Abstract
Abstract 1348
Bone disease is a hallmark of malignancy with osteolytic bone metastasis, including multiple myeloma (MM) and targeting osteoclasts (OCs) to alleviate bone destruction is a component of the standard care for MM. CD26 is a 110-kDa multifunctional membrane-bound glycoprotein, with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular domain and is critical in T-cell activation and several tumor developments, including malignant lymphoma. However, little is known about the role of CD26 in regulating bone remodeling. In this study, we show that CD26 is expressed on normal human osteoclasts and moreover, intensely expressed on activated human osteoclasts with osteolytic bone metastasis, including MM. We explore the function of CD26 in osteoclastgenesis (OCG) and investigate the effects of humanized anti-CD26 monoclonal antibody (huCD26mAb), which has shown promising clinical activity in T-cell lymphoma, on human OC differentiation, maturation and function. We further define the molecular targets of CD26 signaling cascade in OCG and explore the therapeutic potential of huCD26mAb for treating osteolytic bone metastasis. Human bone marrow mononuclear cells (BMMs) were cultured with human M-CSF (25ng/ml) plus sRANKL (50ng/ml) in the absence or presence of huCD26mAb for the indicated times. Then, M-CSF and sRANKL stimulate CD26 expressions during OCG, in a dose-dependent manner. The expression of CD26 up-regulates mitogen-activated protein kinase14 (p38MAPK) phosphorylation. P38MAPK phosphorylation also occurs downstream of RANK signaling in OCs and stimulates its downstream activation of microphthalmia-associated transcription factor (mi/Mitf), which plays an important role in OC function. Importantly, huCD26mAb decreased the number of multinucleated OCs (>3 nuclei) by tartrate-resistant phosphates (TRAP)/CD26 staining and the secretion of TRAP-5b and type 1 collagen; specific mature OC markers. It decreased the size of OCs and the number of nuclei per OC, with significantly defective bone resorption activity, as evidenced by diminished pit formation on fluoresceinated calcium phosphate-coated plates. In contrast, huCD26mAb added after 4- or 7- days' BMM cultures with M-CSF plus sRANKL did not have significant effects on mature osteoclast formation and function. Given these dual roles of CD26 in OCG, we next examined the effects of huCD26mAb on the phosphorylation of p38MAPK in OC precursor cells and mature OCs. At first, in the absence of huCD26mAb, similar amounts of p38MAPK and MKK3/6 (a molecule that is upstream of p38MAPK) were present in OC precursor cells and OCs. In response to RANKL, MKK3/6-p38MAPK was phosphorylated within 15 minutes in OC precursor cells and reached a maximal level within 30 minutes, and was maintained up to 60 minutes. Moreover, mi/Mitf was subsequently rapidly activated and persisted for 24hours. In the presence of huCD26mAb, when huCD26mAb bound to CD26 on OC precursor cells, only the MKK3/6-p38MAPK pathway was specifically rapidly inactivated, as shown by the persistent decrease in the phosphorylation of p38MAPK, together with MKK3/6, starting within 15 minutes of RANKL stimulation. Subsequent mi/Mitf phosphorylation was also persistently inhibited. In contrast, MKK3/6-p38MAPK was not phosphorylated at all in mature OCs after RANKL stimulation, regardless of the absence or presence of huCD26mAb. These results suggest that huCD26mAb suppressed RANKL induced p38MAPK phosphorylation in OC precursor cells, but not in OCs. The activation of other MAPKs including ERK and SAPK/JNK, or NFκB were rapidly induced in response to RANKL both in OC precursor cells and OCs, regardless of the absence or presence of huCD26mAb. Moreover, p38MAPK inhibitor also strongly inhibited OC formation and function through the suppression of p38 MAPK phosphorylation and subsequent mi/Mitf activation in OC precursor cells, but not in OCs. In conclusion, these data demonstrate that targeting CD26 on OC precursor cells with huCD26mAb suppressed human osteoclast differentiation, via the inhibition of MKK3/6-p38MAPK-mi/Mitf phosphorylation pathway and impaired subsequent mature osteoclast formation and function. Our results strongly suggest that targeting OCs with huCD26mAb has a promising alternative therapeutic potential for the treatment of osteolytic bone metastasis, including MM, to reduce the occurrence of total skeletal-related events.
Disclosures:
No relevant conflicts of interest to declare.
Regulation of Osteoclast Differentiation at Multiple Stages by Protein Kinase D Family Kinases
