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

Blood ◽  
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.

scholarly journals Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4481-4481
Author(s):  
Denise Toscani ◽  
Martina Chiu ◽  
Giuseppe Taurino ◽  
Emanuela Vicario ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Bone Disease ◽  
Research Funding ◽  
Bone Cells ◽  
Bone Microenvironment ◽  
Advisory Committees ◽  
Osteolytic Lesions ◽  
Osteolytic Bone Disease ◽  
The Relationship

Abstract Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions. Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions. We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression. Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested. Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. Disclosures Aversa: Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

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

Blood ◽  
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.

scholarly journals Effect of the Wnt-Signaling Inhibitor Sclerostin and Bone Marrow Adipocytes on Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2077-2077
Author(s):  
Cristina Panaroni ◽  
Ka Tat Siu ◽  
Keertik S Fulzele ◽  
Janani Ramachandran ◽  
Noopur Raje
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Board Of Directors ◽  
Research Funding ◽  
Neutralizing Antibody ◽  
Fold Increase ◽  
Advisory Committees ◽  
Cell Engraftment ◽  
Msc Differentiation

Abstract Multiple Myeloma (MM) is a plasma cell tumor that originates and expands within bone marrow (BM). MM occurs primarily later in life with a median age at diagnosis in the seventh decade characterized by loss of bone tissue due to osteolytic lesions. Recent studies have suggested a positive correlation of MM with obesity. BM fat cells originate, as bone cells, from a common progenitor called Mesechymal Stem Cell (MSC). MSC commitment into osteoblastic or adipose lineage is regulated by two major signaling pathways: Wnt signaling, which promotes bone formation, and PPARγ signaling, responsible for adipocyte differentiation. Adipogenesis and osteogenesis are inversely correlated processes, thus a signaling imbalance favoring differentiation towards one lineage will tilt this critical balance. In fact, the amount of marrow adiposity increases with age such that it is 30% of the BM volume in young adults but rises up to 70% in elderly people while bone formation reduces with age. We have recently demonstrated that MM patients have increased levels of a Wnt-signaling inhibitor sclerostin (SOST) and patient MSC differentiation into osteoblastic cells is improved in the presence of a SOST neutralizing antibody (Eda et al. JBMR 2015). Sclerostin is secreted by osteolineage cells and has also been shown to increase adipogenesis of an adipogenic cell line 3T3-L1 (Ukita et al. JCB 2016). However, the role of SOST on BM adipose tissue in MM patients has not been investigated. Here we show that elevated SOST levels, induced by MM cells, increase BM adipogenesis which, in turn, supports MM progression. To assess the role of MM cells on MSC differentiation into adipocytes, MSCs from patients and normal donors (ND) were differentiated in vitro in the presence or absence of MM.1S, a human MM cell line. Presence of MM.1S cells significantly reduced osteogenic differentiation of MSCs as assessed by quantitative and qualitative Alizarin Red S staining; by contrast, the presence of MM.1S cells significantly enhanced adipogenesis in the MSCs as assessed by Oil Red O staining quantification. At the molecular level, we observed a 2-fold increase in PPARγ gene expression in MSCs from MM patients when compared to ND samples at baseline and a 5-fold increase when MSCs from patients and NDs were cultured for 72 hours in the presence of MM.1S in trans-well. To evaluate the role of SOST on BM adiposity, we intraperitoneally injected recombinant SOST, or PBS as control, into wild-type (WT) mice daily for 3 weeks. At the end of the treatment, mice treated with SOST showed a significant increase in BM adiposity. To validate MM cell induced adipogenesis in in-vivo settings, we injected MM.1S cells into the SCID-hu immunodeficient mouse model. Four weeks after cell injection, increased BM adipocytes were observed in MM.1S treated immunodeficient mice. Interestingly, when mice injected with MM.1S cells were treated for 3 weeks with SOST neutralizing antibody the level of BM adiposity returned to the healthy control levels. Finally, MM cell engraftment and tumor development was analyzed in SOST knock-out (KO) mice. While both SOST KO and WT animals showed MM cell engraftment and extramedullary plasmacytoma formation, preliminary results suggest a lower level of MM cell engraftment in BM of SOST KO mice. Our data suggest that sclerostin secretion increases BM adipogenesis supporting MM cell growth and survival and therefore may play a critical role in the development and progression of MM. Disclosures Raje: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.

Selective HDAC6 Inhibition Via ACY-1215, Either Alone or in Combination with Bortezomib, Restores Osteoblast Function and Suppresses Osteoclast Differentiation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2908-2908
Author(s):  
Loredana Santo ◽  
Teru Hideshima ◽  
Andrew L. Kung ◽  
Jen-Chieh Tseng ◽  
David Tamang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Bone Formation ◽  
Board Of Directors ◽  
Bone Disease ◽  
Research Funding ◽  
Bone Homeostasis ◽  
Advisory Committees ◽  
Synergistic Cytotoxicity

Abstract Abstract 2908 Bone disease in multiple myeloma (MM) is due to the disruption of the delicate balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. Agents that target both tumor cells and restore normal bone homeostasis can improve long-term disease control and prolong MM patient survival. It has been demonstrated that in vitro pan HDAC inhibitors accelerate OB maturation and suppress OC maturation, while bortezomib triggers OB activation and inhibits osteoclastogenesis. However it has recently been shown that vorinostat (SAHA), a non-selective HDAC inhibitor, causes bone loss in vivo by inhibiting immature OB. Here, we evaluated effects of a selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib, on MM cell growth and related bone disease. ACY-1215 in combination with bortezomib has synergistic cytotoxicity due to simultaneous inhibition of the proteasome and aggresome pathways. We confirm the in vivo anti-MM activity of ACY-1215 in combination with bortezomib in two different xenograft mouse models: human MM injected subcutaneously; and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall host survival significantly prolonged in animals treated with combined therapy (34 vs 22 days, n=7, p<0.0011) in plasmacytoma model and (40 vs 17 days, n=12, p<0.0001) in disseminated model. Importantly, we show that ACY-1215 alone and in combination with bortezomib overcomes the proliferative effect of bone marrow stromal cells (BMSCs) and cytokines. MM cells stimulate OC formation and function, while inhibiting OB differentiation via both cell-to-cell contact and cytokine secretion. Therefore, osteoclastogenesis is an important therapeutic target in MM. In this context, we evaluated the effect of ACY-1215 (1μM) and bortezomib (2.5nM) on OCs generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B ligand (RANKL). ACY-1215 alone and in combination with bortezomib inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, ACY-1215 (1μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines. We next examined the effect of ACY-1215, alone and in combination with bortezomib, on downstream targets in RANKL/RANK signaling. ACY-1215 plus bortezomib inhibits transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS and NFATC1. Since there is decreased OB function and new bone formation in MM, we next assessed the effect of ACY-1215 on OB differentiation. ACY-1215, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase enzyme activity and alizarin red staining. In addition, we show increased mRNA expression of b-catenin, osteocalcin, Runx2 and Sp7 (OB differentiation markers) in immature OB triggered by ACY-1215. Finally, ACY-1215 was not toxic to PHA stimulated PBMCs, suggesting a favorable side effect profile and therapeutic index. Our studies therefore demonstrate that ACY-1215, alone and in combination with bortezomib, can inhibit osteoclastogenesis enhance osteoblastogenesis, and inhibit MM cell growth. Based upon these studies, ongoing clinical trials are examining the efficacy of ACY-1215 in relapsed MM and associated bone disease. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals, Inc.: founder; Merck: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. 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.

Matrix Metalloproteinses-13 (Collagenase-3) Secreted by Multiple Myeloma Cells Enhances the Bone Resorptive Activity of Osteoclasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2853-2853
Author(s):  
Rentian Feng ◽  
Noriyoshi Kurihara ◽  
Huihui Ma ◽  
G. David Roodman ◽  
Suzanne Lentzsch
Keyword(s):  
Multiple Myeloma ◽  
Bone Resorption ◽  
Bone Disease ◽  
Expression Profiles ◽  
Critical Role ◽  
Tissue Array ◽  
Cellular Interactions ◽  
Serum Samples ◽  
Osteolytic Lesions ◽  
Osteolytic Bone Disease

Abstract Abstract 2853 Poster Board II-829 Introduction: Osteolytic bone disease is a frequent complication of multiple myeloma (MM) that is characterized by reduced bone formation and increased bone resorption. MM cells produce several cytokines causing dysregulation of the bone remodeling process that results in excessive bone resorption mediated by activated osteoclasts (OCLs). Matrix metalloproteinases (MMPs) play a critical role in tumor invasion and progression in many solid tumors such as breast cancer but the role of MMPs in MM has have not been well elucidated. Methods and Results: We analyzed the MMP expression profiles (MMP1, 2, 3, 7, 8, 12, 13) of MM cells and found very high expression (400-fold higher) of MMP13 compared to other MMPs. Tissue array analysis for MMP-13 expression in MM tumor samples and normal control tissue indicated that MMP13 was only highly expressed in MM tumor samples. ELISA analysis of serum samples revealed a positive correlation of MMP13 expression with MM bone disease. MMP13 was not detectable in the sera of healthy donors, whereas 62% of MM patients expressed MMP13 in their serum samples, among which 87% had extensive bone disease. Co-culture of MM cells with primary bone marrow stromal cells (BMSC) resulted in a significant (P<0.001) increase (up to 14-fold) of MMP13 secretion by MM cells. RT-PCR analysis indicated that the expression of MMP13 gene in MM cells was markedly enhanced by BMSC conditioned medium, suggesting that BMSC-secreted soluble factors, instead of direct cellular interactions, induce MMP13. Functional inactivation of different cytokines with neutralizing antibodies identified IL-6 as a critical cytokine for the induction of MMP13. Electromobility shift assays showed that IL-6 mediated AP-1 activation promotes MMP13 transcription. Importantly, MMP13 significantly enhanced osteoclastic bone resorption on dentin slices induced by RANKL plus M-CSF. However, the strong induction of bone resorption was not derived from increased OCL formation since MMP13 only slightly increased OCL numbers. MMP13 significantly increased the OCL size as well as the nuclear number/OCL which increases the bone resorption activity per osteoclast. Conclusions: Our results suggest that MMP13 augments the pathogenic process of osteolytic lesions in MM by increasing the bone resorption capacity/OCL. These results suggest that targeting MMP13 may represent a novel approach to ameliorate osteolytic lesions in MM. Disclosures: Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4052-4052
Author(s):  
Deepika Kassen ◽  
Neil Rabin ◽  
Darren Lath ◽  
Peter Croucher ◽  
Kwee Yong
Keyword(s):  
Bone Loss ◽  
Cell Line ◽  
Bone Disease ◽  
Bone Remodelling ◽  
Bone Lesions ◽  
Bone Homeostasis ◽  
Bone Biology ◽  
Trabecular Thickness ◽  
Osteolytic Lesions ◽  
Osteolytic Bone Disease

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.

Role of Selective HDAC6 Inhibition On Multiple Myeloma Bone Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 328-328
Author(s):  
Loredana Santo ◽  
Diana Cirstea ◽  
Bin Wang ◽  
Tso-Pang Yao ◽  
Joy Y. Wu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Advisory Committees ◽  
Vitro Data ◽  
In Vitro Data

Abstract Abstract 328 In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro. Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2. Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC. In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

scholarly journals 18F-FDG-PET/CT and Osteolytic Bone Disease in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5622-5622
Author(s):  
Brian Østergaard ◽  
Julie R. Mortensen ◽  
Anne L Nielsen ◽  
Jon T. Asmussen ◽  
Oke Gerke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Metabolic Activity ◽  
Bone Disease ◽  
Fdg Pet ◽  
Focal Lesions ◽  
Osteolytic Lesions ◽  
Fdg Uptake ◽  
Pet Ct ◽  
Osteolytic Bone Disease ◽  
Fdg Pet Ct

Abstract Introduction: FDG-PET/CT is a promising methodology for staging, prognostication, and response evaluation in multiple myeloma (MM). The number of focal FDG lesions and the intensity of FDG uptake (standard uptake value, SUV) at diagnosis are informative of disease aggressiveness. Osteolytic bone disease is a hallmark of MM. Hypothetically; increased focal metabolic activity will precede, induce and correlate to osteolytic lesions. We aimed to elucidate the association between focal FDG positive lesions and osteolytic bone disease in MM patients. Methods: Twenty-two newly diagnosed MM patients, 16 males and 6 females aged 53-81years, were prospectively enrolled and studied with a standardized baseline FDG-PET/CT at diagnosis. A nuclear medicine and a radiology specialist evaluated the PET and CT parts separately and independently. Focal FDG positive lesions were assessed by dedicated software (ROVER™, ABX, Germany) to yield volume and SUV measures. All osteolytic lesions identified on CT were noted by size and localization. Results: FDG-PET and CT together identified a total of 390 lesions. We found more osteolytic lesions on CT (335) than FDG-positive lesions (169); the concordance between the modalities was 30%. 34% (114/335) of lytic lesions were FDG-positive, whereas ⅔ (114/169) of focal FDG-PET positive lesions were lytic according to CT. We found a significant correlation between higher FDG uptake measured as SUVpeak and osteolytic lesions >10 mm, and focal lesions with high FDG uptake had more pronounced osteolysis than focal lesions with less FDG uptake. Still sparse follow-up data did not allow analysis of the predictive value of focal PET positivity and the risk of development of future osteolysis. More compulsive data will be presented at the meeting. Conclusion: FDG-PET/CT offers dual information including metabolic activity (FDG-avidity) and degree of lytic bone disease (low dose whole body CT) in MM. About ⅔ (221/335) of osteolytic lesions were FDG-negative, whereas ⅔ (114/169) of FDG-positive lesions had a lytic component. Disclosures Plesner: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Relationship between Bone Marrow PD-1 and PD-L1 Expression and the Presence of Osteolytic Bone Disease in Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3183-3183
Author(s):  
Federica Costa ◽  
Marina Bolzoni ◽  
Rosanna Vescovini ◽  
Fabrizio Accardi ◽  
Anna Benedetta Dalla Palma ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Bone Disease ◽  
Research Funding ◽  
Advisory Committees ◽  
Osteolytic Lesions ◽  
Cd8 Cells ◽  
Monoclonal Gammopathies ◽  
Hla Dr ◽  
Osteolytic Bone Disease

Abstract Alterations of the bone marrow (BM) immune-microenvironment characterize the progression of monoclonal gammopathies and the development of osteolytic bone disease in multiple myeloma (MM). MM patients exhibit immune dysfunctions as impaired dendritic, NK and T cells, whereas the onset of MM osteolytic lesions is associated to an increased prevalence of Th17 cells. Recently, the pathophysiological role of the programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) pathway together with an increase of myeloid derived suppressor cells (MDSCs) in the induction of tumor tolerance and immune evasion has been underlined with a therapeutic relevance. However, unclear data on the expression profile of PD-1/PD-L1 axis in MM patients have been reported and it is not known if this axis could be related with the presence of osteolytic bone disease. In order to elucidate these aspects, we analyzed a total cohort of 80 consecutives patients with monoclonal gammopathies, including 15 monoclonal gammopathy of undetermined significance (MGUS), 23 smoldering MM (SMM), 21 newly diagnosed MM (MMD) and 21 relapsed/refractory MM (MMR) patients. The presence of bone disease was checked by low-dose computerized tomography (CT) with or without positron emission tomography (PET) scan and by X-rays skeletal survey in 11 MM patients. 87% of MM patients displayed osteolytic lesions. High bone disease (HBD) was defined by the presence of 3 or more osteolytic lesions (62% of our cohort). Patients without bone lesions or with minus of 3 lesions were defined as low bone disease (LBD). BM mononuclear cells were analyzed by flow cytometry, evaluating plasma cells (PCs) (CD138+), monocytes (CD14+) and T cells (total CD3+, CD3+CD4+ and CD3+CD8+). PD-L1 (CD274) expression was evaluated on CD138+ and CD14+ cells, and PD-1 (CD279) on CD3+, CD4+ and CD8+ cells. Lastly, in a subgroup of patients we analysed MDSC populations, including both granulocytic (gMDSCs) (CD11b+HLA-DR-CD14-CD15+) and monocytic MDSCs (mMDSCs), (CD11b+HLA-DR-/lowCD14+CD15-). The percentage of BM CD3+PD-1+ cells increased from MGUS to MMR patients with a significant trend (p=0.004). Indeed, patients with active MM (MMD and MMR) showed both higher % of CD3+PD-1+ cells (median value: 48.5% vs 40.6%, p=0.001) and PD-1 median fluorescence intensity (MFI) on CD3+ (median value: 18.9 vs 16.7 MFI, p=0.024) as compared to patients with SMM and MGUS. CD4+PD-1+, but not CD8+PD-1+ cells are increased in active MM compared to SMM and MGUS patients (p=0.023). On the other hand, any significant difference was not observed in the PD-L1 expression on both BM CD138+ and CD14+ cells across patient groups. The percentage of BM MDSC populations did not significantly change across the different monoclonal gammopathies (p=0.14); moreover, comparing MM with SMM and MGUS patients, the % of gMDSCs was significantly reduced (median %: 12.5% vs 17%, p=0.04) and the % of mMDSCs was increased (median %: 0.36% vs. 0.24%) without reaching a statistical significance. Focusing on MM bone disease, we found that osteolytic MM patients displayed higher CD4+/CD8+ ratio compared to non-osteolytic ones (p=0.043). Regarding the PD-1 expression, the % of CD3+PD-1+ cells did not change (p=0.192), whereas the % of CD8+PD-1+ cells was significantly reduced in osteolytic patients compared to non-osteolytic ones (p=0.016). Consistently, among the BM CD8+ cells, a significant decrease of %PD-1+ cells was found in HBD vs LBD MM patients (median value: 46.9% vs 57.4%, p=0.045). Interestingly, when compared to LBD MM patients, HBD MM patients displayed a significant reduction of PD-L1 expression on both BM CD138+ PCs (median MFI value: 13.3 vs 21.6 MFI, p=0.007) and CD14+ cells (median MFI value: 15.4 vs 23.8 MFI, p=0.007). In a multivariate analysis, PD-1+ expression on CD8+ cells and PD-L1 MFI on CD138+ were significant independent factors related to the presence of HBD. In conclusion, our study indicates that the frequency of PD-1+ T cells increases across the progression of the monoclonal gammopathies. On the other hand, for the first time, we show in MM patients a significant relationship between the presence of extensive osteolytic bone disease and a reduced expression profile of BM PD-1/PD-L1 axis on CD8+ and CD138+ cells. We hypothesize that a less immune-suppressive profile could be related to the development of osteolysis consistent with the negative cross talk existing between PD-1/PD-L1 axis and Th17 cells. Disclosures Aversa: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding.

scholarly journals The Clinical Significance of a Novel microRNA Signature in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5529-5529
Author(s):  
Pinelopi I Artemaki ◽  
Aristea-Maria Papanota ◽  
Paraskevi Karousi ◽  
Christine Liacos ◽  
Christos K. Kontos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Untranslated Region ◽  
Research Funding ◽  
Plasma Cells ◽  
Predictive Biomarker ◽  
Lymphocytic Leukemia ◽  
Osteolytic Lesions ◽  
Expression Levels

Multiple myeloma (MM) is a hematologic malignancy characterized by infiltration of the bone marrow (BM) by malignant plasma cells. Multiple myeloma diagnosis is made by the presence of one or more of the CRAB criteria or one of the recently added biomarkers of malignancy. Smoldering MM (SMM) is a plasma cell dyscrasia preceding multiple myeloma, characterized by bone marrow infiltration of 10-60% and/or serum monoclonal protein ≥3g/dL or urinary monoclonal protein ≥500 mg per 24h, along with the absence of myeloma-defining events. MicroRNAs (miRNA) are single-stranded, small non-coding RNA molecules (~21 nt) that regulate protein-coding gene expression at the post-transcriptional level, mainly through interactions with the 3′-untranslated region of target mRNAs. The results of such interactions can be mRNA degradation and/or translational repression, depending on the complementarity of the miRNA seed sequence with the mRNAs 3′-untranslated region. They can function as oncogenes or tumor suppressors, possessing a vital role in several aspects of all stages of tumorigenesis and cancer progression. In the present study, we have investigated the clinical value of a molecular signature consisting of 10 cancer-related miRNAs in MM: miR-15a, miR-16, miR-21, miR-221, miR-222, miR-25, miR-125, miR-155, miR-223, and miR-181a. These molecules were selected due to their well-documented role and clinical significance in numerous human malignancies. More specifically, miR-15a and miR-16 expression levels have been associated with chronic lymphocytic leukemia. The deletion 13q14, the most prevalent alteration in CLL, leads to the deletion of these miRNAs, which act on cell proliferation and in the process of apoptosis. miR-221 and miR-222 form a cluster that has been correlated with tumorigenesis and unfavorable prognosis in human malignancies, while miR-155 is a pro-inflammatory, oncogenic molecule, with a potential role in chronic lymphocytic leukemia. Bone marrow aspiration samples were collected from 94 patients with MM and SMM, and CD138+ plasma cells were positively selected using magnetic beads coated with an anti-CD138 antibody. Total RNA was isolated using TRIZol. Thereafter, 200ng RNA of each sample were polyadenylated at the 3´ end and reversely transcribed. An in-house developed real-time quantitative PCR assay was conducted and the results were biostatistically analyzed. For the normalization of the expression levels of each miRNA, the mean expression of two small nucleolar RNAs (RNU43 and RNU48) was used as reference. Seventy six out of the 94 BM aspiration samples were derived from MM patients and 18 of them from SMM patients, at the time of diagnosis. The MM patients were classified, according to the R-ISS staging system, as follows: 15 patients had stage I disease, 42 patients had stage II, and 19 patients stage III MM. Forty nine myeloma patients presented with osteolytic lesions at diagnosis. The statistical analysis revealed significantly lower expression levels of miR-16 (p=0.036) and miR-155 (p=0.045) in CD138+ cells of MM patients, compared to those from SMM patients, highlighting their potential value to discriminate MM from SMM. Furthermore, miR-221 and miR-222 expression levels were negatively correlated with R-ISS; thus, miR-221 and miR-222 expression was significantly downregulated in MM patients with R-ISS stage III (p=0.004 and 0.034, respectively). This tendency reveals a potential favorable prognostic value of miR-221/222 cluster in MM. Next, miR-15a and miR-16 expression was shown to be associated with the presence of osteolytic lesions. In this regard, the expression levels of miR-15a (p=0.048) and miR-16 (p=0.047) were decreased in MM patients with bone disease, compared to those without bone disease. The observed decreased expression of these two miRNAs in symptomatic MM patients could constitute a predictive biomarker for the occurrence of bone disease and, hence, a putative predictive biomarker of SMM patients at high risk of evolution to symptomatic disease with bone lesions. We conclude that miR-221/222 correlate with more favorable R-ISS stage, while miR-15a and miR-16 correlate with the presence of osteolytic disease in MM. This ongoing study will further reveal the possible prognostic significance of this 10 miRNAs signature studied, when response to therapy, progression-free and overall survival is available. Disclosures Kastritis: Genesis: Honoraria; Prothena: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Gavriatopoulou:Takeda: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Honoraria; Genesis: Honoraria, Other: Travel expenses. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Takeda: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding.

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