Dasatinib (SprycelTM) Inhibits Osteoclast Activity in Vitro and in Vivo Via a C-Fms-Dependent and C-Src-Independent Mechanism

Abstract

We have previously shown that the tyrosine kinase inhibitor imatinib mesylate potently inhibits osteoclast formation and activity due, in part, to its specificity for the macrophage colony stimulating factor (M-CSF) receptor, c-fms. We have also shown that imatinib therapy is associated with altered bone remodelling and an increase in trabecular bone volume in chronic myeloid leukaemia patients. In the present study, we examined whether a second-generation tyrosine kinase inhibitor, dasatinib, could similarly inhibit c-fms activation and osteoclastogenesis.The effect of dasatinib on c-fms phosphorylation in human peripheral blood CD14+ mononuclear cells (huCD14+) and mouse bone marrow monocytes (mBM) was assessed using Western blotting. M-CSF activation of c-fms phosphorylation was inhibited at pharmacologically-relevant concentrations (5 nM) in huCD14+ and mBM. We next examined the effects of dasatinib treatment on cell proliferation/survival in M-CSFdependent FDC-fms and mBM cells. Dasatinib treatment resulted in a dose-dependent decrease in FDC-fms (IC50 = 57.8 nM) and mBM (IC50 = 5.0 nM) cell numbers. Similarly, in huCD14+ and mBM cultures stimulated with RANKL and M-CSF, the number of TRAP-positive, multinucleated osteoclasts was significantly reduced in the presence of 20 nM dasatinib in huCD14+ (p < 0.001; IC50 = 10.5 nM) and 10 nM dasatinib in mBM (p < 0.01; IC50 = 8.0 nM), relative to vehicle controls. In addition, there was a dose-dependent decrease in bone resorption by osteoclasts at 2.5 nM dasatinib and higher (p < 0.05; IC50 = 2.4 nM [PBMNC] and 3.5 nM [mBM]).Since signal transduction via c-fms is crucial for osteoclastogenesis, we examined whether inhibition of c-fms by dasatinib could account for these results. Dasatinib decreased proliferation of FDC-c-fms (IC50 = 57.8 nM) and mBM (IC50 = 5.0 nM) cultured with M-CSF, but not IL-3-stimulated FDC-c-fms cells. Furthermore, dasatinib inhibited phosphorylation of c-fms at 5 nM in huCD14+ and mBM, while c-Src was inhibited at 20 nM dasatinib.We next performed a comprehensive investigation of dasatinib's effects on bone remodelling in a rat model of physiological bone turnover. In this study, 18 adult (8 months old) Sprague Dawley rats were administered dasatinib (5 mg/kg/day) or vehicle control (10% DMSO/90% polyethylene glycol 300 [v/v]) by daily oral gavage. Following 4, 8 and 12 weeks of treatment, 6 animals from each group were sacrificed for serum biochemical analysis, bone morphometry, and histological analysis of tibiae and lumbar vertebrae.While no significant changes in serum calcium levels were observed, hypophosphataemia was induced in dasatinib-treated animals by 4 weeks. Micro-computed tomographic (micro-CT) analysis of a region of cancellous bone at the proximal tibiae, 2.4 mm distal to the growth plate, revealed that trabecular bone volume (BV/TV) was increased in the dasatinib-treated group after 4 weeks of treatment. The increase in BV/TV was attributed to an increase in trabecular number and not an increase in trabecular thickness. Furthermore, there were no observed changes in tibial cortical thickness or in trabecular BV/TV in the lumbar vertebrae.These studies demonstrate that therapeutic concentrations of dasatinib inhibit OC formation and activity in vitro and in vivo and this effect is attributable to an inhibition of signal transduction via c-fms rather than c-Src. The anti-resorpitive properties of dasatinib were further confirmed in a rat model of normal bone remodelling, with dasatinibtreated animals exhibiting an increase in trabecular bone volume. These data suggest that decreased bone resorption is a likely side-effect of dasatinib therapy. In addition, they highlight the possibility that dasatinib may represent a possible treatment for diseases of low bone mass or metastasis-associated bone loss.