Stephen Fitter

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.

Heat Shock Protein-90 beta Is Expressed at the Surface of Multipotential Mesenchymal Precursor Cells: Generation of a Novel Monoclonal Antibody, STRO-4, With Specificity for Mesenchymal Precursor Cells From Human and Ovine Tissues

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

NVP-BEZ235, A Dual Pan Class I PI3 Kinase and mTOR Inhibitor, Promotes Osteogenic Differentiation in Human Mesenchymal Stromal Cells

Osteoblasts are bone‐forming cells derived from mesenchymal stromal cells (MSCs) that reside within the bone marrow. In response to a variety of factors, MSCs proliferate and differentiate into mature, functional osteoblasts. Several studies have shown previously that suppression of the PI3K and mTOR signaling pathways in these cells strongly promotes osteogenic differentiation, which suggests that inhibitors of these pathways may be useful as anabolic bone agents. In this study we examined the effect of BEZ235, a newly developed dual PI3K and mTOR inhibitor currently in phase I–II clinical trials for advanced solid tumors, on osteogenic differentiation and function using primary MSC cultures. Under osteoinductive conditions, BEZ235 strongly promotes osteogenic differentiation, as evidenced by an increase in mineralized matrix production, an upregulation of genes involved in osteogenesis, including bone morphogenetic proteins (BMP2, ‐4, and ‐6) and transforming growth factor β1 (TGF‐β1) superfamily members (TGFB1, TGFB2, and INHBE), and increased activation of SMAD signaling molecules. In addition, BEZ235 enhances de novo bone formation in calvarial organotypic cultures. Using pharmacologic inhibitors to delineate mechanism, our studies reveal that suppression of mTOR and, to a much lesser extent PI3K p110α, mediates the osteogenic effects of BEZ235. As confirmation, shRNA‐mediated knockdown of mTOR enhances osteogenic differentiation and function in SAOS‐2 osteoblast‐like cells. Taken together, our findings suggest that BEZ235 may be useful in treating PI3K/mTOR‐dependent tumors associated with bone loss, such as the hematologic malignancy multiple myeloma.

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.

Brief Report: The Differential Roles of mTORC1 and mTORC2 in Mesenchymal Stem Cell Differentiation

Adipocytes (AdCs) and osteoblasts (OBs) are derived from mesenchymal stem cells (MSCs) and differentiation toward either lineage is both mutually exclusive and transcriptionally controlled. Recent studies implicate the mammalian target of rapamycin (mTOR) pathway as important in determining MSC fate, with inhibition of mTOR promoting OB differentiation and suppressing AdC differentiation. mTOR functions within two distinct multiprotein complexes, mTORC1 and mTORC2, each of which contains the unique adaptor protein, raptor or rictor, respectively. While compounds used to study mTOR signaling, such as rapamycin and related analogs, primarily inhibit mTORC1, prolonged exposure can also disrupt mTORC2 function, confounding interpretation of inhibitor studies. As a result, the relative contribution of mTORC1 and mTORC2 to MSC fate determination remains unclear. In this study, we generated primary mouse MSCs deficient in either Rptor (RapKO) or Rictor (RicKO) using the Cre/loxP system. Cre‐mediated deletion of Rptor or Rictor resulted in impaired mTORC1 and mTORC2 signaling, respectively. Under lineage‐inductive culture conditions, RapKO MSCs displayed a reduced capacity to form lipid‐laden AdCs and an increased capacity to form a mineralized matrix. In contrast, RicKO MSCs displayed reduced osteogenic differentiation capacity and enhanced adipogenic differentiation potential. Taken together, our findings reveal distinct roles for mTORC1 and mTORC2 in MSC lineage commitment. Stem Cells 2015;33:1359–1365

The differential roles of mTORC1 and mTORC2 in mesenchymal stem cell differentiation

Adipocytes (AdCs) and osteoblasts (OBs) are derived from mesenchymal stem cells (MSCs) and differentiation toward either lineage is both mutually exclusive and transcriptionally controlled. Recent studies implicate the mammalian target of rapamycin (mTOR) pathway as important in determining MSC fate, with inhibition of mTOR promoting OB differentiation and suppressing AdC differentiation. mTOR functions within two distinct multiprotein complexes, mTORC1 and mTORC2, each of which contains the unique adaptor protein, raptor or rictor, respectively. While compounds used to study mTOR signaling, such as rapamycin and related analogs, primarily inhibit mTORC1, prolonged exposure can also disrupt mTORC2 function, confounding interpretation of inhibitor studies. As a result, the relative contribution of mTORC1 and mTORC2 to MSC fate determination remains unclear. In this study, we generated primary mouse MSCs deficient in either Rptor (RapKO) or Rictor (RicKO) using the Cre/loxP system. Cre‐mediated deletion of Rptor or Rictor resulted in impaired mTORC1 and mTORC2 signaling, respectively. Under lineage‐inductive culture conditions, RapKO MSCs displayed a reduced capacity to form lipid‐laden AdCs and an increased capacity to form a mineralized matrix. In contrast, RicKO MSCs displayed reduced osteogenic differentiation capacity and enhanced adipogenic differentiation potential. Taken together, our findings reveal distinct roles for mTORC1 and mTORC2 in MSC lineage commitment. Stem Cells 2015;33:1359–1365

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