The Notch pathway controls multiple myeloma crosstalk with the osteoclastogenic niche

Introduction Multiple myeloma (MM) is an incurable hematological tumor stemming from malignant plasma cells that accumulate in the bone marrow (BM) and establish interactions with BM stroma, promoting tumor survival and bone disease due to unbalanced bone deposition and resorption. The Notch family consists of 4 receptors, Notch1 to 4, which once activated, act as transcription factors. The activation is triggered by Jag1-2 and DLL1-3-4 ligands. Notch plays a key role in bone tissue remodeling and skeletal development. Notch signaling is deregulated in MM and plays a role in MM pathogenesis by modulating tumor cell biology, as well as pathological interactions with BM niche. The myeloma-associated alteration of Notch signaling consists in the aberrant expression of the ligands Jag1 and Jag2 by MM cells, resulting in Notch signaling activation in both tumor cells and BM stroma cells. Aims The aim of this work was to investigate the role of Notch signaling in MM-driven osteoclastogenesis. To address this issue we assessed: · The role of Notch in osteoclast (OCL) differentiation and activity · The contribute of different Notch isoforms in OCL development · The involvement of Notch pathway in MM cell osteoclastogenic properties. Methods Cells were maintained in complete DMEM medium with 10% heat inactivated FBS. DAPT was added to the medium at a final concentration of 50μM. Recombinant mouse RANKL was used at the final concentration of 50ng/ml. Jagged1 recombinant peptide was used at 0.5μg/ml. anti-RANKL neutralizing antibody was used at 0.1μg/ml. OCL differentiation of Raw264.7 cells was induced by treating them with mRANKL or co-culturing with MM cells or their conditioned medium (CM). After 5-7days cells were stained using the TRAP Kit and counted. For bone resorption assay, Raw264.7 cells were cultured on Osteo Assay Surface plates under differentiation conditions. After 7-10 days, the plates were washed in 5% sodium hypochlorite solution. Images of the resorbed areas on the plates were captured and the percentage of resorbed area was measured by using the Wimasis image analysis software (Wimasis GmbH). Select RNAiTM siRNA system (Invitrogen) was used according to the manufacturer’s guidelines for the selective knock-down of Jag1 and Jag2. Transfection was performed by electroporation using two plasmid carrying intracellular Notch1 (ICN1) and Notch2 (ICN2). Total RNA was isolated using TRI-Reagent. cDNA was prepared through MMLV reverse transcriptase, then quantitative PCR (qPCR) was performed by Maxima SYBR Green qPCR Master Mix. ELISA Assay was performed using biotin-conjugated goat anti-human RANKL (Merck-Millipore) and Streptavidin-HRP-labeled secondary antibody. Results We demonstrates that Notch signaling drives MM cell-induced osteoclastogenesis. The underlying molecular mechanisms is based on MM cell-derived Jagged ligands ability to efficiently drive osteoclastogenesis by contemporaneously activating Notch signaling on tumor cells and osteoclasts. Notch signaling activation in MM cell promote the release of the osteoclastogenic receptor activator of NF-kB ligand (RANKL). RANKL, in turn, promotes within OCL precursors Notch2 signaling which drives osteoclastogenesis completion by promoting the transcription of osteoclastogenic master genes, such as Tartrate-resistant acid phosphatase (TRAP) and Receptor Activator of Nuclear Factor κ B (RANK) and the autonomous secretion of RANKL by OCL precursors. Remarkably, MM-induced osteoclastogenesis can be disrupted by silencing Jagged1 and Jagged2 Notch ligands in MM cells. Conclusions Our findings make Jagged1 and Jagged2 new promising therapeutic targets to hamper MM-associated bone disease and co-morbidities, lacking the toxicity of the currently used drugs which contemporaneously affect the signaling of all Notch receptors.