽

Bone Disease ◽

Osteoclast Differentiation ◽

Inhibitory Effect ◽

Bone Diseases ◽

Ros Generation ◽

Bone Homeostasis ◽

Disease Treatment ◽

And Migration

In bone homeostasis, bone loss due to excessive osteoclasts and inflammation or osteolysis in the bone formation process cause bone diseases such as osteoporosis. Suppressing the accompanying oxidative stress such as ROS in this process is an important treatment strategy for bone disease. Therefore, in this study, the effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (BAG), an arylbutanoid glycoside isolated from Betula platyphylla var. japonica was investigated in RANKL-induced RAW264.7 cells and LPS-stimulated MC3E3-T1 cells. BAG inhibited the activity of TRAP, an important marker of osteoclast differentiation and F-actin ring formation, which has osteospecific structure. In addition, the protein and gene levels were suppressed of integrin β3 and CCL4, which play an important role in the osteoclast-induced bone resorption and migration of osteoclasts, and inhibited the production of ROS and restored the expression of antioxidant enzymes such as SOD and CAT lost by RANKL. The inhibitory effect of BAG on osteoclast differentiation and ROS production appears to be due to the inhibition of MAPKs phosphorylation and NF-κβ translocation, which play a major role in osteoclast differentiation. In addition, BAG inhibited ROS generated by LPS and effectively restores the mineralization of lost osteoblasts, thereby showing the effect of bone formation in the inflammatory situation accompanying bone loss by excessive osteoclasts, suggesting its potential as a new natural product-derived bone disease treatment.

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Reprogrammed marrow adipocytes contribute to myeloma-induced bone disease

Science Translational Medicine ◽

10.1126/scitranslmed.aau9087 ◽

2019 ◽

Vol 11 (494) ◽

pp. eaau9087 ◽

Cited By ~ 13

Author(s):

Huan Liu ◽

Jin He ◽

Su Pin Koh ◽

Yuping Zhong ◽

Zhiqiang Liu ◽

...

Keyword(s):

Bone Disease ◽

Unique Feature ◽

Histone H3 ◽

Bone Lesions ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Osteolytic Lesions ◽

Therapeutic Implications ◽

Polycomb Repressive Complex 2 ◽

Bone Changes

Osteolytic lesions in multiple myeloma are caused by osteoclast-mediated bone resorption and reduced bone formation. A unique feature of myeloma is a failure of bone healing after successful treatment. We observed adipocytes on trabecular bone near the resorbed area in successfully treated patients. Normal marrow adipocytes, when cocultured with myeloma cells, were reprogrammed and produced adipokines that activate osteoclastogenesis and suppress osteoblastogenesis. These adipocytes have reduced expression of peroxisome proliferator–activated receptor γ (PPARγ) mediated by recruitment of polycomb repressive complex 2 (PRC2), which modifies PPARγ promoter methylation at trimethyl lysine-27 histone H3. We confirmed the importance of methylation in the PPARγ promoter by demonstrating that adipocyte-specific knockout of EZH2, a member of the PRC2, prevents adipocyte reprogramming and reverses bone changes in a mouse model. We validated the strong correlation between the frequency of bone lesions and the expression of EZH2 in marrow adipocytes from patients in remission. These results define a role for adipocytes in genesis of myeloma-associated bone disease and that reversal of adipocyte reprogramming has therapeutic implications.

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Inhibition of p38α MAPK Reduces Tumor Burden, Prevents the Development of Myeloma Bone Disease, and Increases Survival in the 5T2 and 5T33 Murine Models of Myeloma.

Blood ◽

10.1182/blood.v108.11.3436.3436 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3436-3436 ◽

Cited By ~ 7

Author(s):

Karin Vanderkerken ◽

Satya Medicherla ◽

Les Coulton ◽

Benjamin Van Camp ◽

Andy Protter ◽

...

Keyword(s):

Bone Disease ◽

Critical Role ◽

Disease Free Survival ◽

Myeloma Cell ◽

Bone Lesions ◽

Myeloma Cells ◽

Growth And Survival ◽

Myeloma Bone Disease ◽

Osteolytic Bone Disease

Abstract The bone microenvironment plays a critical role in supporting the growth and survival of myeloma cells and the development of osteolytic bone disease. Signalling through p38 α MAPK mediates synthesis of myeloma cell survival factors by stromal cells; whereas, inhibiting p38 α MAPK reduces myeloma cell proliferation and inhibits osteoclast formation in vitro. However, it is unclear whether p38 α MAPK inhibition will prevent the growth and survival of myeloma cells and the bone disease in vivo. The aim of this study was to determine whether SCIO-469, a selective p38 α MAPK inhibitor, would inhibit myeloma growth and prevent the development of bone disease in the 5TMM syngeneic models of myeloma. Treatment of 5TMM cells, in vitro, with SCIO-469 resulted in a clear inhibition of p38 phosphorylation, as assessed by Western blotting and an inhibition up to 35% of stromal cell induced 5T33MM proliferation. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the growth of myeloma in bone and the development of bone disease characterized by increased osteoclast surface (p<0.05), a reduction in cancellous bone (p<0.01) and the presence of osteolytic bone lesions on x-ray (p<0.01). Treatment of 5T2MM-bearing mice with SCIO-469 (150mg/kg in the diet, therapeutical treatment from paraprotein detection) resulted in a 42% decrease in serum paraprotein and prevented development of osteolytic lesions (p<0.01). Injection of 5T33MM cells into C57Bl/KaLwRij mice also resulted in the development of myeloma but not associated bone disease. Treatment of 5T33MM-bearing mice from the time of tumor cell injection with SCIO-469 resulted in a decrease in serum paraprotein (8.8+/−1.4g/dl to 0.04+/− 0.03g/dl, p<0.001) and a reduction in the proportion of tumor cells in the bone marrow (67 +/− 8.1% to 1.09 +/− 0.58%, p<0.001). Kaplan-Meier analysis demonstrated an increase in disease-free survival (veh=27.5 days vs 96 days, p<0.001) after treatment of the mice with SCIO-469. These data demonstrate that targeting p38 α MAPK with SCIO-469 is associated with an anti-myeloma effect, which indirectly prevents the development of myeloma bone disease.

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Circulating microRNAs Correlate with Multiple Myeloma and Skeletal Osteolytic Lesions

Cancers ◽

10.3390/cancers13215258 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5258

Author(s):

Sara Reis Moura ◽

Hugo Abreu ◽

Carla Cunha ◽

Cláudia Ribeiro-Machado ◽

Carla Oliveira ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Disease ◽

Area Under The Curve ◽

Genetic Alterations ◽

Disease Stage ◽

Bone Lesions ◽

Healthy Controls ◽

Osteolytic Lesions ◽

Circulating Micrornas ◽

Myeloma Bone Disease

Multiple myeloma (MM) is the second most frequent hematological disease and can cause skeletal osteolytic lesions. This study aims to evaluate the expression of circulating microRNAs (miRNAs) in MM patients and to correlate those levels with clinicopathological features, including bone lesions. A panel of miRNAs associated with MM onset and progression, or with bone remodeling, was analyzed in the plasma of 82 subjects (47 MM patients; 35 healthy controls). Results show that miR-16-5p, miR-20a-5p, and miR-21-5p are differently expressed between MM patients and healthy controls. Receiver operating characteristic analyses indicate that their combined expression has potential as a molecular marker (Area Under the Curve, AUC of 0.8249). Furthermore, significant correlations were found between the analyzed miRNAs and disease stage, treatment, β2 microglobulin, serum albumin and creatinine levels, but not with calcium levels or genetic alterations. In this cohort, 65.96% of MM patients had bone lesions, the majority of which were in the vertebrae. Additionally, miR-29c-3p was decreased in patients with osteolytic lesions compared with patients without bone disease. Interestingly, circulating levels of miR-29b-3p correlated with cervical and thoracic vertebral lesions, while miR-195-5p correlated with thoracic lesions. Our findings suggest circulating miRNAs can be promising biomarkers for MM diagnosis and that their levels correlate with myeloma bone disease and osteolytic lesions.

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The Src Inhibitor AZD0530 Prevents the Development of Osteolytic Bone Disease in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3018.3018 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3018-3018

Author(s):

Roy Heusschen ◽

Joséphine Muller ◽

Marilène Binsfeld ◽

Erwan Plougonven ◽

Nadia Mahli ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Disease ◽

Osteoblast Differentiation ◽

Osteoclast Differentiation ◽

Disease Stage ◽

Bone Lesions ◽

Mrna Levels ◽

Osteoblast Function ◽

Src Inhibitor ◽

Osteolytic Bone Disease

Abstract Destructive bone lesions due to osteolytic bone disease are a major cause of morbidity and mortality in multiple myeloma patients, occurring in more than 80% of cases. Underlying osteolytic bone disease is an uncoupling of the bone remodeling process, with an increased activity of osteoclasts and a decreased activity of osteoblasts. Current strategies to treat osteolytic bone disease focus on anti-resorptive agents, which do not rebuild bone loss. Src kinase has been implicated in both osteoclast and osteoblast function. In this study, we assessed the effect of Src inhibition with AZD0530 (saracatinib, Astra Zeneca) on the development of multiple myeloma and its associated osteolytic bone disease. We first determined Src family kinase expression in the multiple myeloma microenvironment and found that patient-derived myeloma cells express Src at low levels but disease stage does not correlate with Src expression levels. In accordance with the literature, Src mRNA expression was found to increase during osteoclast differentiation and decrease during osteoblast differentiation in publicly available microarray datasets. Next, we validated an inhibitory role of AZD0530 on osteoclast differentiation and function. At a pharmacological relevant concentration of 1 micromolar, AZD0530 inhibited the differentiation of RAW264.7 osteoclasts (Oc.N/FOV: 15.5+-1.6 treated vs. 53+-1.5 non-treated). AZD0530 treatment appeared to hamper efficient progenitor cell fusion and osteoclast polarization, reflected by a decrease of CTSK and DC-STAMP mRNA levels and a defective actin ring formation in treated cultures, which culminated in a complete inhibition of bone resorption. When assessing the effect of AZD0530 on osteoblast function we found that AZD0530 inhibits osteoblast differentiation, with a decreased expression of OSX and OCN, and alters osteoblast morphology. In vivo, AZD0530 did not alter myeloma cell bone marrow infiltration in both the 5TGM.1 (37+-6.3% AZD0530 treated vs. 25.2+-6.7% non-treated) and 5T2MM (26.1+-7.7% vs. 29.1+-6.4%) murine multiple myeloma models. However, bone health was significantly improved in both models following treatment with AZD0530. In the 5TGM.1 model multiple trabecular bone parameters were restored to levels observed in healthy control mice following AZD0530 treatment, including BV/TV (11.7+-0.3% treated vs. 6.4+-0.3% non-treated), Tb.N. (2.5+-6x10^-2/mm vs. 1.7+-9x10^-2/mm) and Tb.Th (46.2+-1micron vs. 37+-0.8micron). These results were confirmed in the 5T2MM model, which displays a more severe osteolytic bone disease. In addition, AZD0530 treatment resulted in an increase in cortical thickness (157.8+-0.8micron treated vs. 151.4+-0.7micron non-treated) and a decrease in the number and size of cortical lesions in 5TGM.1 mice. Finally, our findings were corroborated by histomorphometric analyses. In conclusion, we report a potent inhibitory effect of the Src inhibitor AZD0530 on the development of osteolytic bone disease in multiple myeloma. Our results indicate that AZD0530 exerts this effect via the modulation of both osteoclast and osteoblast function. These findings warrant further study of the feasibility and efficacy of AZD0530 to treat osteolytic bone disease in multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

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Monoclonal immunoglobulins promote bone loss in multiple myeloma

Blood ◽

10.1182/blood.2020006045 ◽

2020 ◽

Vol 136 (23) ◽

pp. 2656-2666

Author(s):

Marita Westhrin ◽

Vlado Kovcic ◽

Zejian Zhang ◽

Siv H. Moen ◽

Tonje Marie Vikene Nedal ◽

...

Keyword(s):

Multiple Myeloma ◽

Sialic Acid ◽

Bone Loss ◽

Bone Disease ◽

Osteoclast Differentiation ◽

Osteolytic Bone Disease ◽

Monoclonal Immunoglobulins ◽

Diagnostic Criterion ◽

Acid Precursor

Abstract Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient’s sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

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Ixazomib Improves Bone Remodeling and Counteracts Sonic Hedgehog Signaling Inhibition Mediated by Myeloma Cells

Cancers ◽

10.3390/cancers12020323 ◽

2020 ◽

Vol 12 (2) ◽

pp. 323 ◽

Cited By ~ 5

Author(s):

Daniele Tibullo ◽

Anna Longo ◽

Nunzio Vicario ◽

Alessandra Romano ◽

Alessandro Barbato ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Bone Remodeling ◽

Sonic Hedgehog ◽

Bone Disease ◽

Human Mscs ◽

Bone Homeostasis ◽

Shh Signaling ◽

Shh Pathway ◽

Pathway Activation ◽

Osteolytic Bone Disease

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of plasma cells (PC) in the bone marrow (BM), leading to bone loss and BM failure. Osteolytic bone disease is a common manifestation observed in MM patients and represents the most severe cause of morbidity, leading to progressive skeletal damage and disabilities. Pathogenetic mechanisms of MM bone disease are closely linked to PCs and osteoclast (OCs) hyperactivity, coupled with defective osteoblasts (OBs) function that is unable to counteract bone resorption. The aim of the present study was to investigate the effects of Ixazomib, a third-generation proteasome inhibitor, on osteoclastogenesis and osteogenic differentiation. We found that Ixazomib was able to reduce differentiation of human monocytes into OCs and to inhibit the expression of OC markers when added to the OC medium. Concurrently, Ixazomib was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs), increasing osteogenic markers, either alone or in combination with the osteogenic medium. Given the key role of Sonic Hedgehog (SHH) signaling in bone homeostasis, we further investigated Ixazomib-induced SHH pathway activation. This set of experiments showed that Ixazomib, but not Bortezomib, was able to bind the Smoothened (SMO) receptor leading to nuclear translocation of GLI1 in human MSCs. Moreover, we demonstrated that PCs act as GLI1 suppressors on MSCs, thus reducing the potential of MSCs to differentiate in OBs. In conclusion, our data demonstrated that Ixazomib regulates bone remodeling by decreasing osteoclastogenesis and prompting osteoblast differentiation via the canonical SHH signaling pathway activation, thus, representing a promising therapeutic option to improve the complex pathological condition of MM patients.

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Blockade of Mer By the Small Molecule Inhibitor R992 Inhibits Multiple Myeloma and Its Associated Bone Disease By Restoring the Perturbed Bone Homeostasis

Blood ◽

10.1182/blood-2018-99-118273 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1922-1922 ◽

Cited By ~ 1

Author(s):

Janik Engelmann ◽

Isabel Ben Batalla ◽

Hanna Taipaleenmäki ◽

Kristoffer Riecken ◽

Victoria Gensch ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Disease ◽

Osteoblast Differentiation ◽

Biological Effects ◽

Osteoclast Differentiation ◽

Bone Homeostasis ◽

Mineral Density ◽

Trabecular Thickness ◽

Dose Dependent

Abstract Despite many therapeutic advances in recent years Multiple Myeloma (MM) still remains incurable in the majority of the patients. In addition, MM patients suffer significantly from co-morbidities including bone pain and renal insufficiency. Therefore, the development of novel treatments is warranted. The TAMR family consists of Tyro3, Axl and Mer which represent evolving targets in cancer. We demonstrated that the role of TAMR is non-redundant in hematologic malignancies, with Axl exerting an important function in AML, but not in MM, where Mer represents a novel target. Therefore, we tested the therapeutic potential of the Mer-inhibitor R992, which has an 8-fold selectivity over Tyro3 and a 13-fold selectivity over Axl in preclinical MM models (Rigel, San Francisco, USA). R992 exerted a dose-dependent growth inhibition of U266, JJN3 and RPMI8226 cells in vitro (n=3, *p<0.001). Mechanistically, Mer blockade inhibited proliferation in 5-bromo-2′-deoxyuridine assays and induced apoptosis as shown by increased numbers of Annexin V+ cells (n=3,*p<0.05 and *p<0.001, respectively). To delineate signaling pathways mediating the biological effects of Mer blockade in MM cells we investigated key mediators of MM cell proliferation and survival. Here, we found reduced phosphorylation of Akt upon Mer inhibition with R992. Furthermore, R992 inhibited mitogen-activated protein kinase (MAPK) pathways Erk and p38. Subsequently, we investigated whether inhibition of Mer signaling increases chemosensitivity of MM cells. Combination treatment of R992 with bortezomib and cyclophosphamide demonstrated that Mer inhibition significantly increased sensitivity of MM cells to these established MM therapies. Oral administration of 60mg/kg R992 BID to mice significantly reduced tumor burden in the U266 systemic myeloma mouse model. The λ light chain concentration and the CD138+ MM cell load was reduced 2-fold in R992 treated mice compared to placebo-treated mice 8 weeks after injection (n=5/5, *p<0.05 and n=4/5, *p<0.05, respectively). Importantly, treatment with R992 resulted in a significant prolongation of overall survival by 15 days in the U266 model (median OS 73 vs. 88 days (n=13/12, *p<0.05). In addition, treatment with R992 prolonged survival in the more aggressive JJN3 model (median OS 24 vs. 27 days, n=9/7, *p<0.005). For further phenotyping of the effects of R992 we performed microcomputed tomography (µCT) and histological analysis of the tibias in the U266 model. µCT analysis of proximal tibia metaphyses revealed, that bone volume and bone mineral density (BMD) were significantly increased by R992 (n=7/7, *p<0.05). Moreover, analysis of the metaphyseal spongiosa showed that R992 could retard myeloma-mediated destruction of trabecular bone area measured by increased trabecular number and increased trabecular thickness (n=7/7, *p<0.05). Interestingly, R992 could also enlarge the metaphyseal diameter due to thickened cortical bone. Vice versa, overexpression of the Mer ligands Gas6 and Pros1 in U266 and JJN3 cells led to increased osteoclast and decreased osteoblast differentiation in vitro and more rapid and destructive myeloma bone disease in vivo. These data suggest that the expression of Mer ligands represent thus far unrecognized mediators of MM-induced perturbed bone homeostasis. To directly assess the effect of R992 on osteoclasts, we treated osteoclast cultures with R992 and observed an inhibition of osteoclast differentiation by R992 alone and in co-culture with myeloma cells. Western blot analysis confirmed, that Mer phosphorylation was reduced by R992, whereas the phosphorylation of Tyro3 was not altered. Concomitantly, phosphorylation of p38 and activation of non-canonical NFκB pathway showed a dose dependent reduction after Mer blockade. Interestingly, R992 led also to increased osteoblast differentiation and could restore myeloma mediated osteoblast inhibition in co-cultures of MM cells and osteoblasts. In summary, our data suggest that Mer blockade leads to inhibition of MM and its associated bone disease. Furthermore, the function of Mer in bone homeostasis promoting osteoclast and inhibiting osteoblast activity leads to the potential application of Mer inhibitors also in osteolytic bone metastases or osteoporosis. Disclosures Darwish: Rigel Pharmaceuticals: Employment. Bhamidipati:Rigel Pharmaceuticals: Employment. Masuda:Rigel Pharmaceuticals: Employment, Equity Ownership. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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