ALBERT

Description: Multiple myeloma (MM) is an incurable cancer of plasma cells (PC), with a median survival of 5-7 years. Osteolytic bone disease and skeletal complications occur in more than 80% of MM patients and significantly contribute to the morbidity and mortality of these patients. Glycosphingolipid (GSL), an essential constituent of the outer leaflet of the cellular membrane, is altered in MM and other hematological cancers. We previously reported that GM3, a subtype of GSL promotes osteoclastogenesis. On the other hand, the GSL synthase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) reduces myeloma bone disease in the 5TGM1 mouse model of MM. Mechanistically, NB-DNJ prevents osteoclast (OC) development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of the transcriptional activator NFATc1. Although NB-DNJ is an FDA-approved drug treating Gaucher's disease, it has many undesired off-target effects, such as inhibiting lysosomal and plasma membrane Beta-glucocerebrosidase and interfering with intestinal glucosidases which leads to gastrointestinal toxicities and severe weight loss. Therefore, more specific GSL inhibitors are required to minimize the side effects. Here we report a novel GSL inhibitor called Genz112638 with comparable effects as NB-DNJ but reduced side effects. Genz112638 inhibits both OC formation (p 〈 0.01) and MM cell growth (p 〈 0.0001) in vitro in a dose-dependent manner. Moreover, compared to NB-DNJ, Genz112638 more significantly improved bone condition and potentially reduced MM burden, as evidenced by the amelioration of bone loss in the 5TGM1 model of myeloma, and a reduction in the proportion of MM within bone marrow and spleen without obvious adverse effects (n=6) (p 〈 0.01). As excessive malignant PC in MM normally arise from germinal centre, we also checked the effects of Genz112638 on germinal centre reactions in wildtype mice. We found that Genz112638 suppresses the formation of germinal centre B cells in mouse spleen induced by sheep red blood cells (n=7). Thus, Genz112638 may affect the pathogenesis of MM disease at the initial stage. Taken together, our data elucidate a novel specific GSL inhibitor as a promising candidate drug relieving two main features of MM: bone destruction and tumour burden with negligible side effects. In vitro, it decreases OC differentiation and proliferation, and meanwhile decreases MM viability and proliferation. In vivo, it may suppress B cell formation in germinal centre, ameliorate bone destruction, and potentially interfere with the vicious cycle between increased OC and susceptibility to MM. In short, we provide a preclinical platform for GSL inhibition as a new tool against MM and its related complications. Figure. Figure. Disclosures Horwood: Genzyme: Research Funding.

Description: The contributions of the host microenvironment to the pathogenesis of multiple myeloma, including progression from the non-malignant disorder monoclonal gammopathy of undetermined significance, are poorly understood. In the present study, microarray analysis of a murine model requiring a unique host microenvironment for myeloma development identified decreased host-derived adiponectin compared with normal mice. In support, clinical analysis revealed decreased serum adiponectin concentrations in monoclonal gammopathy of undetermined significance patients who subsequently progressed to myeloma. We investigated the role of adiponectin in myeloma pathogenesis and as a treatment approach, using both mice deficient in adiponectin and pharmacologic enhancement of circulating adiponectin. Increased tumor burden and bone disease were observed in myeloma-bearing adiponectin-deficient mice, and adiponectin was found to induce myeloma cell apoptosis. The apolipoprotein peptide mimetic L-4F was used for pharmacologic enhancement of adiponectin. L-4F reduced tumor burden, increased survival of myeloma-bearing mice, and prevented myeloma bone disease. Collectively, our studies have identified a novel mechanism whereby decreased host-derived adiponectin promotes myeloma tumor growth and osteolysis. Furthermore, we have established the potential therapeutic benefit of increasing adiponectin for the treatment of myeloma and the associated bone disease.

Description: There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of β-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

Description: In phases of myeloma dormancy such as MGUS (monoclonal gammopathy of uncertain significance) or post chemotherapy remission, certain characteristics of the bone marrow niche promote quiescence of the tumor. Myeloma cell dormancy has been proposed to be induced by contact with 'bone-lining' endosteal niche cells, which are destroyed by active disease. Preservation of dormancy could prevent disease relapse or MGUS progression to myeloma, however the molecular mechanisms and signaling pathways that maintain this bone-lining niche are unknown. We addressed this in vivo by sorting endosteal niche components (osteoblasts, mesenchymal stem cells (MSCs), endothelial cells and tumor) from myeloma-bearing and control mice, followed by RNA-Seq transcriptome profiling and gene set enrichment analysis (GSEA). Endosteal MSCs showed greatest transcriptome differences between myeloma-bearing and control groups. MSCs from myeloma-bearing mice showed positive enrichment (p = 0.004) for a 200-gene bone remodeling gene set. The leading edge (highest contributors to enrichment) of this gene set contained 24% BMP pathway genes, far higher than the next nearest pathway represented (TGFb signaling and AP-1 complex, both 6.5%). Whereas various other signaling pathways identified in the leading edge are known to be of high importance in myeloma bone disease (e.g. RANKL, TGFb, Wnt, hedgehog), BMP signaling has not hitherto been reported as deregulated. To assess the role of BMP signaling in myeloma in vitro and in vivo, we used the BMP pathway inhibitor LDN-193189 (LDN), which has high affinity for type I BMP receptors Alk2, Alk3 and Alk6. In the myeloma-bearing KaLwRij/5TGM1 mouse model, LDN significantly improved trabecular (p=0.02) and cortical bone volume (p=0.004) and reduced serum TRAP levels (p=0.003). Histomorphometric analysis demonstrated that LDN reduced osteoclast numbers (p

Description: When cancer cells are resident in bone, they initiate a vicious cycle with osteoclasts (OCs) which perpetuates their growth and aggressive behavior. OCs are critical for the maintenance of the vicious cycle, since they control not only bone destruction associated with cancer, but also the aggressive behavior of tumor cells. It has recently been recognized that tumor cells grow in distant sites because they induce non-malignant cells to establish a “pre-metastatic niche” for tumor cells to later engraft. But nothing is yet known for bone. Primitive bone marrow progenitor cells, called myeloid immune suppressor cells (MISCs), which suppress immune reactivity, are important niche components. MISCs belong to the myelomonocytic lineage with surface markers of Gr-1 and CD11b. We hypothesize that MISCs are precursors of OCs recruited by tumors to assist in the establishment of the vicious cycle. To test this hypothesis, we used the well-characterized 5TGM1 murine myeloma model. 5TGM1-GFP tagged myeloma cells were inoculated via tail vein. The proportion of Gr-1+CD11b+ cells in bone marrow and spleen were assessed by FACS. On week 4 after tumor cell inoculation, %Gr-1+CD11b+ cells were significantly greater in tumor-bearing mice compared with controls (60.9±7.8% vs 37.7±8.6% p

Description: The proteasome inhibitor bortezomib (VELCADE®) represents an important advance in the treatment of multiple myeloma. Despite its very significant beneficial effects, its precise mechanism of action remains unclear. There is currently evidence to suggest that bortezomib has the potential to directly affect both myeloma cells and osteoblasts. However, it is not possible to discern the precise mechanism of action of bortezomib in patients with myeloma. To address this we used a well-characterized murine model of myeloma combined with MALDI mass spectrometry (MALDI MS) to study the effect of bortezomib on bone formation and tumor burden in vivo and protein profiles of myeloma cells. To more closely reflect the clinical setting, we used an established treatment protocol, in which bortezomib treatment was initiated upon development of myeloma. 5TGM1-GFP myeloma cells were inoculated into C57BlKaLwRij mice, resulting in tumor growth within the bone marrow and the development of an osteolytic bone disease. Tumor burden was monitored by measurement of serum IgG2bκ, and a significant increase was detected 14 days following tumor inoculation. Mice were then randomized to receive either bortezomib (0.5mg/kg, 3x week) or vehicle control for the remainder of the experiment. Treatment with bortezomib resulted in a significant reduction in tumor burden, as determined by serum IgG2bκ concentrations and by flow cytometric analysis of GFP-positive cells in the bone marrow. The tibia was analyzed by microCT, and bortezomib was found to significantly increase trabecular bone volume and reduce the number of osteolytic bone lesions. In order to study bone formation, mice were treated with 2 doses of calcein at a 7 day interval. Myeloma-bearing mice were associated with a significant reduction in rates of bone formation, which was prevented by treatment with bortezomib. GFP-positive myeloma cells were isolated from bone marrow of control- and bortezomib-treated mice and purified by fluorescence-activated cell sorting. Protein profiling by MALDI MS identified a number of proteins, both known and unknown, which were regulated by bortezomib treatment in vivo. Free ubiquitin (m/z 8565) was down-regulated, which is a known effect of proteasome inhibition resulting from the inhibition of protein degradation and release of free ubiquitin. Regulated proteins also included the up-regulation of thymosin beta-10 (m/z 4936) following bortezomib treatment. Thymosin beta 10 was undetectable in myeloma cells from control-treated mice. In addition, the calcium binding protein calgranulin A (S100A8) (m/z 10163) was up-regulated in myeloma cells isolated from bortezomib-treated mice. Neither thymosin-beta 10 nor calgranulin A have been previously linked to the activity of the proteasome, and thus may indicate novel molecular mechanisms involved in the anti-myeloma effect of bortezomib. Our data demonstrate that treatment with bortezomib from the time of established myeloma not only reduces myeloma tumor burden but also significantly increases bone formation in vivo. In addition, proteomic analysis of myeloma cells following in vivo treatment with bortezomib reveals a number of regulated proteins, providing novel insights into the molecular mechanism of action of proteasome inhibition in multiple myeloma in vivo.

Description: Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.