Ixazomib Modulates Bone Remodeling and Actives Sonic Hedgehog Pathways
Authors: Tibullo Daniele , Longo Anna , Romano Alessandra , Barbato Alessandro , Giallongo Cesarina , Di Rosa Michelino , Anfuso Carmelina Daniela , Lupo Gabriella , Conticello Concetta , Vicario Nunzio , Gulino Rosario , Parenti Rosalba , Palumbo Giuseppe A. , Li Volti Giovanni , Di Raimondo Francesco 
Published: November 2019

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p<0.001). In addition, IXA treatment reduced CHIT1 enzymatic activity and downregulated CHIT1 and YKL40 (both mRNA and proteins). Immunofluorescence evaluation confirmed that IXA inhibited the mature OCs formation with five or more nuclei. Moreover, IXA was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs). After 21 days of treatment, IXA alone or added to osteogenic medium increased the osteogenic markers genes (BMP2, RUNX2, Osteocalcin and Osteonectin). Immunofluorescence assay confirmed the increase of BMP2 after IXA treatment alone or in combination with osteogenic medium. Sonic Hedgehog (Shh) is one of the intricate signal transduction mechanisms that govern the precisely regulated developmental processes of multicellular organisms. Its signaling cascade plays an important role in bone homeostasis, and reducing Shh pathway protects against age-related bone loss. In this work we observed that IXA, but not Bortezomib, was able to bind the Smoothened (Smo) receptor which is the transducing molecule of the extracellular signal of Shh. Interestingly, using cellular thermal shift assay (CETSA), we demonstrated that IXA was able to bind directly Smo receptor activating the Shh cascade that leads to the nuclear translocation of Gli1 an effector of this pathway. Inhibiting Shh signaling by using an inhibitor of Smo activation (Cyclopamine), IXA-activated ostegenic differentiation-related genes were down-regulated. In conclusion, our data demonstrated that IXA regulates bone remodeling decreasing osteoclastogenesis and promoting osteogenic differentiation and therefore represents a good therapeutic option to improve the complex pathological condition of patients with MM.

Disclosures
Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.