Bone Morphogenetic Protein 4 Gene Therapy in Mice Inhibits Myeloma Tumor Growth, But Has a Negative Impact on Bone.

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre- and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4-treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2-/- γC-/-mice. Mice were treated with adeno-associated virus serotype 8 BMP4 vectors (AAV8-BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4-treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non-myeloma femurs by μCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8-CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

Serum protein N-glycosylation changes in multiple myeloma

Proteomic

Glycosylation of immunoglobulins determine bone loss in multiple myeloma

Proteomic

Monoclonal immunoglobulins promote bone loss in multiple myeloma

Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.Proteomic

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism