Abstract
Bone disease is a hallmark of multiple myeloma (MM) due to increased osteoclast (OC)-mediated bone resorption and decreased osteoblast (OB)-mediated bone formation. CD26 is a 110-kDa cell surface glycoprotein with dipeptidyl peptidase IV (DPPIV) activity and has a well-defined role in T cell activation and several tumor developments including malignant lymphoma, whereas its expression in OCs further suggests a role in osteoclastgenesis (OCG). Interestingly, CD26 is associated with increased phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) which also occurs downstream of RANK signaling in OCG and plays an important role in OC function. Our previous studies showed that CD26 is preferentially expressed in normal human OCs, and is also intensely expressed in activated OCs in MM, but not in MM cells themselves. In this study, we investigated the efficacy of humanized anti-CD26 monoclonal antibody (YS110) on OCs derived from MM patient bone marrow (BM) and MM growth within the BM microenvironment.
OCs derived from MM patient BM mononuclear cells were assayed with macrophage-colony stimulating factor (M-CSF) (25ng/ml) plus receptor activator of NF- κB ligand (RANKL) (50ng/ml) with or without YS110 for OC formation and maturation for TRAP/CD26 staining and functional assay with bone resorptive pit formation assay. To assess the mechanisms of action of YS110 against OC differentiation in MM, RANK signaling proteins were examined by immunoblotting.
Significantly reduced numbers of multinucleated TRAP/CD26 positive mature OCs (>3 nuclei per OC) were observed in YS110-treated versus non-treated MM OC culture. YS110 blocked RANKL/M-CSF induced phosphorylation of p38MAPK and its downstream activation of mi/Mitf in MM-derived OC precursor cells, which otherwise increased OC differentiation. In contrast, YS110 did not show significant inhibition against other MAPKs involved in RANK signaling pathway in OC precursor cells. Importantly, YS110 remarkably diminished bone erosion area formed by mature OCs on dentine slices in pit formation assay. YS110 also consistently induced changes of cell supernatants in a dose-dependent manner in MM OC culture by ELISA. In fact, OC- or MM- related growth factors such as TRAP5b, collagen type 1, APRIL and BAFF which are secreted by OCs were also down-regulated. DPPIV inhibitor had no significant inhibitory effects on OCG in MM. These results suggest that the mechanism of action by YS110 on OCG in MM result from the blockade of RANKL induced p38MAPK-mi/Mitf phosphorylation in MM-derived OC precursor cells. Next, although YS110 did not demonstrate direct inhibition of proliferation of MM cells, to further investigate the role of CD26 in MM cells in the BM microenvironment, co-cultures of MM cell lines (KMS11, KMS12, KMS18, KMS20, KMS21, KMS26, KMS27, KMS28, KMS34, KMM1, U266) with MM-derived CD26-stained OCs were performed. We examined the expression of CD26 in co-cultured MM cells and co-cultured OCs by flow cytometry and immunoblotting. On day 1 of the co-culture, the expression of CD26 was barely detected in MM cells. However, on day 5, the expression level of CD26 was shown to be further increased in OCs. Moreover, interestingly, the expression of CD26 was also significantly up-regulated in MM cells. By immunohistochemistry, co-cultured MM cells were intensely stained positive for CD26 as well as co-cultured OCs. P38MAPK was activated in co-cultured CD26-stained MM cells; it regulates OC differentiation and bone resorption activity in MM. MM cells in the BM microenvironment may induce functional changes which promote tumor development and progression, as well as microenvironmental alternations. Now on going studies are confirming the anti-myeloma efficacy of YS110 on co-cultures of MM cells with OCs.
Our data strongly demonstrate that targeting CD26 with YS110 inhibits MM-derived OC differentiation and function through blocking p38MAPK-mi/Mitf cascade of RANK signaling in MM-derived OC precursor cells. These results suggests that CD26 in OCs may be a novel therapeutic target for the treatment of myeloma-assoiated bone disease. It was further confirmed that the direct interaction between MM cells and OCs results in the up-regulation of CD26 expression in MM cells and that targeting CD26-p38MAPK signaling cascade may have a novel therapeutic potential not only in MM-derived OCs but in MM cells.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
