CD26 Is a Novel Target for the Treatment of Tumor Progression and Its Related Osteolytic Bone Disease in Multiple Myeloma

Background: CD26, a 110-kDa transmembrane glycoprotein with DPPIV activity, has been implicated in tumorigenesis and shown to be expressed in several tumor cells including malignant lymphoma, whereas its role has not been characterized in plasma cell malignancies, yet. We have recently shown that CD26 is intensely expressed in activated osteoclasts (OCs) in osteolytic bone tumors including multiple myeloma (MM). CD26 is not expressed in hematopoietic stem cells, but M-CSF and sRANKL induced human OC differentiation with the upregulation of CD26 expression in monocyte-macrophage lineage cells, OC precursor cells and OCs. Blockade of CD26 signaling with humanized anti-CD26 monoclonal antibody (YS110) impaired OC differentiation via inhibiting RANKL-induced MKK3/6-p38MAPK-mi/Mitf phosphorylation in OC precursor cells (Nishida et al., 2014). In the present study, we characterize the biological function of CD26 in MM cells in the bone marrow (BM) and investigate the therapeutic potential of YS110 to treat MM cell growth and reduce MM-induced osteolytic lesions.

Methods and Results: Although CD26 expression was not detected any of 11 MM cell lines in mono-culture, CD26 expression levels were upregulated in all MM cell lines when co-cultured with OCs for 72 hours by flow cytometry and immunohistochemistry analysis. CD26 protein levels in MM cell lines were also enhanced in co-culture with OCs by immunoblotting. To determine the factors responsible for CD26 upregulation in MM cells, we examined CD26 expression in MM cell lines under transwell co-culture conditions with OCs or under stimulation with anti-apoptotic cytokines produced by OCs. CD26 expression in MM cell lines was reduced under transwell co-culture conditions with OCs. TNFα, BAFF, APRIL and SDF-1 slightly upregulated CD26 expression in MM cell lines. To further explore CD26 expression in BM of MM patients, we performed immunohistochemical stainig on decalcified bone sections of biopsy specimens of MM cases. CD26/CD138 positive plasma cells were detected around CD26 positive OCs and certain endothelial vascular cells in several cases. Next, to clarify the role of CD26 in MM cell survival, we examined the effects of YS110 on MM cell growth and related osteolytic bone lesions in vitro and in vivo. YS110 had no significant effects on viability of MM cell lines in mono-culture, but dose-dependently inhibited growth of MM cell lines in co-culture with OCs. YS110 immediately inhibited p38 activation and its downstream Hsp27 phosphorylation in MM cells. Contitnued treatment of MM cells with YS110 also led to the reduction of total Hsp27 protein at 12 hours and 16 hours after the treatment of YS110, with commensurate decrease of phospho-Hsp27 and to increased MM apoptosis correlated with the induction of p53, increased activation of caspases and PARP, as well as the reduction of Mcl-1 and c-Myc. In parallel, we constructed a xenograft of murine model of human MM. A total of 5x10⁶ CD26 positive MM cell lines by co-culture with OCs were inoculated to mice (NOD/SCID-s.c. mice) or directly injected into the implanted human bone chips in mice (NOD/SCID-hu mice). Mice treated with YS110 (500μg/dose) showed a significant effect in inhibiting MM tumor weight compared with control mice 4 weeks after tumor cell inoculation. To further investigate whether YS110 could reduce MM-induced osteolysis as well as suppress MM cell growth, we used NOD/SCID-hu mice model. Mice were treated with YS110 after the first detection of tumor growth. Continuous YS110 treatment significantly suppressed MM cell growth after 4 weeks. Immunohistochemistry for CD138 and TRAP staining showed decreased numbers of MM cells and TRAP positive OCs, with decreased bone resorption activity in human bones from YS110-treated versus control mice. Lastly, we examined the effects of targeting CD26 with YS110 in CD26 positive myeloma stem-like cells (SP cells). CD26 expression was demonstrated in SP cells of several MM cell lines in co-culture with OCs. YS110 dose-dependently inhibited cell viability of CD26 positive SP cells co-cultured with OCs, indicating targeting CD26 might reduce chemoresistance in MM.

Conclusions: Theses results suggest that CD26 may be a promising novel target in MM, strongly supporting YS110 to inhibit MM cell growth in the BM as well as its related osteolytic bone lesions. Our results provide the framework for clinical development of YS110 as a novel therapeutic option in MM.