Immunomodulatory Thalidomide Analogs Do Not Affect Osteoprogenitor Differentiation and Low Concentration of Bortezomib Promotes It, While Both Agents Suppress Osteoclast Differentiation.
Authors: Munemasa Shoso , Sakai Akira , Kuroda Yoshiaki , Okikawa Yoshiko , Katayama Yuta , Asaoku Hideki , Kubo Tadahiko , Shimose Shoji , Kimura Akiro 
Published: October 2019

Abstract
Bone disease (BD) is a frequent characteristic of multiple myeloma (MM) and leads to severe clinical complications. BD in MM is due to not only the activation of osteoclast but also the impairment of osteoblast differentiation. Treatment for MM using high-dose melphalan followed by tandem auto-stem cell transplantation (tandem ASCT) and new drugs such as bortezomib (PS-341), thalidomide, and lenalidomide have improved OS. Interestingly, some reports have shown high serum bone-specific alkaline phosphatase levels after bortezomib-combined therapy in MM patients, and administration of proteasome inhibitor induced bone formation accompanied by the expression of bone morphogenetic protein-2 (BMP-2) in mice. However, the effects of immunomodulatory compounds on osteoblast or osteoclast differentiation remain unknown. We investigated the effects of bortezomib (PS-341) and immunomodulatory thalidomide analogs (immunomodulatory compounds; CC-4047, CC-6032, and CC-5013, or lenalidomide) on osteoblast and osteoclast differentiation in vitro. We used a human mesenchymal stem cell (MSCs) culture system for osteoblast differentiation and normal peripheral blood mononuclear cells for osteoclast differentiation. First, we tried to determine the working concentration of bortezomib and immunomodulatory compounds in our culture system of osteoblast and osteoclast differentiation by treatment of MM cell lines (KMS21BM, U266, and RPMI8226) with bortezomib (0.5–5.0nM) and immunomodulatory compounds (10μM). Low bortezomib concentrations of not more than 2.5nM did not show any apoptotic effect on these MM cell lines, and the apoptotic effects of immunomodulatory compounds on MM cells differed among MM cells and these compounds themselves. Subsequently, these agents (bortezomib; 0.5–5.0nM, immunomodulatoy compounds; 10μM) were added into the osteoprogenitor cell culture media or the media for osteoclast differentiation with M-CSF and receptor activator of NF-κB ligand (RANKL). We analyzed ALP activity and mineralized nodular formation by von Kossa staining as markers of osteoblast differentiation, and analyzed TRAP-positive multinucleated cells as mature osteoclasts. Furthermore, we analyzed the expression of BMP2 on osteoprogenitor cells in the treatment with bortezomib. The low bortezomib concentrations (0.5 and 1.0nM) increased ALP activity compared with that of the control, and mineralized nodular formation at less than 2.5nM was equal to that in the control. BMP2 mRNA content was found to increase in a time-dependent manner in osteoprogenitor cell culture medium that was independent of the treatment with bortezomib. However, these low bortezomib concentrations did not show either anti-myeloma or fixed anti-osteoclast effects despite the possibility of suppression of osteoclast differentiation even at doses of 1.0 and 2.5nM bortezomib according to cases. In contrast, immunomodulatory compounds at 10μM showed an anti-osteoclast effect without cytotoxicity to osteoblast differentiation, at which dose myeloma cell lines underwent apoptosis. These findings might improve the treatment strategy for MM patients without damage to BM stromal cells by combining of bortezomib with immunomodulatory compounds such as lenalidomide.