Improving bone health via modulation of glycosphingolipid metabolism and autophagy


 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.


Introduction
Multiple Myeloma (MM) is a haematological cancer caused by abnormal plasma cell 53 expansion in the bone marrow (BM) 1  to suppression of bone turnover and increased fracture risk 10 . Novel ways to alleviate both pain 66 and disability caused by these bone lesions without causing severe side effects represent an 67 ongoing unmet clinical need. 68 Autophagy maintains eukaryotic cell homeostasis by degrading and recycling cellular waste 69 including protein aggregates and damaged organelles 11 . During OC differentiation Beclin-1 is 70 upregulated 12 and Atg7 knockdown inhibits the expression of key OC proteins such as tartrate-71 resistant acid phosphatase (TRAP) and cathepsin K 13 . Additionally, autophagy-related proteins 72 including ATG5, ATG7, ATG4B, and LC3 regulate a number of OC functions 14 . Concordantly, 73 autophagy inhibitors such as chloroquine (CQ) can inhibit OC formation and restore bone 74 mass 8 . Molecularly, TRAF3, as a suppressor of OC differentiation, is degraded by autophagy upon RANKL induction in BM OC precursors 8 . Based on these findings, it has been proposed 76 that autophagy inhibitors may be beneficial in treating bone loss diseases in the clinic. 77 Glycosphingolipids (GSLs) are constituents of the cellular membrane that are expressed in 78 varying ratios and combinations. We previously observed that GM3, a ganglioside abnormally 79 expressed in MM cells, promotes osteoclastogenesis 15 and that this can be blocked using 80 miglustat, an iminosugar and an analogue of D-glucose 16 . However, miglustat is involved in 81 various biological and pathological pathways and can cause deleterious side effects in the 82 gastrointestinal track including severe diarrhea and weight loss 17 . Eliglustat, a small synthetic 83 molecule, specifically prevents the synthesis of all GSLs by inhibition of glucosylceramide 84 synthase (GCS) 18 and is used for the treatment of Gaucher disease type 1 in adults 19 . Gaucher 85 patients suffer from osteoporosis/osteopenia and treatment to reduce excess bioactive GSL 86 associated with the disease has been shown to alleviate bone loss 20 . Interestingly, patients with 87 Gaucher disease have a 6-50 times increased risk of developing MM or the pre-MM MGUS 88 condition 21,22,23 . However, it remains to be determined whether the reduction in bone symptoms 89 is due to the treatment of Gaucher disease or if eliglustat acts directly on bone cells, which 90 raised the possibility that eliglustat may be of benefit in treating MM bone complications. 91 Here we demonstrate that eliglustat reduces bone disease by directly acting on bone cells,   Figure S1C and E), indicating that increased trabecular bone was a consequence 120 of OC inhibition rather than an increase in OB.

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Eliglustat treatment did not change the population of OC precursors as demonstrated by 122 expression of surface markers (CD11b-CD3-B220-CD115+) 24 in MM-bearing mice ( Figure   123 S2A-C) suggesting that eliglustat blocks OC formation rather than inhibiting the generation of 124 OC progenitors. Flow cytometry analysis of BM and spleen showed that there was no 125 difference in GFP+ 5TGM1 cells found in BM or spleen between MM-bearing mice and mice 126 treated with eliglustat ( Figure S2D-F). Consistently, serum paraprotein IgG2b! showed that 127 eliglustat did not reduce systemic tumor burden ( Figure S2G). Therefore, eliglustat ameliorates 128 MM-induced bone disease by specifically inhibiting OC without exacerbating tumor burden.   ****P<0.0001. Statistical analysis was performed using One-way ANOVA. Prior to 5TGM1-GFP cell injection, C57BL/6J mice were fed with 42% HFD or control diet 150 for 7 weeks, resulting in a significant increase in body weight in HFD group (Figure 2A-B).

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As previously reported, C57BL/6J mice on a control diet inoculated with 5TGM1 cells showed  Statistical analysis was performed using One-way ANOVA. 182 ZA leads to OC apoptosis via blockade of the mevalonate pathway 27 , whilst our data show that 183 eliglustat inhibits OC via an as yet unknown mechanism. The efficacy of combining these two 184 OC inhibitors was evaluated to ascertain any potential additive effect that would also allow ZA 185 to be used at lower concentrations to mitigate the potential to develop ONJ.

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Neither eliglustat nor ZA demonstrated a significant effect on OB parameters ( Figure S4A).

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Statistical analysis was performed using One-way ANOVA. 273 The degradation of TRAF3 in RANKL-induced OC formation is mediated by autophagy 274 machinery 8 . Therefore, whether eliglustat acts as an autophagic inhibitor was investigated.   311 Autophagy is critical to osteoclastogenesis and gangliosides are involved in autophagosomal 312 biogenesis 32,33 . It was hypothesized that eliglustat prevents the conversion of ceramide to 313 certain GSLs that are required for autophagy.

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To evaluate the overall lipid changes on the cellular membranes, the lipid organization of 315 eliglustat-treated RAW264.7 cells was evaluated by spectral imaging ( Figure 6A). The      This finding is particularly important as a known complication of high dose ZA is osteonecrosis of the jaw in MM patients 9 . Combination of eliglustat with ZA for optimal bone protection can 389 be achieved with lower amounts of ZA leading to a potential novel clinical strategy with 390 reduced side effects and thus accelerate eliglustat's translational use into the clinic. In addition, 391 following work will be conducted to combine eliglustat with anabolic drugs to further evaluate 392 eliglustat's role in bone disease models.

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However, CQ has considerable ophthalmic side effects and its substitute hydroxychloroquine, 415 now used more widely in the clinic, had no inhibitory effect on human OC differentiation 8,46 .

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On the other hand, BafA1 is not an FDA approved drug and its OC inhibition effect was only 417 observed in vitro 45 . Therefore, eliglustat is a more suitable drug than either BafA1 or CQ to 418 treat bone lesions in patients. Our findings pave the way to further investigate if the autophagy 419 inhibitor eliglustat can be used in combination with other compounds to treat diseases beyond 420 bone including refractory myeloma and melanoma 47,48 .

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The unprecedented finding that LacCer and GlcCer are essential for functional autophagy, Gaucher disease, could be repurposed for patients who suffer from various bone loss diseases.

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Experimental design 436 The objective of the study was to investigate if eliglustat increases bone mass and decreases 437 MM bone disease in several pre-clinical models, and to decipher the underlying mechanism.   Table S1. Briefly, BM mononuclear cells were isolated by Ficoll Paque (GE 454 Healthcare) density centrifugation from fresh BM aspirates as previously described 50 .