The notch ligands Jagged1 and 2 are a potential therapeutic target in multiple myeloma- associated bone disease

Background: Bone disease is still a relevant issue in multiple myeloma (MM), since it not only affects patients quality of life but it also promote tumor growth and survival, finally contributing to the fatal outcome of this disease. The Notch oncogenic pathway is dysregulated in MM, due to the hyperexpression of the Jagged 1 and 2 ligands. This alteration improve the ability of MM cells to establish pathological interactions with the bone marrow (BM) niche, finally promoting tumor progression. Notch is also a key regulator of bone tissue remodeling and skeletal development. Aims: The aim of this work was to provide a rationale for the targeting of the two Notch ligands Jagged 1 and 2 in MM- driven bone disease. To address this issue we assessed: • The role of the Notch pathway in MM-associated osteoclastogenesis • The effects of Jagged1/2 silencing on the ability of MM cells to activate the Notch pathway in OCLs and boost bone resorption Methods: OCL differentiation of Raw264.7 cells was induced by treating them with 50ng/ml mRANKL or co-culturing with MM cells or their conditioned medium (CM). After 5-7days cells were stained using the TRAP Kit and counted. DAPT was used at a concentration of 50μM. Select RNAi TM siRNA system (Invitrogen) was used according to the manufacturer’s guidelines for the selective knock-down of Jag1 and Jag2. Jagged1 recombinant peptide was used at 0.5μg/ml. anti- RANKL neutralizing antibody was used at 0.1μg/ml.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.RANKL was quantify by ELISA Assay and flow cytometry. Results: Our findings indicate that the autonomous release of RANKL (Receptor activator of nuclear factor kappa-B ligand) by MM cells is essential for their ability to boost osteoclastogenesis. Interestingly, RANKL release is Notch- dependent, since Jagged1/2 silencing in MM cells causes the inhibition of the Notch pathway and impairs their ability to secrete RANKL and to stimulate osteoclasts (OCLs) differentiation and activity. MM-derived Jagged are also able to directly activate the pro-osteoclastogenic Notch signaling in neighboring pre-OCLs, boosting their differentiation. Moreover, Jagged1/2 are essential for the interaction of MM cells with BM stromal cells (BMSCs), that can further enhance the osteoclastogenic potential of tumor cells. Jagged1/2 withdrawal blocks the cross-talk between MM cells and the surrounding BMSCs and pre-OCLs, finally causing a decrease in the formation of mature OCLs and in bone resorption. Summary/Conclusion: Our study provided the first evidence that two Notch ligands dysregulated in MM, Jagged 1 and 2, play an essential role in myeloma-induced osteoclast differentiation and bone resorption activity. Jagged ligands can trigger Notch signaling in the same MM cells, resulting in the release of the key osteoclastogenic factor RANKL, and may also activate Notch signaling in the neighboring osteoclast progenitor further promoting their differentiation. Finally, we demonstrated that MM cells are able to crosstalk with BMSCs which are able to stimulate low-RANKL expressing myeloma cells, to release higher amount of RANKL and acquire osteoclastogenic ability. Importantly, BMSC support can be prevented by silencing Jagged ligands on myeloma cells. All together, our results demonstrate that the two Notch ligands Jagged1 and 2 represents two new promising therapeutic targets in MM-associated bone disease.