ALBERT

Description: Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p

Description: Megakaryocytes are often expanded in bone marrow (BM) areas infiltrated with myeloma (MM) cells while thrombocytopenia is uncommon in untreated MM patients. The aim of this study was to investigate the reciprocal interaction between primary megakaryocytes and MM cells. Cultures of megakaryocytes and their precursors were prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (〉90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. We initially demonstrated that purified MM cell-conditioned media from 7 patients increased migration of megakaryocyte precursors across a 8 μM pore size membrane 3.6±0.9 fold (p=0.001), an effect that was inhibited by anti-CXCR4 neutralizing antibody (p

Description: The development of lytic bone lesions in myeloma (MM) is caused by the activation of osteoclasts through direct and indirect interactions with MM cells and by inactivation of osteoblasts by MM cells. Megakaryocytes have been shown to promote osteoblast differentiation and produce critical factors associated with osteoclast activity. Cells of megakaryocytic linage are often expanded in myelomatous bone. The aim of this study was to investigate the impact of myeloma cells on regulation of bone remodeling by megakaryocytes. Culture of megakaryocytes was prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (〉90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. Mesenchymal stem cells (MSCs), osteoclast precursors and CD138-selected MM cells were isolated as previously described (Pittenger et al., Science, 1999; Yaccoby et al., Cancer Res., 2004). Our study revealed that MSCs expressed TPO and that expression of TPO was upregulated 5 fold in MSCs after co-culture with MM cells, as determined by quantitive real time PCR (qRT-PCR). To study the effect on osteoblastogenesis, MSCs were co-cultured with megakaryocytes in the presence and absence of primary MM cells (n=5), in media lacking growth factors. Megakaryocytes promoted osteoblast differentiation as determined by elevated expression of alkaline phosphatase, an effect that was completely abrogated by adding MM cells to the cultures. To study the involvement of megakaryocytes on osteoclastogenesis, we initially examined expression of RANKL and OPG by these cells cultured with and without MM cells, using qRT-PCR. OPG and RANKL were expressed by megakaryocytes and gene expression of both factors was upregulated in co-cultured megakaryocytes. However, whereas OPG was insignificantly upregulated by 1.6±0.3 fold, RANKL expression was increased by 5.1±0.4 folds after co-culture with MM cells (p

Description: Recent studies have indicated that certain proteasome inhibitors have bone anabolic activity in vivo. Our clinical study have shown a significant association between anti-myeloma (MM) response of bortezomib (Millennium Pharmaceuticals) and increased serum bone alkaline phosphatase in patients with MM (Zangari et al., BJH 2005, In press). The aim of this study was to investigate the effect of bortezomib on bone remodeling and the association between anti-myeloma response and increased bone formation in our established SCID-rab model for primary MM. In this system rabbit bones were implanted S.C. in unconditioned SCID mice. After 6 weeks, primary MM cells were injected directly into the implanted bone. MM cells from 〉85% of patients (n〉70) were successfully engrafted, grew exclusively in the implanted bone and produced typical disease manifestations including increased osteoclast activity, reduced osteoblast numbers and induction of osteolytic bone disease (Yata & Yaccoby, Leukemia 2004). In the present study, SCID-rab mice were engrafted with MM cells from 5 patients. Following establishment of MM growth, as monitored by weekly measurement of human monoclonal immunoglobulins (hIg) in mice sera (587±276 μg/ml) and by x-rays, mice were injected subcutaneously twice a week with 0.5 mg/kg bortezomib or PBS for 4–8 weeks. Whereas all PBS-treated mice had increased hIg levels during the experimental period, bortezomib treatment resulted in marked reduction of hIg in 2 experiments by 95% and 73% from pretreatment levels, respectively, retardation of myeloma growth in an additional experiment and no response in 2 experiments. Overall, tumor burden in control PBS- and bortezomib-treated mice was increased by 298%±51 and 110%±88 from pretreatment levels, respectively (p

Description: We have previously demonstrated that myeloma (MM) cells produce the Wnt signaling antagonist, Dickkopf-1 (DKK1), and that DKK1 inhibits differentiation of osteoblasts (Tian et al. NEJM 2003). This is thought to result in an uncoupling process that may lead to induction of lytic bone disease in MM and possibly promote tumor growth. The aim of this study was to investigate the role of DKK1 in our established SCID-rab model for primary MM. Growth of primary MM in this system is restricted to the implanted bone and associated with typical disease manifestations including increased osteoclast activity, reduced osteoblast numbers and induction of osteolytic bone disease (Yata & Yaccoby, Leukemia 2004). In this study, SCID-rab mice were engrafted with MM cells expressing DKK1 (assessed by global gene expression profiling) from 8 patients. The level of DKK1 expression was correlated with numbers of MRI and x-ray focal lesions in these patients. Following establishment of MM growth, as monitored by weekly measurement of human monoclonal immunoglobulins (hIg) in mouse sera and radiographically, mice were injected subcutaneously into the surrounding area of the implanted bone with neutralizing antibody (AB, R&D) against DKK1 (polyclonal AB: n=4, 50 μg/injection/2 days; monoclonal AB: n=4, 100 μg/injection/day) or control IgG AB, for 4–6 weeks. Whereas bone mineral density (BMD) in control mice was reduced by 7.1%±4.6% from pre-treatment levels, BMD in mice treated with anti-DKK1 was increased by 5.6%±6.7% from pre-treatment levels (p

Description: We have recently demonstrated the inhibitory effect of osteoblasts on myeloma (MM) ex vivo and in vivo (Yaccoby et al., Haematologica 2006) and that anti-MM response of bortezomib is associated with osteoblast activation in patients with MM (Zangari et al., BJH 2005). The aims of this study were to investigate the effect of intermittent PTH and bortezomib on bone remodeling and tumor growth, and the consequences of PTH pretreatment on MM progression in our SCID-rab model for primary MM (Yata & Yaccoby, Leukemia 2004). In nonmyelomatous hosts, both PTH and bortezomib significantly increased bone mineral density (BMD) of the implanted bone. SCID-rab mice were engrafted with MM cells from 13 patients. Following establishment of MM growth, as monitored by bi-weekly measurement of human monoclonal immunoglobulins (hIg) in mice sera and by x-rays, mice were injected subcutaneously with bortezomib (0.5 mg/kg twice a week, n=10), PTH (0.3 mg/kg/day, n=5) or PBS for 4–8 weeks. Whereas all PBS-treated mice had increased hIg levels during the experimental period, bortezomib treatment resulted in marked reduction of hIg in 5/10 experiments by 73±10% from pretreatment levels (responding hosts) and stabilized or delayed growth in additional 5 experiments. Overall, tumor burden in control- and bortezomib-treated mice was increased by 447±118% and 157±97% from pretreatment levels, respectively (p

Description: Myeloma cells induce bone marrow angiogenesis and increased microvessel density (MVD) is associated with poor prognosis in this disease. The aim of this study was to investigate whether neovascularization is required for myeloma growth by testing the anti-angiogenesis and anti-tumor efficacy of thrombospondin-1 (TSP-1)-derived mimetic peptide, ABT-510 (Abbott Laboratories), in the SCID-hu model for primary myeloma. TSP-1 is a matricellular glycoprotein with multiple biological functions, including antiangiogenic activity. ABT-510 is a capped nonapeptide based on the linear TSP-1 heptapeptide sequence. Like TSP-1, ABT-510 can be targeted to a specific receptor on vascular endothelial cells and efficiently inhibits angiogenesis in vitro and after systemic injection in experimental models of human malignancies. SCID-hu mice were constructed as previously described (Yaccoby et al., Blood 1998; 1999; 2002). Myelomatous SCID-hu mice were prepared by injection of myeloma cells from 6 patients into the implanted human bone in the hosts. Changes in levels of tumor burden were monitored by weekly measurements of human monotypic immunoglobulins (hIg) using ELISA and histologically confirmed by immunohistochemical staining of human bone sections for cIg. MVD, visualized by CD34 staining, were counted in 4 non-overlapping areas. Upon establishment of myeloma growth (401±146 μg/ml pre-treatment levels) mice were intraperitoneally injected with ABT-510 (100 mg/kg, twice a day) or with the vehicle (5% dextrose) for 4–10 weeks. Treatment was well tolerated and no sign of toxicity or reductions in body weight were observed. Whereas all dextrose -treated mice had increased hIg levels during the experimental period, ABT-510 treatment resulted in marked reduction of tumor burden in 2 experiments by 48% and 20%, respectively, retardation of myeloma growth in 3 additional experiments and no response in one experiment. Overall, tumor burden in control- and ABT-510-treated mice was increased by 3696%±3264 and 448%±169 from pre-treatment levels, respectively (p

Description: Two TNF family members known to play key roles in normal B cell biology, BLyS/BAFF (B-lymphocyte stimulator/B cell activating factor) and APRIL (A PRoliferation-Inducing Ligand), also promote the survival of various malignant B cell types, including multiple myeloma (MM). BLyS binds to 3 TNF-R-related receptors, BCMA (B-cell maturation antigen, TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF-receptor), whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as CD138. All MM cells express HSPG and one or more of these 3 receptors, and incubation of MM cells with BLyS and/or APRIL leads to enhanced survival of these malignant cells in vitro. Stromal cells and osteoclasts residing in the bone marrow (BM) produce BLyS and APRIL and thus provide a paracrine source of these survival factors MM cells. Inhibition of BLyS and APRIL in vitro using a soluble receptor, TACI-Ig, causes cultured MM cells to die rapidly. It has been shown (Moreaux et al, Blood 106:1021) that differences in TACI gene expression can distinguish tumors with a BM microenvironment dependence signature (TACIhigh) from those with a plasmablastic signature (TACIlow), suggesting that TACIhigh MM cells may be more sensitive to growth factor withdrawal. We tested the ability of atacicept (TACI-Ig) to inhibit MM growth in the SCID-hu model of MM (Yaccoby et al, Blood 92:2908). Myelomatous SCID-hu mice were constructed by implanting a human fetal bone into which primary MM cells were directly injected. Changes in levels of tumor burden were monitored by weekly measurements of serum human monotypic immunoglobulins (hIg), and confirmed by histology. We compared the ability of atacicept and BAFFR-Ig (which binds BLyS, but not APRIL) to inhibit MM cell growth in this model. Upon establishment of myeloma growth, mice were injected intraperitoneally with atacicept (5 or 10 mg/kg, 3 times a wk; n=7), BAFFR-Ig (10 mg/kg, 3 times a wk; n=5) or with vehicle (PBS; n=7) for 6 wks. Compared to controls, in which tumor burden (hIg) increased by 1007±260% (avg±SEM) from pre-treatment levels, atacicept treatment markedly reduced tumor burden in 5 experiments in which TACIhigh MM cells were used, and delayed growth or had no effect in 2 experiments using TACIlow MM cells (avg increase in tumor burden of 302±184% for all 7 experiments; p

Description: The cytosolic malic enzyme (ME1) plays an important role in insulin-induced lipogenesis and has profound effects on colon cancer progression. ME1 generates pyruvate, NADPH and carbon dioxide from malate, a Krebs cycle intermediate. NADPH has an important role in de novo synthesis of long-chain fatty acids whereas pyruvate is cycled back into mitochondria. The pyruvate cycle has been hypothesized to be a necessary component of glucose-stimulated insulin secretion. Increased insulin signaling in liver and adipose tissues leads to higher accumulation of fat mass increasing the risk for obesity. NADPH and fatty acids also support cancer cell proliferation and migration. Thus ME1 may be a suitable drug target to counter obesity and prevent cancer progression. In the current work, computer-aided drug design techniques were used to identify possible ME1 inhibitors with therapeutic value. The software package SYBYL was used for defining the binding pocket and virtual screening was performed to mine through large databases (ZINC-UCSF) containing drug-like molecules in order to identify molecules that could form hydrogen bonds to the enzyme and fit into the active site. The molecules so obtained were then used for docking using the software packages SURFLEX DOCK (SYBYL) and AutoDock Vina (Scripps Research Institute). Lead molecules having minimum binding energy score were identified and two in vitro assays were carried out on the top hit molecules. We tested a total of 11 compounds for activity using an enzyme assay and 4 of these compounds were found to diminish NADPH production significantly. Additionally we performed a cell proliferation assay with colorectal cancer cell line (HCT-116) using the above 4 compounds and three of these compounds exhibited strong activity against cancer cell growth. Supported in part by NIH grant CA136493.