MM-induced Bone Disease Research Articles

Investigating the Relationship between Primary Bone Marrow Adipocytes and Myeloma Cells with Three Cell Culture Systems

Introduction & Aims of the Study: The unique properties of the bone marrow allow for migration and proliferation of multiple myeloma (MM) cells, while also providing the ideal environment for development of quiescent, drug-resistant MM cell clones. Recent studies suggest that bone marrow adipocytes (BMAds) can contribute to myeloma-induced bone disease, and reciprocally, that BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes differentiated from bone marrow-derived mesenchymal stromal cells (MSCs) in vitro due to the technical challenges associated with isolating and culturing primary adipocytes. To that end, we investigated the MM-BMAd relationship ex vivo utilizing primary adipocytes (pBMAds), which overcome many of the drawbacks associated with using MSC-derived adipocytes. Methods: pBMAds were isolated from the top of human bone marrow biopsies after centrifugation and seeded onto one of three in vitro systems. To develop an in vitro model with a tissue-like structure to mimic the bone marrow microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human bone marrow. Following adherence of pBMAds, MM cells were added to investigate bidirectional tumor-host signaling. Viable pBMAds were measured utilizing CellTiterGlo and RealTime Glo (bioluminescence), alone and in culture with MM cells (MM.1Sgfp+luc+ and RPMI-8226). MM.1Sgfp+luc+ cell number was determined by luciferin spike-in and measurement of bioluminescence. To investigate the effects of myeloma cells on pBMAds, we used fluorescence and confocal microscopy to image cell morphology, and mass spectrometry proteomics of conditioned media samples to specifically interrogate soluble signals. Gene expression in both cell types was investigated by qPCR. Results: Utilizing adipocytes isolated from 12 different human donors, we found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for short-terms (~2 weeks) or long-terms (1 month) in vitro (Figure 1A). We monitored stable pBMAd presence in 2D transwells via Cell TiterGlo and on 3D scaffolds via RealTime Glo up to 9 days post-seeding, and by calcein staining after 10 days in culture. Using bioluminescence in pBMAs (Cell TiterGlo) and MM.1S (luciferin spike-in) we were able to detect viability of both cell types in co-culture. Utilizing mass spectrometry proteomics, in pBMAds alone, we detected secretion of CXCL12 (SDF1; p<0.01), as well as TGFB1 (p<0.01) which has implications for bone remodeling. Interestingly, in conditioned media collected from the co-culture condition, we found increased secretion of the WNT inhibitor DKK3 (p<0.05) and detection of proteins known to be associated with extracellular exosomes including: CNTN1, ACP1, OMD, RPS18, RPL24, PGAM1, LAMA4, CTSB, PLOD2 and ARHGDIA (Figure 1B), suggesting that pBMAds and MM cells likely interact via exosomes. Conclusions: In sum, we developed three in vitro cell culture systems to study primary bone marrow adipocytes and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the bone marrow, including other bone-homing tumor types. Our use of these models to examine the interaction between MM cells and pBMAds has revealed new roles for pBMAds in the MM bone marrow microenvironment, with implications for MM-induced bone disease and pBMAd-MM bidirectional signaling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 6374: BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans

Abstract Multiple myeloma (MM), the second most common hematologic malignancy in adults, is characterized by clonal proliferation of malignant plasma cells and tumor-induced complications, including MM-induced bone disease (MBD). Up to 90% of MM patients develop MBD during the course of their disease, which is a major cause of morbidity and mortality. MM therapy focuses primarily on agents that seek to eradicate the tumor cells and others which inhibit bone resorption. In contrast, no drugs are currently available to promote bone formation in MM patients and reverse MBD. Towards addressing this unmet therapeutic need, we applied a modified version of our “humanized” bone marrow (BM)-like scaffold (huBMsc)-based xenograft model, where MM cells and bone marrow mesenchymal stromal cells (BMMSCs) are co-implanted. In this system, we investigated the impact of MM cells on BMMSCs to identify markers that can be targeted with the intent to reverse MBD. After confirming that MM cells indeed inhibit bone formation in vivo, we performed transcriptional analysis of ex vivo expanded “unexposed” control BMMSCs vs. “MM-exposed” BMMSCs and found 332 differentially expressed genes. Next, we compared these data with transcriptional profiles of BMMSCs after in vitro differentiation towards osteoblasts, as well as publicly available data of transcriptional profiles of BMMSCs obtained from MM, ALL, and AML patients. Combined, this analysis identified 8, MM-specific, differentially expressed genes, including the small leucine-rich proteoglycans (SLRPs), ASPN, OGN and OMD, that are 1) suppressed in the presence of MM (xenograft model and patient-derived BMMSCs); 2) induced during in vitro osteogenic differentiation; and 3) not dysregulated in BMMSCs in other hematological malignancies. As reported previously, we observed that bone morphogenetic protein (BMP)6 can upregulate the expression of these SLRPs in BMMSCs, while inducing osteogenic differentiation. Moreover, incubation of MM cells with BMP6 in either mono- or co-cultures with stromal cell lines, resulted in reduced MM cell viability. In addition, pretreatment of BMMSCs with BMP6 completely abolished their support of MM proliferation. Interestingly, BMP6, in contrast to BMP2 and BMP4, did not induce, but inhibited receptor activator of nuclear kappa-B ligand (RANKL)-dependent osteoclast generation. Collectively, our analyses identify BMP6 as a master regulator that promotes osteogenic differentiation, inhibits RANKL-dependent osteoclastogenesis and can directly induce MM cell death. Therefore, by reshaping the BM microenvironment to become less tumor-supportive, BMP6 represents an attractive candidate for MM treatment. Citation Format: Jiaxian Wang, Thomas Baardemans, Ricardo de Matos Simoes, Olga Dashevsky, Huipin Yuan, Joost D. de Bruijn, Anton C. Martens, Constantine S. Mitsiades, Richard W. Groen. BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6374.

Acid Ceramidase (ASAH1) Mediates Intrinsic and Intercellular Transfer of Proteasome Inhibitor Resistance in Multiple Myeloma

INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells.METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models.RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls.CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. DisclosuresHampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Aplidin (Plitidepsin) Regulates Viability and Function of Myeloma Cells and Bone Cells in Combination with Other Anti-MM Drugs

In multiple myeloma (MM) the clonal expansion of malignant plasma cells within the bone marrow results in the development of lytic bone lesions due to exacerbated bone resorption. Despite recent progress in its treatment, MM remains incurable and the associated bone disease persists even after complete remission. Thus, identification of new therapeutic approaches that suppress MM cell growth and protect the bone is an unmet need. Aplidin, a novel anti-cancer compound isolated from the marine tunicate Aplidium albicans, exhibits anti-MM activity even in MM cells resistant to conventional chemotherapy and is currently in phase III clinical trials in Europe. However, the effects of Aplidin on bone cells and MM-induced bone disease are unknown. The goal of the current study was to examine the effect of Aplidin, alone or in combination with other anti-MM drugs, on myeloma cells and bone cells (osteocytes, osteoblasts, and osteoclasts) using in vitro approaches.Aplidin decreased the number of human JJN3 and murine 5TGM1 MM cells with an EC50~10nM, determined by MTT assay, due to decreased cell viability, assessed by Trypan blue exclusion, reaching a 20% increase in MM cell death after 48h. Treatment with glucocorticoids (GC) (10-5M) or Bortezomib (3nM) also decreased MM viability and enhanced the anti-MM activity of Aplidin by 1.6-fold and 2.5-fold, respectively. In addition, Aplidin at >10nM induced MLO-A5 osteocytic dead up to 15% and in combination with GC up to 50%; whereas, in contrast, Bortezomib did not affect osteocyte viability and partially prevented the increase in MLO-A5 cell death induced by Aplidin. Further, 1nM Aplidin increased OB-6 osteoblastic cell death up to 20% and decreased their matrix mineralization capacity, quantified by Alizarin red. These effects were enhanced when Aplidin was combined with GC, but partially prevented by combination of Aplidin with Bortezomib. Cells of the osteoclastic lineage exhibited higher sensitivity to Aplidin in comparison to MM cells or osteoblastic cells, and thus it was tested at lower concentrations. Specifically, 50 pM Aplidin decreased the number of hematopoietic precursors by 15-20%, as quantified by enumerating GM-CSF colonies in cultures established from murine bone marrow. Moreover, Aplidin, at concentrations as low as 0.01 pM, reduced osteoclast precursor commitment and differentiation by 10%, as quantified by enumerating the number of osteoclast precursors maintained in the presence of M-CSF for 3 days with or without Aplidin. In addition, Aplidin decreased by 40% the number of TRAP positive osteoclasts generated after 4 days in the presence of M-CSF and sRANKL; and blunted the rapid (5-15min) increased in ERK phosphorylation induced by Rankl, but did not affect RANKL-induced p38 phosphorylation, suggesting that Aplidin inhibited RANKL-induced differentiation of osteoclast precursors by preventing ERK activation. Lastly, to test the effects of Aplidin on osteoclasts function, mature osteoclasts generated in the presence of M-CSF/sRANKL were detached by trypsinization, replated on bovine dentin slides and exposed to Aplidin for 3 and 7 days. 10pM Aplidin reduced the number of mature osteoclasts by 60% and markedly decreased the number of resorption pits.In summary, Aplidin (1nM and up) decreases MM cell viability and GC or Borteozmib enhances its anti-MM activity. In addition, Aplidin (10nM and up) decreases osteocytic cell viability and this effect is exacerbated by GC but partially prevented by Bortezomib. Moreover, Aplidin (1nM and up) induces osteoblastic cell death and decreases their mineral production, and these effects are increased by GC, but partially prevented by Bortezomib. Furthermore, Aplidin (0.01pM and up) reduces osteoclast precursor commitment and differentiation, and inhibits mature osteoclast resorption activity. Taken together these findings suggest that combining Aplidin with proteosomal inhibitors may be a novel therapy for MM to enhance anti-tumor responses, decrease bone destruction by inhibiting bone resorption, and prevent potential side effects of Aplidin on cells of the osteoblastic lineage. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Galmarini:PharmaMar: Employment. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

The HGF/Met/NF-κB Pathway Regulates RANKL Expression in Osteoblasts and Bone Marrow Stromal Cells.

Multiple myeloma (MM)-induced bone disease occurs through hyperactivation of osteoclasts by several factors secreted by MM cells. MM cell-secreted factors induce osteoclast differentiation and activation via direct and indirect actions including enhanced expression of receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts and bone marrow stromal cells (BMSCs). Hepatocyte growth factor (HGF) is elevated in MM patients and is associated with MM-induced bone disease, although the mechanism by which HGF promotes bone disease remains unclear. In the present study, we demonstrated that HGF induces RANKL expression in osteoblasts and BMSCs, and investigated the mechanism of induction. We found that HGF and MM cell supernatants induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. In addition, HGF increased phosphorylation of Met and nuclear factor κB (NF-κB) in ST2 cells, MC3T3-E1 cells, or mouse BMSCs. Moreover, Met and NF-κB inhibitors suppressed HGF-induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. These results indicated that HGF promotes RANKL expression in osteoblasts and BMSCs via the Met/NF-κB signaling pathway, and Met and NF-κB inhibitors suppressed HGF-induced RANKL expression. Our findings suggest that Met and NF-κB inhibitors are potentially useful in mitigating MM-induced bone disease in patients expressing high levels of HGF.

Emerging Insights Into the Role of the Hippo Pathway in Multiple Myeloma and Associated Bone Disease

Multiple myeloma (MM) is an incurable plasma-cell dyscrasia with numerous treatment options currently available; however, drug resistance is usually inevitable, so there is a constant need for novel treatment approaches. The Hippo pathway has emerged as an important mediator of oncogenesis in solid tumors. More recently, its key role in regulating apoptosis and mediating resistance in MM and other hematologic malignancies has been demonstrated in preclinical studies, which provides a strong basis for further clinical investigation. The Hippo pathway is also implicated in the pathogenesis of MM-induced bone disease, as it regulates both osteoblast and osteoclast function. We provide an overview of the available data regarding the role of the Hippo signaling components in the pathophysiology of MM. A better understanding of the underlying interactions at the molecular and cellular levels will lead to novel and promising treatment approaches.

Semaphorin 4D correlates with increased bone resorption, hypercalcemia, and disease stage in newly diagnosed patients with multiple myeloma

Multiple myeloma (MM) is characterized by bone destruction due to increased bone resorption and decreased bone formation. Semaphorin 4D (CD100, Sema4D) is expressed by osteoclasts, binds to its receptor Plexin-B1, and acts as a mediator of osteoclast–osteoblast interaction that ultimately inhibits osteoblastic bone formation. Preclinical data suggest that Sema4D/Plexin-B1 pathway is implicated in MM-induced bone disease. However, there is no information on the role of Sema4D in MM patients. Thus, we evaluated Sema4D and Plexin-B1 in six myeloma cells lines in vitro; in the bone marrow plasma (BMP) and serum of 72 newly diagnosed symptomatic MM (NDMM) patients and in 25 healthy controls. Only one myeloma cell line produced high Sema4D. BMP and circulating Sema4D and Plexin-B1 levels were significantly higher in MM patients compared to controls (p < 0.01). Sema4D correlated with serum calcium levels (p < 0.001), increased bone resorption (as assessed by CTX; p < 0.01), and ISS (p < 0.001). There was a trend for higher Sema4D levels in patients with osteolysis (p = 0.07), while patients with diffuse MRI pattern had higher BMP Sema4D levels (p = 0.02). Our data suggest that Sema4D is elevated in MM patients and correlate with adverse myeloma features and increased bone resorption, providing a possible target for novel therapeutic approaches in MM.

Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl) levels, an osteocyte-derived inhibitor of Wnt/β-catenin signaling, are elevated in MM patient sera and are increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Genetic Sost Deletion and Pharmacological Inhibition of Sclerostin Prevent Multiple Myeloma-Induced Bone Loss without Affecting Tumor Growth

In Multiple myeloma (MM) plasma cells grow in the bone marrow and induce localized lytic lesions due to increased bone resorption and suppressed bone formation. High levels of Sclerostin, a potent inhibitor of bone formation, are found in sera of MM patients. The expression of Sost/Scleorstin by osteocytes is also increased in mice bearing MM tumors, suggesting that Sclerostin might play a role in MM-induced bone disease. The goal of this study was to examine the impact of Sost/Sclersotin on tumor growth and MM-induced bone disease.We first examined the effect of genetic deletion of Sost. Therefore, we generated global Sost KO mice in an immunodeficient background (SCID), which exhibited the expected high bone mass phenotype associated with Sost deficiency. 6-wk-old SostKO or WT control littermates mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline (n=7-10/group), and sacrificed after 4wks. Sost KO and WT mice injected with hMM had equivalent tumor engraftment as demonstrated by serum human kappa light chain levels. hMM-injected WT mice exhibited ~50% decrease in tibia cancellous bone volume (BV/TV) and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). In contrast, hMM-injected Sost KO mice displayed no changes in BV/TV or bone architecture. Importantly, X-ray analysis revealed that the number and area of osteolytic lesions was reduced in Sost KO by 60% and 74%, respectively, compared to WT mice.We next examined the effect of pharmacological inhibition of Sclerostin in an immunocompetent preclinical model of established MM. 6-wk-old C57BLKalwRij mice were injected intratibially with 105 5TGM1 murine MM cells (mMM) or saline. After 4wks mMM-injected mice had a 2-fold increase in the serum tumor engraftment marker IgG2b. Saline or mMM-injected mice were then treated with a Sclerostin neutralizing antibody (Scl-Ab; 15mg/kg/wk) or control antibody (IgG; n=6-10/group). After 4wks of treatment, serum IgG2b levels were similar in mMM-injected mice receiving Scl-Ab or IgG. mMM-injected mice receiving IgG injections had ~35% decreased BV/TV, Tb.N, and increased Tb.Sp. In contrast, mMM-injected mice receiving Scl-Ab displayed increased trabecular BV/TV (52%), Tb.N (22%), Tb.Th (33%) and decreased Tb.Sp (14%), results that did not differ from saline-injected mice treated with Scl-Ab. Moreover, the number of osteolytic lesions was reduced by 46% in Scl-Ab treated mice when compared to the IgG-treated group. Further, mMM-injected mice treated with IgG or Scl-Ab showed similar increases in the bone resorption markerCTX in the circulation, whereas mMM-injected mice treated with Scl-Ab had a smaller decrease in the bone formation marker P1NP in sera compared to IgG-treated mice (22 vs 45%).Consistent with the lack of effect of the Scl-Ab on MM tumor growth in vivo, Scl-Ab did not affect the proliferation or viability of MM cells in vitro. We then examined next if Scl-Ab alters the anti-myeloma activity of dexamethasone (DEX), bortezomib (BOR) and the Notch inhibitor GSIXX. As expected, DEX, BOR and GSIXX increased the number of dead mMM and hMM cells in a time- and dose-dependent manner. Importantly, the increase in the number of dead hMM and mMM cells induced by DEX, BOR and GSIXX remained unchanged when Scl-Ab was co-administered.Taken together, these results demonstrate that increased Scl production by osteocytes inhibits bone formation and contributes to MM-induced bone loss. Further, our data shows that pharmacological inhibition of Scl does not alter tumor growth or the activity of anti-myeloma drugs. These findings provide the rationale for combining Scl-Ab with anti-tumor drugs to simultaneously prevent tumor growth and the bone diseases in MM patients. DisclosuresRoodman:Amgen: Consultancy.

Multiple myeloma in the marrow: pathogenesis and treatments.

Multiple myeloma (MM) is a B cell malignancy resulting in osteolytic lesions and fractures. In the disease state, bone healing is limited owing to increased osteoclastic and decreased osteoblastic activity, as well as an MM-induced forward-feedback cycle where bone-embedded growth factors further enhance tumor progression as bone is resorbed. Recent work on somatic mutation in MM tumors has provided insight into cytogenetic changes associated with this disease; the initiating driver mutations causing MM are diverse because of the complexity and multitude of mutations inherent in MM tumor cells. This manuscript provides an overview of MM pathogenesis by summarizing cytogenic changes related to oncogenes and tumor suppressors associated with MM, reviewing risk factors, and describing the disease progression from monoclonal gammopathy of undetermined significance to overt MM. It also highlights the importance of the bone marrow microenvironment (BMM) in the establishment and progression of MM, as well as associated MM-induced bone disease, and the relationship of the bone marrow to current and future therapeutics. This review highlights why understanding the basic biology of the healthy and diseased BMM is crucial in the quest for better treatments and work toward a cure for genetically diverse diseases such as MM.

Notch- and TNFα-Activated Signaling Pathways Mediate Osteocyte Apoptosis Triggered By Multiple Myeloma Cells

Osteocytes comprise 95% of all bone cells and are central regulators of bone homeostasis and skeletal integrity. However the role of osteocytes in MM bone disease is unknown. We have previously shown that interactions with multiple myeloma cells have a profound effect on osteocytic gene expression, increasing Sost and RANKL transcripts and decreasing OPG. More recently, we and others have shown that myeloma cells increase the prevalence of osteocyte apoptosis, which might contribute to MM induced bone disease by increasing resorption in specific areas of bone. However, the mechanisms responsible for and the potential for targeting osteocyte apoptosis in myeloma are unknown. Osteocytes extensively communicate with each other and with cells on the bone surface and in the marrow, through cytoplasmic connections that run within canaliculi, which allows direct cell-to-cell contact and the distribution of secreted molecules among all bone and marrow cells including myeloma cells. Therefore, to explore the mechanism underlying osteocyte apoptosis induced by myeloma cells we used a co-culture system that allows both cell-to-cell contact and exchange of soluble factors between osteocytic MLO-A5 cells and the human JJN3 MM cell line or primary CD138+ cells isolated from MM patients. Osteocyte apoptosis was quantified by trypan blue uptake and chromatin condensation/nuclear fragmentation in the absence or presence of the caspase3 specific inhibitor DEVD. We found that osteocyte apoptosis was increased 2-3 fold when osteocytes were co-cultured with JJN3 cells compared to osteocytes cultured alone. Apoptosis occurred within 8h of co-culture and gradually increased for up to 48h. Osteocyte apoptosis was completely inhibited by DEVD. Importantly, co-culture with primary CD138+ MM cells from 5 different patients also increased osteocyte apoptosis, which was also blocked by DEVD. We next determined if direct cell-to-cell contact was required for MM cells to induce osteocyte apoptosis and whether Notch signaling, a signaling pathway with profound effects on the skeleton activated by such interactions, was involved. We found that osteocytes co-cultured with JJN3 cells exhibited 3-7 fold higher levels of expression of the Notch target genes Hes1 and Hey1, detected as early as 4h and maintained up to 48h of co-culture. The pharmacological specific Notch inhibitor GSIXX completely blocked osteocyte apoptosis induced by either JJN3 cells or primary CD138+ MM cells measured at 8h and 24h. Moreover, cultures of osteocytes grown on plates coated with the Notch ligand Delta 1 fused to IgG2 exhibited 2-5 times higher levels of apoptosis compared to osteocytes cultured on IgG2 control, and this effect was inhibited by GSIXX. In addition, overexpression of the Notch intracellular domains 1 or 2, known to activate Notch signaling, increased osteocyte apoptosis by 2 fold. These findings demonstrate that rapid activation Notch signaling in osteocytes triggered by direct cell-to-cell contact with myeloma cells induces osteocyte apoptosis. Interestingly, Notch inhibition by GSIXX only partially prevented osteocyte apoptosis induced by JJN3 cells measured at 48h, suggesting the involvement of other mechanisms. To further investigate this finding, we measured soluble levels of TNFα in JJN3 cultures since TNFα is a recognized inducer of osteocyte apoptosis. The levels of TNFα secreted by JJN3 cells increased 5 fold (from 2 to 9 pg/ml) during 4 to 48h of culture. Conditioned medium (CM) from JJN3 cells cultured alone for 48h increased osteocyte apoptosis, and this effect was blocked by DEVD and by a neutralizing anti-human TNFα antibody, but not by GSIXX. Moreover, combination of GSIXX and anti-TNFα antibody completely inhibited osteocyte apoptosis induced by co-culture with JJN3 cells, while each agent added separately only partially inhibited osteocyte apoptosis measured at 48h. These results demonstrate that direct interactions with MM cells induces caspase3-dependent osteocyte apoptosis, triggered by rapid activation of Notch signaling through cell-cell contact and is maintained by accumulation of MM-derived TNFα. Our findings suggest both Notch and TNFα signaling pathways are potential targets to reverse or prevent myeloma induced osteocyte apoptosis. DisclosuresNo relevant conflicts of interest to declare.

The Effects of CCL3 on Osteoblast Inhibition in Myeloma-Induced Bone Disease

Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. A common manifestation of the disease is bone destruction that caused by increased osteoclastic bone resorption and decreased bone formation. Suppression of osteoblast activity appears to persist even in patients enjoying long-term complete remission that without any detectable MM tumor cells. CCL3, also known as MIP-1α, is a pro-inflammatory and chemoline. To investigate if CCL3, a recently described osteoclast stimulatory factor,was possibly involved in the inhibition of osteoblast in MM-induced bone disease,osteoblast from BM of MM patients and healthy donors were cultured in vitro. Our results showed the proliferation and osteogenic potential of osteoblasts from MM patients were suppressed. MM-derived OBs expressed higher levels of CCR1 compared with normal controls. It’s an indication that CCL3 is able to influence OBs in MM bone disease (MBD).We cultured MM-derived OBs in osteogenic media and then stimulated them with CCL3 in the absence or presence of neutralizing antibody against CCL3. Our data suggested that CCL3 impaired matrix mineralization and suppressed osteocalcin (OCN).Gene expression analysis of osteoblastic transcription factors in CCL3-treated OBs revealed a Runx2 and Osterix downregulation. CCL3 antibody at least partially restored OBs activity with an upregulation of OCN levels and Runx2 and Osterix expression. These results showed that CCL3 may contribute to OB/OC imbalance by inhiting OBs’ dedifferentiation and function in MBD. DisclosuresNo relevant conflicts of interest to declare.

Mathematical modelling of the pathogenesis of multiple myeloma-induced bone disease.

Multiple myeloma (MM) is the second most common haematological malignancy and results in destructive bone lesions. The interaction between MM cells and the bone microenvironment plays an important role in the development of the tumour cells and MM-induced bone disease and forms a ‘vicious cycle’ of tumour development and bone destruction, intensified by suppression of osteoblast activity and promotion of osteoclast activity. In this paper, a mathematical model is proposed to simulate how the interaction between MM cells and the bone microenvironment facilitates the development of the tumour cells and the resultant bone destruction. It includes both the roles of inhibited osteoblast activity and stimulated osteoclast activity. The model is able to mimic the temporal variation of bone cell concentrations and resultant bone volume after the invasion and then removal of the tumour cells and explains why MM-induced bone lesions rarely heal even after the complete removal of MM cells. The behaviour of the model compares well with published experimental data. The model serves as a first step to understand the development of MM-induced bone disease and could be applied further to evaluate the current therapies against MM-induced bone disease and even suggests new potential therapeutic targets. © 2014 The Authors. International Journal for Numerical Methods in Biomedical Engineering published by John Wiley & Sons Ltd

Investigating the efficacy of bisphosphonates treatment against multiple myeloma induced bone disease using a computational model

Multiple myeloma (MM)-induced bone disease is mortal for most MM patients. Bisphosphonates are first-line treatment for MM-induced bone disease, since it can inhibit osteoclast activity and the resultant bone resorption by suppressing the differentiation of osteoclast precursors into mature osteoclasts, promoting osteoclast apoptosis and disrupting osteoclast function. However, it is still unclear whether bisphosphonates have an anti-tumour effect. In our previous work, a computational model was built to simulate the pathology of MM-induced bone disease. This paper extends this proposed computational model to investigate the efficacy of bisphosphonates treatment and then clear the controversy of this therapy. The extended model is validated through the good agreement between simulation results and experimental data. The simulation results suggest that bisphosphonates indeed have an anti-tumour effect.

Direct Cell-To-Cell Interactions Between Osteocytes and Multiple Myeloma (MM) Cells Upregulate Sost and Downregulate OPG Expression In Osteocytes: Evidence For Osteocytic Contributions To MM-Induced Bone Disease

Osteocytes are the most abundant bone cells, comprising more than 95% of the cells in bone. They are embedded into the bone matrix, but extensively communicate among themselves and with cells on the bone surface and the bone marrow through the osteocytic lacunar-canalicular network. Osteocytes secrete sclerostin, the product of the Sost gene, an antagonist of Wnt signaling that potently inhibits bone formation. Osteocytes are also a major source of pro- and anti-osteoclastogenic cytokines that regulate osteoclastogenesis and bone resorption, including RANKL and osteoprotegerin (OPG). Recent evidence suggests that the bone remodeling compartment is disrupted in multiple myeloma (MM) allowing close contact of MM cells with bone cells including osteocytes. However, the consequences of these interactions and the contribution of osteocytes to MM bone disease are unclear. Therefore, we determined if interactions between MM cells and osteocytes regulate osteocytic gene expression. We found that co-culture of murine MLO-A5 osteocytic cells with human JJN3 MM cells up-regulated murine Sost mRNA expression 2-3 fold as early as 4h, which remained elevated up to 24h. Consistent with Sost upregulation induced by MM cells, the expression of OPG, a Wnt target gene, was decreased by 30-50% in MLO-A5 cells, resulting in an increased RANKL/OPG at 4h. Culture of JJN3 cells in the top and MLO-A5 cells in the bottom of Boyden chambers abolished both upregulation of Sost and downregulation of OPG mRNA expression in osteocytic cells, demonstrating the requirement of direct contact between MM cells and osteocytic cells. Human Sost and OPG mRNA transcripts were not detected in any of these experiments, demonstrating lack of contribution of MM JJN3 cells. These findings demonstrate that direct interactions between osteocytes and MM cells upregulate the expression of the bone formation inhibitor Sost in osteocytes, which in turn decreases Wnt signaling, reduces osteocytic OPG expression increasing the RANKL/OPG ratio. We propose that increased Sost/Sclerostin expression contributes to the exacerbated bone resorption and the decreased bone formation that characterizes MM induced bone disease. Disclosures:No relevant conflicts of interest to declare.

Adenosine regulates bone metabolism via A 1 , A 2A , and A 2B receptors in bone marrow cells from normal humans and patients with multiple myeloma

Multiple myeloma (MM) is characterized by osteolytic bone lesions with uncoupled bone remodeling. In this study, we examined the effects of adenosine and its receptors (A1R, A2AR, A2BR, and A3R) on osteoblast and osteoclast differentiation of cells derived from patients with MM and healthy control subjects. Mesenchymal stem cells and bone marrow-derived mononuclear cells were isolated from bone marrow and differentiated into osteoblasts and osteoclasts, respectively. A1R antagonist rolofylline and A2BR agonist BAY60-6583 inhibit osteoclast differentiation of cells from patients with MM in a dose-dependent manner, as shown by TRAP staining (IC50: 10 and ∼10 nM, respectively). BAY60-6583 and dipyridamole, a nucleoside transport inhibitor, stimulate osteoblast differentiation of cells from patients with MM, as measured by ALP activity at d 14 and Alizarin Red staining at d 21 (by 1.57±0.03- and 1.71±0.45-fold, respectively), which can be blocked by A2BR antagonist MRS1754. Consistently, real-time PCR showed a significant increase of mRNA of osteocalcin and osterix at d 14. The effect of adenosine and its receptors is consistent in patients with MM and healthy subjects, suggesting an intrinsic mechanism that is important in both MM bone metabolism and normal physiology. Furthermore, the effect of dipyridamole on osteoblast differentiation is diminished in both A2BR- and CD39-knockout mice. These results indicate that adenosine receptors may be useful targets for the treatment of MM-induced bone disease.

In silico investigations of potential anabolic treatments in multiple myeloma-induced bone disease

No anabolic drugs are currently approved to treat multiple myeloma (MM)-induced bone disease and the anti-MM agent bortezomib exhibits the anabolic effects in the clinic. In this study, we focus on investigating potential anabolic treatments of MM-induced bone disease using our previously proposed MM–bone model, with the goal for clarifying the underlying molecular/cellular mechanisms. Firstly, a variety of virtual drug treatments are explored by the parametric study to clarify the anabolic-related molecular/cellular mechanisms. The real drug (i.e., bortezomib) treatments are further examined by developing an integrated model with bortezomib to validate the clarified anabolic-related molecular/cellular mechanisms. The simulated responses to the bortezomib treatments that are validated by the clinical data are consistent with the simulated responses to the virtual drug treatments. Our study clarifies that the anabolic effects in the treatment of MM-induced bone disease are associated with promoting the differentiation of bone marrow stromal cells (BMSC) and inhibiting the apoptosis of active osteoblasts, while promoting the differentiation of osteoblast precursors is instead suggested to be associated with the anti-catabolic effects. Compared with the individual anabolic therapies, the anabolic therapies that promote the differentiation of BMSC in combination with the anti-MM/anti-catabolic therapies are found to induce a greater increase in the bone volume, while the anabolic therapies that inhibit the apoptosis of active osteoblasts in combination with the anti-MM/anti-catabolic therapies induce a lower increase in the bone volume. The simulations also suggest that the direct inhibition of bortezomib on the osteoclast activity is probably a redundant mechanism.

Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Abstract 4977 Background:Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients. Methods:Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity. Results:We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both). Conclusions:These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease. Disclosures:No relevant conflicts of interest to declare.

In Silico Investigations of the Anti-Catabolic Effects of Pamidronate and Denosumab on Multiple Myeloma-Induced Bone Disease

It is unclear whether the new anti-catabolic agent denosumab represents a viable alternative to the widely used anti-catabolic agent pamidronate in the treatment of Multiple Myeloma (MM)-induced bone disease. This lack of clarity primarily stems from the lack of sufficient clinical investigations, which are costly and time consuming. However, in silico investigations require less time and expense, suggesting that they may be a useful complement to traditional clinical investigations. In this paper, we aim to (i) develop integrated computational models that are suitable for investigating the effects of pamidronate and denosumab on MM-induced bone disease and (ii) evaluate the responses to pamidronate and denosumab treatments using these integrated models. To achieve these goals, pharmacokinetic models of pamidronate and denosumab are first developed and then calibrated and validated using different clinical datasets. Next, the integrated computational models are developed by incorporating the simulated transient concentrations of pamidronate and denosumab and simulations of their actions on the MM-bone compartment into the previously proposed MM-bone model. These integrated models are further calibrated and validated by different clinical datasets so that they are suitable to be applied to investigate the responses to the pamidronate and denosumab treatments. Finally, these responses are evaluated by quantifying the bone volume, bone turnover, and MM-cell density. This evaluation identifies four denosumab regimes that potentially produce an overall improved bone-related response compared with the recommended pamidronate regime. This in silico investigation supports the idea that denosumab represents an appropriate alternative to pamidronate in the treatment of MM-induced bone disease.

Bruton's tyrosine kinase: oncotarget in myeloma

Our findings therefore provide a strong rationale for investigating Btk inhibitors in MM and WM to target both tumor cells and their supporting BM microenvironment and thereby both suppress tumor cell growth and abrogate MM-induced bone disease.