Glucose Ceramide Synthase Inhibitors Inhibit Osteoclast Activation Induced by Myeloma-Derived and De Novo Synthesized Glycosphingolipids.

Abstract 424

In Multiple myeloma (MM), a malignancy of plasma cells, autocrine and paracrine networks involving the malignant cells and their microenvironment, especially osteoclasts (OC), play a crucial role in disease pathogenesis. OC activation and bone destruction are common and devastating events in this disease. Tumour-derived glycosphingolipids (GSL) have been shown to modify the tumour microenvironment by promoting tumour growth, angiogenesis, immune evasion and metastasis. The aim of this work was two-fold: 1) to investigate the role of MM-derived GSL in OC development and activation and 2) to define the role of de novo GSL synthesis in OC development. Using MALDI-TOF MS and MS-MS, we first determined the GSL repertoire of primary CD138+ myeloma cells (n=3) and compared it with non-myeloma bone marrow cells (i.e., without CD138+ cells) (n=3), and various myeloma cell lines (n=5). We found that GM3 was the dominant GSL in primary myeloma cells and GM2/GM3 in myeloma cell lines; by contrast, in non-myeloma marrow the non-polar LacCer was the dominant GSL. As GM3 was the dominant GSL in myeloma cells, we tested its effect on osteoclast function. We found that exogenous GM3 synergistically enhanced the ability of M-CSF and RANKL to induce maturation of murine bone marrow OC in vitro. This, as shown by immunoblotting, was associated with increased ERK1/2, p38, JNK phosphorylation and NFATc dephosphorylation, signal transduction and transcriptional events respectively, required for OC differentiation and maturation in response to RANKL. Furthermore, GM3 further enhanced OC maturation in synergy with IGF-1, a growth factor known to promote myeloma growth and OC activation. Next we tested the effect of inhibition of de novo GSL biosynthesis on osteoclastogenesis. We found that the glucose ceramide synthase (GCS) inhibitors NB-DNJ inhibited RANKL- and M-CSF-dependent development of murine as well as human, monocyte-derived OC in a dose dependent manner when added either in the beginning or during OC differentiation cultures. This effect was associated with significantly reduced RANKL- and M-CSF-dependent phosphorylation of ERK, JNK and p38 as well as reduced localisation of NFATc in the nucleus. OC development in response to RANKL-RANK interaction requires movement of RANK into lipid rafts where it interacts with TRAF6, an adaptor crucial for downstream signalling and with cSrc which is required for actin ring formation and OC resorptive activity. Using sucrose gradient membrane fractionation and GM1 as a marker of rafts we found that GCS inhibitors partially disrupt the integrity of lipid rafts in developing osteoclasts and prevent RANKL-induced localisation of TRAF6 and Src in lipid rafts. Next we tested the ability of NB-DNJ to block OC activation caused by a single injection of alpha-galactosylceramide (αGC), an invariant NKT cell ligand known to rapidly activate the innate immune response. We found that in mice receiving αGC, serum C-telopeptide Type I collagen (CTX) levels increased by ∼50% (p<0.01), while in mice co-injected with αGC plus NB-DNJ (daily for 3 days i.p) CTX levels returned to baseline (p<0.01); in mice receiving only NB-DNJ, CTX levels were not significantly different to vehicle controls (p>0.05). Taken together, our data demonstrate a novel role of GSL in promoting OC differentiation and activation. Thus, GCS inhibitors may be of benefit in reducing OC activation and bone destruction in MM by preventing generation of tumour-derived, pre-osteoclastogenic GSL as well as by inhibiting de novo OC GSL synthesis and thus OC activation.