Search

Patients with multiple myeloma (MM), an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions. In this study, glycosphingolipids were essential in promoting autophagic degradation of the signaling molecule TRAF3, a key step in bone-resorbing osteoclast differentiation. Specifically altering the glycosphingolipid composition with eliglustat, an FDA approved glucosylceramide synthase inhibitor, arrested osteoclast differentiation; this could be rescued by exogenous addition of the missing glycosphingolipids. Eliglustat significantly reduced bone disease in several preclinical models of MM by inhibiting osteoclastogenesis and, due to its unique mode of action, it was able to act in combination with existing bone protective drugs. Furthermore, eliglustat arrested osteoclast differentiation from the bone marrow of MM patients in a glycosphingolipid-dependent way. This work identifies both the mechanism by which glucosylceramide synthase inhibition blocks autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the translational potential of eliglustat to be combined with current treatments.

Multiple myeloma (MM) is a fatal hematological malignancy, where the majority of patients are diagnosed with, or develop, destructive and debilitating osteolytic bone lesions. Current treatments for MM bone disease such as the bisphosphonate zoledronic acid can result in deleterious side effects at high doses. In this study, eliglustat, an FDA approved glycosphingolipid inhibitor, was shown to reduce MM bone disease in preclinical models of MM. Mechanistically, eliglustat alters the lipid composition and plasma membrane fluidity and acts as an autophagy flux inhibitor in bone-resorbing osteoclasts (OC). Autophagic degradation of the signaling molecule TRAF3 is key step in OC differentiation; this was prevented by eliglustat in OC precursors. In addition, eliglustat works depend on TRAF3 in vivo. Furthermore, the combination of eliglustat and zoledronic acid was found to have an additive effect to reduce MM bone disease, suggesting the potential for combination therapies that would allow for drug dose reductions. Taken together, this project identifies a novel mechanism in which glycosphingolipid inhibition reduces osteoclastogenesis via autophagy and highlights the translational potential of eliglustat for the treatment of bone loss disorders such as MM.One Sentence SummaryTranslational use of eliglustat as an autophagy inhibitor to limit bone lesions in multiple myeloma.

Alterations in glycosphingolipid production results in lysosomal storage disorders associated with neurodegenerative changes. In Gauchers disease, the patients also develop osteoporosis that is ameliorated upon treatment for the underlying defect in glycosphingolipid metabolism. The role of glycosphingolipids in osteoclast and osteoblast formation is discussed here as well as the potential therapeutic uses of already approved drugs that limit glycosphingolipid production in bone loss disorders such as multiple myeloma and periodontal disease.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

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.

The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening.