Myeloma-induced Bone Disease Research Articles

Evidence for peri-lacunar remodeling and altered osteocyte lacuno-canalicular network in mouse models of myeloma-induced bone disease.

Myeloma bone disease (MBD) affects ~90% of multiple myeloma patients, but current treatment options are suboptimal. Therefore, to successfully develop new therapies or optimize current ones, we must improve our fundamental knowledge of how myeloma affects bone microstructure and function. Here, we have investigated the osteocyte lacuno-canalicular network (LCN) in MBD, as bone porosity affects bone quality and resilience. We used the syngeneic 5TGM1-C57BL-Kalwrij and the xenograft U266-NSG models at end stage and compared them to healthy controls (naïve). Micro-computed tomography (μCT) and histomorphometry indicated the 5TGM1 and U266 models developed mild and extensive MBD, respectively, with the U266 model producing large osteolytic lesions. High-resolution synchrotron micro-CT (SR-μCT) revealed significant osteocyte lacunae changes in U266 bones but not 5TGM1, with a reduction in lacunae number and sphericity, and an increase in lacunae volume compared with naïve. Canalicular length, visualized using histological Ploton silver staining, appeared significantly shorter in 5TGM1 and U266 bones compared with naïve. Canalicular area as a proportion of the bone was also decreased by 24.2% in the U266 model. We observed significant upregulation of genes implicated in peri-lacunar remodeling (PLR), but immunohistochemistry confirmed that the osteocyte-specific protein sclerostin, a known driver of PLR, was unchanged between MBD and naïve bones. In summary, we have demonstrated evidence of PLR and altered organization of the osteocyte LCN in MBD mouse models. The next step would be to further understand the drivers and implications of PLR in MBD, and whether treatments to manipulate PLR and the LCN may improve patient outcomes.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

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.

Multi-Targeting DKK1 and LRP6 Prevents Myeloma-Induced Bone Disease

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodeling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting their quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. Therapeutic strategies aimed at promoting bone formation are therefore required to overcome the loss of skeletal integrity and subsequent fractures in MM patients.Therapeutic agents that target the canonical Wnt signaling pathway, a potent regulator of bone formation, have the potential to address these skeletal complications, where they could rebuild lost bone and improve bone strength in affected individuals. We have demonstrated a novel anti-LRP6 agent, which potentiates Wnt signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. However, since MM patients present with both increased bone resorption and decreased bone formation, we hypothesised that combining anti-LRP6 with the bone anabolic anti-DKK1 (100mg/kg twice weekly intravenously) would lead to more robust improvements in bone structure than single treatment approaches.MicroCT analysis demonstrated a 74% increase in femoral trabecular bone volume per tissue volume (BV/TV) in naïve, non-tumour bearing mice given the combination treatment compared to control agents (p<0.0001). Mice injected with 5TGM1eGFP murine myeloma cells had a 34% reduction in femoral BV/TV compared to naïve controls (p<0.0001). Combination treatment drastically improved BV/TV in 5TGM1-bearing mice by 111% (p<0.0001), compared to control, and this improvement with the combination treatment strategy was 25% greater than anti-LRP6 single treatment approaches (p<0.001). MicroCT analysis in L4 lumbar vertebrae demonstrated similar bone structural changes in 5TGM1-bearing mice treated with the combination strategy. Consequently, this combination significantly improved resistance to fracture in L4 vertebrae in 5TGM1eGFP-bearing mice compared to their controls (p<0.001), and it provided greater protection against fracture compared to anti-LRP6 single agent treatment. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1eGFP-bearing mice treated with the combination strategy (p<0.001) compared to control. Importantly, tumour activity was not altered with either single or combination Wnt-promoting treatment strategies.This study defines a novel therapeutic strategy, which will reduce fractures and improve quality of life in patients with MM when used in combination with tumour-targeted treatments. [Display omitted] DisclosuresCong: Novartis Institutes for Biomedical Research: Current Employment. Daley: Novartis Institutes of Biomedical Research: Current Employment.

Myeloma Bone Disease: The Osteoblast in the Spotlight.

Lytic bone disease remains a life-altering complication of multiple myeloma, with up to 90% of sufferers experiencing skeletal events at some point in their cancer journey. This tumour-induced bone disease is driven by an upregulation of bone resorption (via increased osteoclast (OC) activity) and a downregulation of bone formation (via reduced osteoblast (OB) activity), leading to phenotypic osteolysis. Treatments are limited, and currently exclusively target OCs. Despite existing bone targeting therapies, patients successfully achieving remission from their cancer can still be left with chronic pain, poor mobility, and reduced quality of life as a result of bone disease. As such, the field is desperately in need of new and improved bone-modulating therapeutic agents. One such option is the use of bone anabolics, drugs that are gaining traction in the osteoporosis field following successful clinical trials. The prospect of using these therapies in relation to myeloma is an attractive option, as they aim to stimulate OBs, as opposed to existing therapeutics that do little to orchestrate new bone formation. The preclinical application of bone anabolics in myeloma mouse models has demonstrated positive outcomes for bone repair and fracture resistance. Here, we review the role of the OB in the pathophysiology of myeloma-induced bone disease and explore whether novel OB targeted therapies could improve outcomes for patients.

Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche

BackgroundMultiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct...

Checkpoint Inhibitor PD-1H/VISTA Mediates MMP-13 Induced Osteoclast Activation and Multiple Myeloma Bone Disease

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

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

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

Hyaluronan hydrogels delivering BMP-6 for local targeting of malignant plasma cells and osteogenic differentiation of mesenchymal stromal cells

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm2, corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad 1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to minimize disease relapse or fractures. Statement of SignificanceMultiple myeloma is a hematological cancer characterized by the accumulation of clonal plasma cells in the bone marrow and local suppression of bone formation, resulting in osteolytic lesions and fractures. Despite recent advances in systemic treatment of multiple myeloma, it is rare to achieve a targeted suppression of myeloma cells and healing of bone lesions. Here we present hydrogels which mimic the physico-chemical properties of the bone marrow, consisting of hyaluronic acid with crosslinked heparin for the controlled presentation of bioactive BMP-6. The hydrogels decrease the viability of myeloma cell lines and primary myeloma cells and induces osteogenic differentiation of mesenchymal stromal cells. The presentation of BMP-6 in the hyaluronan hydrogels enhances the phosphorylation of Smad1/5 while reducing the activity of the BMP-6 antagonist sclerostin. As such, BMP-6 functionalized hyaluronan hydrogels represent a promising material for the localized eradication of myeloma cells.

Abstract 359: Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma

Abstract The bromodomain and extra-terminal (BET) family of proteins are important epigenetic regulators of critical oncogenes, including c-Myc. JQ1 is the most potent BRD4 inhibitor, currently used in several clinical trials and effective in preventing multiple myeloma progression in vivo. Currently, clinical data is showing that patients often develop acquired resistance, indicating that single agent therapies targeting BRD4 such as JQ1 may not provide durable therapeutic responses. To overcome this limitation, we have developed a new class of dual inhibitors, capable to simultaneously target BRD4 and a panel of tyrosine kinases highly expressed in cancer including JAK2, FLT3, RET, FGFR1, ULK1 and ULK3. A preliminary in vitro screening identified SG3-014 as the lead compound, showing BRD4 and c-Myc inhibition activity similar to JQ1, but additionally, it is capable of inhibiting kinases that are over-activated in cancer and responsible for cytokine-mediated drug resistance pathways. In vitro assays on MM cell lines reveals higher SG3-014 sensitivity (5TGM1, 0.85μM; U266, 0.99μM) compared to JQ1 (5TGM1, 4.7μM; U266, 16μM). In vivo studies using 1x106 5TGM1-Luc cells demonstrate that SG3-014 treatment (25mg/Kg) significantly contributes to overall survival compared to JQ1 and the vehicle control cohort (n=10/group) (median survival CTRL=40.5; SG3-014=50.5; JQ1=46 days). Post-study analyses demonstrated a significant reduction in myeloma induced bone disease in the SG3-014 treated mice as measured by X-ray/ μCT/ histomorphometry. Clinical datasets identified ULK3 as the candidate kinase of SG3-014 targeting activity and correlated its expression with MM disease stages. Since ULK3 is involved in cellular senescence and autophagy regulation, a key mechanism by which several cancer cell lines protect themselves against apoptosis and become resistant to standard treatments, we explored the role of SG3-014 inhibition in the autophagy pathway. Preliminary data confirm that SG3-014 is an autophagy inhibitor and, as opposed to JQ1, downregulates ATG proteins level, leading to dysfunctional autophagosome formation. We noted that within 6 h, SG3-014 completely shut down autophagy with decrease levels of ULK1, beclin-1, ATG16L-1, ATG12, ATG3, p62 and increased LC3I/II ratios (additionally confirmed by microscopy and flow cytometry). These effects were not noted with JQ1. Further investigation is clarifying the mechanism by which SG3-014 inhibitory activity on ULK3 is responsible for autophagy mediated cancer cell death. In order to validate this target in the progression of the human disease, our future directions will analyze ex vivo multiple myeloma patient samples and examine the efficacy of the inhibitor in CD138 isolated multiple myeloma cells derived from patient biopsies. We propose dual BRD4/autophagy inhibition as a resilient strategy to prevent multiple myeloma by-pass of single targeted therapies. Citation Format: Marilena Tauro, Muhammad Ayaz, Harshani R. Lawrence, Nicholas J. Lawrence, Kenneth H. Shain, Ernst Schonbrunn, Conor C. Lynch. Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 359.

Reprogrammed marrow adipocytes contribute to myeloma-induced bone disease.

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

Hyaluronan Hydrogels Delivering BMP-6 for Local Targeting of Malignant Plasma Cells and Osteogenic Differentiation of Mesenchymal Stromal Cells

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm2, corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to prevent disease relapse or fractures.

Preventing and Repairing Myeloma Bone Disease by Combining Conventional Antiresorptive Treatment With a Bone Anabolic Agent in Murine Models.

ABSTRACTMultiple myeloma is a plasma cell malignancy, which develops in the bone marrow and frequently leads to severe bone destruction. Current antiresorptive therapies to treat the bone disease do little to repair damaged bone; therefore, new treatment strategies incorporating bone anabolic therapies are urgently required. We hypothesized that combination therapy using the standard of care antiresorptive zoledronic acid (Zol) with a bone anabolic (anti‐TGFβ/1D11) would be more effective at treating myeloma‐induced bone disease than Zol therapy alone. JJN3 myeloma‐bearing mice (n = 8/group) treated with combined Zol and 1D11 resulted in a 48% increase (p ≤ 0.001) in trabecular bone volume (BV/TV) compared with Zol alone and a 65% increase (p ≤ 0.0001) compared with 1D11 alone. Our most significant finding was the substantial repair of U266‐induced osteolytic bone lesions with combination therapy (n = 8/group), which resulted in a significant reduction in lesion area compared with vehicle (p ≤ 0.01) or Zol alone (p ≤ 0.01). These results demonstrate that combined antiresorptive and bone anabolic therapy is significantly more effective at preventing myeloma‐induced bone disease than Zol alone. Furthermore, we demonstrate that combined therapy is able to repair established myelomatous bone lesions. This is a highly translational strategy that could significantly improve bone outcomes and quality of life for patients with myeloma. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Bone Research Society Abstracts

Bone Research Society Abstracts

ARQ-197, a small-molecule inhibitor of c-Met, reduces tumour burden and prevents myeloma-induced bone disease in vivo.

The receptor tyrosine kinase c-Met, its ligand HGF, and components of the downstream signalling pathway, have all been implicated in the pathogenesis of myeloma, both as modulators of plasma cell proliferation and as agents driving osteoclast differentiation and osteoblast inhibition thus, all these contribute substantially to the bone destruction typically caused by myeloma. Patients with elevated levels of HGF have a poor prognosis, therefore, targeting these entities in such patients may be of substantial benefit. We hypothesized that ARQ-197 (Tivantinib), a small molecule c-Met inhibitor, would reduce myeloma cell growth and prevent myeloma-associated bone disease in a murine model. In vitro we assessed the effects of ARQ-197 on myeloma cell proliferation, cytotoxicity and c-Met protein expression in human myeloma cell lines. In vivo we injected NOD/SCID-γ mice with PBS (non-tumour bearing) or JJN3 cells and treated them with either ARQ-197 or vehicle. In vitro exposure of JJN3, U266 or NCI-H929 cells to ARQ-197 resulted in a significant inhibition of cell proliferation and an induction of cell death by necrosis, probably caused by significantly reduced levels of phosphorylated c-Met. In vivo ARQ-197 treatment of JJN3 tumour-bearing mice resulted in a significant reduction in tumour burden, tumour cell proliferation, bone lesion number, trabecular bone loss and prevented significant decreases in the bone formation rate on the cortico-endosteal bone surface compared to the vehicle group. However, no significant differences on bone parameters were observed in non-tumour mice treated with ARQ-197 compared to vehicle, implying that in tumour-bearing mice the effects of ARQ-197 on bone cells was indirect. In summary, these res ults suggest that ARQ-197 could be a promising therapeutic in myeloma patients, leading to both a reduction in tumour burden and an inhibition of myeloma-induced bone disease.

Maternal embryonic leucine zipper kinase inhibitor OTSSP167 has preclinical activity in multiple myeloma bone disease.

Multiple myeloma bone disease is characterized by an uncoupling of bone remodeling in the multiple myeloma microenvironment, resulting in the development of lytic bone lesions. Most myeloma patients suffer from these bone lesions, which not only cause morbidity but also negatively impact survival. The development of novel therapies, ideally with a combined anti-resorptive and bone-anabolic effect, is of great interest because lesions persist with the current standard of care, even in patients in complete remission. We have previously shown that MELK plays a central role in proliferation-associated high-risk multiple myeloma and its inhibition with OTSSP167 resulted in decreased tumor load. MELK inhibition in bone cells has not yet been explored, although some reports suggest that factors downstream of MELK stimulate osteoclast activity and inhibit osteoblast activity, which makes MELK inhibition a promising therapeutic approach. Therefore, we assessed the effect of OTSSP167 on bone cell activity and the development of myeloma-induced bone disease. OTSSP167 inhibited osteoclast activity in vitro by decreasing progenitor viability as well as via a direct anti-resorptive effect on mature osteoclasts. In addition, OTSSP167 stimulated matrix deposition and mineralization by osteoblasts in vitro. This combined anti-resorptive and osteoblast-stimulating effect of OTSSP167 resulted in the complete prevention of lytic lesions and bone loss in myeloma-bearing mice. Immunohistomorphometric analyses corroborated our in vitro findings. In conclusion, we show that OTSSP167 has a direct effect on myeloma-induced bone disease in addition to its anti-multiple myeloma effect, which warrants further clinical development of MELK inhibition in multiple myeloma.

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.

Effects of time delays in a mathematical bone model**Project supported by the National Natural Science Foundation of China (Grant No. 11474117).

In this paper we propose a mathematical model of bone remodeling with time delays of both osteoclast-derived paracrine signaling of tumor and tumor-derived paracrine signaling of osteoclast. The effects of time delays on the growth of tumor cells and bone system are studied in multiple myeloma-induced bone disease. In the case of small osteoclast-derived paracrine signaling, it is found that the growth of tumor cells slows down, the oscillation period of the ratio of osteoclasts to osteoblasts is extended with increasing time delay, and there is a competition between the delay and osteoclast-derived paracrine signaling. In the case of large tumor-derived paracrine signaling, the tumor-derived paracrine signaling can induce a more significant decline in tumor growth for long time delay, and thus slowing down the progression of bone disease. There is an optimal coupling between the tumor-derived paracrine signaling of osteoclasts and time delay during the progressions of bone diseases, which suppresses the tumor growth and the regression of bone disease.

Pinpointing a Factor in Myeloma Bone Disease.

A recent study implicates thymidine phosphorylase in myeloma-induced bone disease and suggests that inhibiting the enzyme may be a viable therapeutic option. Thymidine phosphorylase provokes osteolytic lesions by disrupting the balance between bone resorption and formation, shifting it toward a net loss of bone tissue.