Kate Vandyke

Dysregulation of bone remodeling by imatinib mesylate

Imatinib mesylate is a rationally designed tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Although the efficacy and tolerability of imatinib are a vast improvement over conventional chemotherapies, the drug exhibits off-target effects. An unanticipated side effect of imatinib therapy is hypophosphatemia and hypocalcemia, which in part has been attributed to drug-mediated changes to renal and gastrointestinal handling of phosphate and calcium. However, emerging data suggest that imatinib also targets cells of the skeleton, stimulating the retention and sequestration of calcium and phosphate to bone, leading to decreased circulating levels of these minerals. The aim of this review is to highlight our current understanding of the mechanisms surrounding the effects of imatinib on the skeleton. In particular, it examines recent studies suggesting that imatinib has direct effects on bone-resorbing osteoclasts and bone-forming osteoblasts through inhibition of c-fms, c-kit, carbonic anhydrase II, and the platelet-derived growth factor receptor. The potential application of imatinib in the treatment of cancer-induced osteolysis will also be discussed.

The tyrosine kinase inhibitor dasatinib dysregulates bone remodeling through inhibition of osteoclasts in vivo

Dasatinib is a potent tyrosine kinase inhibitor that is used to treat chronic myeloid leukemia in patients resistant or intolerant to imatinib mesylate. While designed to inhibit Abl and Src kinases, dasatinib shows multitarget effects, including inhibition of the macrophage colony‐stimulating factor (M‐CSF) receptor c‐fms. We have shown previously that dasatinib abrogates osteoclast formation and activity in vitro owing, in part, to its specificity for c‐fms. In this study we examined whether dasatinib could significantly alter bone volume in a model of physiologic bone turnover. Sprague‐Dawley rats were administered dasatinib (5 mg/kg/day) or vehicle by gavage or zoledronic acid (ZOL; 100 µg/kg/6 weeks) subcutaneously. Following 4, 8, and 12 weeks of treatment, serum biochemical, bone morphometric, and histologic analyses were performed. Whole‐body bone mineral density and tibial cortical thickness where unchanged in the dasatinib‐ or ZOL‐treated animals relative to controls. However, micro–computed tomographic (µCT) analysis of cancellous bone at the proximal tibias showed that trabecular volume (BV/TV) and thickness (Tb.Th) were increased in dasatinib‐treated animals at levels comparable with those of the ZOL‐treated group. These changes were associated with a decrease in osteoclast numbers (N.Oc/B.Pm) and surface (Oc.S/BS) and decreased serum levels of the osteoclast marker c‐terminal collagen crosslinks (CTX‐1). Mineral apposition rate (MAR), bone‐formation rate (BFR), and levels of the serum osteoblast markers osteocalcin and N‐terminal propeptide of type I procollagen (P1NP) were not altered significantly in the dasatinib‐treated animals relative to controls. These studies show that dasatinib increases trabecular bone volume at least in part by inhibiting osteoclast activity, suggesting that dasatinib therapy may result in dysregulated bone remodeling.

Therapeutic concentrations of dasatinib inhibit in vitro osteoclastogenesis

Dasatinib (BMS-354825, SPRYCEL; Bristol-Myers Squibb, New York, USA) is a second-generation ATP-competitive inhibitor of a subset of protein tyrosine kinases, including abl, Src-family kinases (c-Src, Lck, Hck, Yes, Fgr, Lyn and Fyn), and the plateletderived growth factor family members c-kit and platelet-derived growth factor-a and -b. Dasatinib displays clinical activity in chronic myeloid leukaemia patients who develop resistance to or are intolerant to the frontline chronic myeloid leukaemia chemotherapeutic imatinib mesylate (STI571, Gleevec; Novartis, Basel, Switzerland). Although designed to inhibit abl and/or Src protein kinases, tyrosine kinase inhibitors, like dasatinib and imatinib, show off-target effects. We have shown earlier that therapeutic concentrations of imatinib decrease osteoclast numbers and activity, at least in part through inhibition of the macrophage colony-stimulating factor (M-CSF) receptor, c-fms. Signalling through c-fms plays an essential role in the survival and activity of bone-resorbing osteoclasts, which act in conjunction with bone-forming osteoblasts to maintain skeletal health. In in vitro osteoclast cultures and animal models, such as op/op mice, an absence of signalling through c-fms results in an osteopetrotic phenotype because of deficient osteoclasts and osteoclast precursors. Recent evidence suggests that dasatinib, like imatinib, may potentially inhibit signal transduction through c-fms. In cultures of M-CSF-dependent Ba/F3 cells that ectopically expressed human c-fms, cell numbers were decreased by dasatinib treatment. In light of this observation, this study examined whether dasatinib could modulate osteoclast formation and/or activity, and whether any such effects were attributable to an inhibition of c-fms signal transduction. We examined the effect of dasatinib on osteoclast formation and activity in human and murine systems in vitro. Human CD14þ mononuclear cells (huCD14þ ) were isolated from the peripheral blood of healthy volunteers using Percoll gradient separation and a MACS negative selection monocyte kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured with recombinant human (rh)M-CSF and rhRANKL, to induce osteoclastogenesis, supplemented with dasatinib or vehicle. After 14 days of culture, a significant decrease in the number of tartrate-resistant acid phosphatasepositive multinucleated cells was observed at 20 nM dasatinib (Po0.001; IC501⁄4 10.5 nM; Figure 1a). Murine osteoclastogenesis assays were established by explanting mouse bone marrow from the tibiae and femora of C57 BL/6 mice. Cells were incubated overnight at 37 1C in 5% CO2, to allow stromal cells to adhere, and non-adherent mouse bone marrow cells (mBM) were collected and used as monocyte/macrophage osteoclast precursor cells. In mBM cultures treated with rhM-CSF and rhRANKL for 6 days, osteoclast numbers were decreased at 10 nM dasatinib (Po0.01; Figure 1b) and osteoclast formation was completely abrogated at 20 nM dasatinib (IC501⁄4 8.0 nM; Figure 1b). Osteoclast size was also substantially reduced in cultures treated with 10 nM dasatinib compared with controls, in both human and murine systems (Figure 1c). The effect of dasatinib on osteoclast activity was also assessed in huCD14þ and mBM cultures established on calcium phosphate-coated slides. A significant inhibition of resorption was observed at X1.25 nM dasatinib in huCD14þ cultures (Po0.001; IC501⁄4 2.4 nM; Figures 1d and f), and at X2.5 nM dasatinib in mBM cultures (Po0.05; IC501⁄4 3.5 nM; Figures 1e and f), relative to vehicle controls. Thus, therapeutically relevant concentrations of dasatinib (Cmax1⁄4 110.0 nM) significantly reduced the formation and activity of osteoclasts from huCD14þ and mBM at IC50 concentrations of p10 nM dasatinib. As dasatinib may affect osteoclastogenesis through inhibition of c-fms, we investigated whether dasatinib could specifically inhibit c-fms kinase activity. Initially, the effect of dasatinib on c-fms-dependent cell proliferation was examined using FDC-P1 cells expressing c-fms protein (FDC-cfms). FDC-P1 cells are dependent on interleukin-3 for proliferation and are also rendered M-CSF-responsive by transfection with a c-fms expression construct. The resulting cells are dependent on either interleukin-3 or M-CSF for survival and proliferation. Treatment of rhM-CSF-stimulated FDC-cfms cells with dasatinib for 24 h significantly decreased cell numbers at concentrations of X50 nM (Po0.001; IC501⁄4 57.8 nM; Figure 2a). In contrast, the proliferation of FDC-P1 cells or FDC-cfms cells cultured in the presence of interleukin-3 (0.5 ng/ ml) was not significantly affected by dasatinib treatment at concentrations up to 100 nM (P1⁄4 0.9012 and P1⁄4 0.2008, respectively; Figure 2a). To determine whether dasatinib treatment could also inhibit the M-CSF-dependent proliferation/survival of primary bone marrow cells, the effect of dasatinib on mBM cells was assessed using a WST-1 assay (Tanaka, Madison, WI, USA). The relative number of viable, metabolically active cells per well, as detected by the quantification of mitochondrial dehydrogenase

Suppression of PDGF-induced PI3 kinase activity by imatinib promotes adipogenesis and adiponectin secretion

Improved glucose and lipid metabolism is a unique side effect of imatinib therapy in some chronic myeloid leukaemia (CML) patients. We recently reported that plasma levels of adiponectin, an important regulator of insulin sensitivity, are elevated following imatinib therapy in CML patients, which could account for these improved metabolic outcomes. Adiponectin is secreted exclusively from adipocytes, suggesting that imatinib modulates adiponectin levels directly, by transcriptional upregulation of adiponectin in pre-existing adipocytes, and/or indirectly, by stimulating adipogenesis. In this report, we have demonstrated that imatinib promotes adipogenic differentiation of human mesenchymal stromal cells (MSCs), which in turn secrete high-molecular-weight adiponectin. Conversely, imatinib does not stimulate adiponectin secretion from mature adipocytes. We hypothesise that inhibition of PDGFRα (PDGFRA) and PDGFRβ (PDGFRB) is the mechanism by which imatinib promotes adipogenesis. Supporting this, functional blocking antibodies to PDGFR promote adipogenesis and adiponectin secretion in MSC cultures. We have shown that imatinib is a potent inhibitor of PDGF-induced PI3 kinase activation and, using a PI3 kinase p110α-specific inhibitor (PIK-75), we have demonstrated that suppression of this pathway recapitulates the effects of imatinib on MSC differentiation. Furthermore, using mitogens that activate the PI3 kinase pathway, or MSCs expressing constitutively activated Akt, we have shown that activation of the PI3 kinase pathway negates the pro-adipogenic effects of imatinib. Taken together, our results suggest that imatinib increases plasma adiponectin levels by promoting adipogenesis through the suppression of PI3 kinase signalling downstream of PDGFR.

Plasma Adiponectin Levels Are Markedly Elevated in Imatinib-Treated Chronic Myeloid Leukemia (CML) Patients: A Mechanism for Improved Insulin Sensitivity in Type 2 Diabetic CML Patients?

CONTEXT The mechanism(s) by which imatinib improves glycemic control in chronic myeloid leukemia (CML) patients with type 2 diabetes remains unclear. OBJECTIVE Adiponectin is an important regulator of insulin sensitivity that is secreted exclusively by adipocytes. We previously reported that imatinib promotes adipocytic differentiation of mesenchymal stromal cells. We therefore hypothesized that imatinib therapy would be associated with an increase in peripheral and intramedullary adiposity and elevated plasma adiponectin levels. RESEARCH DESIGN AND METHODS Adiponectin levels in CML patient plasma, at diagnosis and then during imatinib mesylate therapy, was measured using an ELISA. Adiponectin multimers in plasma were analyzed using nondenaturing PAGE and immunoblotting. Intramedullary adiposity and adipose tissue mass was determined using histomorphometry and dual-energy X-ray absorptiometry, respectively. RESULTS In CML patients, an increase in intramedullary and peripheral adiposity was observed after 6 months of imatinib therapy and plasma adiponectin levels, in the form of high- and low-molecular-weight complexes, were elevated 3-fold, compared with pretreatment levels, after 3, 6, and 12 months of therapy. CONCLUSIONS Elevated adiponectin levels in imatinib-treated CML patients provide a possible mechanism for improved glucose and lipid metabolism reported for some imatinib-treated patients.

Imatinib mesylate causes growth plate closure in vivo

K. Vandyke, A.L. Dewar, S. Fitter, D. Menicanin, L.B. To, T.P. Hughes and A.C.W. Zannettino

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1‐EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1‐EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild‐type mice. The fractured bones of Col1‐EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild‐type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1‐EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU‐F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild‐type control mice. Furthermore, Col1‐EphB4 mice had significantly lower numbers of TRAP‐positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones.

SAMSN1 is a tumor suppressor gene in multiple myeloma.

Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138 + PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell–PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.

The tyrosine kinase inhibitor dasatinib (SPRYCEL) inhibits chondrocyte activity and proliferation

Dasatinib (BMS-354825, SPRYCEL, Bristol-Myers Squibb, New York, NY, USA) is an ATP-competitive protein tyrosine kinase inhibitor (TKI), which was originally identified as a potent inhibitor of Src family kinases (including Src, Lck, Hck, Yes, Fgr, Lyn and Fyn) and was subsequently found to have activity against Abl, Kit, the macrophage colony stimulating factor receptor (Fms), the platelet-derived growth factor (PDGF) receptor (PDGFR)-α and -β and the Eph receptor family members EphB1, EphB2 and EphB4.1, 2, 3 Dasatinib is an effective therapy for chronic myeloid leukaemia (CML) in patients who are resistant to front-line imatinib mesylate therapy due to its increased affinity for the CML oncoprotein BCR-Abl and its insensitivity to mutations in the BCR-Abl kinase domain. Furthermore, recent data suggest that dasatinib may be more effective than imatinib as a front-line therapy for chronic phase CML.4

The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma

The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in its capacity to cause osteoclast‐mediated skeletal destruction. The PI3K/Akt/mTOR pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone‐forming osteoblasts and bone‐resorbing osteoclasts. NVP‐BEZ235 is a dual pan class I PI3K and mTOR inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we examined the anti‐tumorigenic effects of BEZ235 in an immunocompetent mouse model of MM and assessed the effects of BEZ235 on osteoblast and osteoclast formation and function. BEZ235 treatment (50 mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BEZ235‐treated animals. Levels of the serum osteoblast marker P1NP were significantly higher in BEZ235‐treated animals, while levels of the osteoclast marker TRAcP5 were reduced. In vitro, BEZ235 decreased MM plasma cell proliferation, osteoclast formation and function and promoted osteoblast formation and function. These findings suggest that, in addition to its anti‐tumour properties, BEZ235 could be useful in treating osteolytic bone disease in MM patients.