Seint T. Lwin

Inhibition of TGF-β signaling by 1D11 antibody treatment increases bone mass and quality in vivo

Transforming growth factor β (TGF‐β) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF‐β represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF‐β inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF‐β. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro–computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole‐bone strength was assessed biomechanically by three‐point bend testing, and tissue‐level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF‐β blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue‐level modulus. In addition, Raman microspectroscopy demonstrated that 1D11‐mediated TGF‐β inhibition in the bone environment led to an 11% increase in the mineral‐to‐collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF‐β with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)–treated rodent studies.

Host-derived adiponectin is tumor-suppressive and a novel therapeutic target for multiple myeloma and the associated bone disease.

The contributions of the host microenvironment to the pathogenesis of multiple myeloma, including progression from the non-malignant disorder monoclonal gammopathy of undetermined significance, are poorly understood. In the present study, microarray analysis of a murine model requiring a unique host microenvironment for myeloma development identified decreased host-derived adiponectin compared with normal mice. In support, clinical analysis revealed decreased serum adiponectin concentrations in monoclonal gammopathy of undetermined significance patients who subsequently progressed to myeloma. We investigated the role of adiponectin in myeloma pathogenesis and as a treatment approach, using both mice deficient in adiponectin and pharmacologic enhancement of circulating adiponectin. Increased tumor burden and bone disease were observed in myeloma-bearing adiponectin-deficient mice, and adiponectin was found to induce myeloma cell apoptosis. The apolipoprotein peptide mimetic L-4F was used for pharmacologic enhancement of adiponectin. L-4F reduced tumor burden, increased survival of myeloma-bearing mice, and prevented myeloma bone disease. Collectively, our studies have identified a novel mechanism whereby decreased host-derived adiponectin promotes myeloma tumor growth and osteolysis. Furthermore, we have established the potential therapeutic benefit of increasing adiponectin for the treatment of myeloma and the associated bone disease.

Osteoclasts in multiple myeloma are derived from Gr-1+CD11b+myeloid-derived suppressor cells.

Osteoclasts play a key role in the development of cancer-associated osteolytic lesions. The number and activity of osteoclasts are often enhanced by tumors. However, the origin of osteoclasts is unknown. Myeloid-derived suppressor cells (MDSCs) are one of the pre-metastatic niche components that are induced to expand by tumor cells. Here we show that the MDSCs can differentiate into mature and functional osteoclasts in vitro and in vivo. Inoculation of 5TGM1-GFP myeloma cells into C57BL6/KaLwRij mice led to a significant expansion of MDSCs in blood, spleen, and bone marrow over time. When grown in osteoclastogenic media in vitro, MDSCs from tumor-challenged mice displayed 14 times greater potential to differentiate into mature and functional osteoclasts than those from non-tumor controls. Importantly, MDSCs from tumor-challenged LacZ transgenic mice differentiated into LacZ+osteoclasts in vivo. Furthermore, a significant increase in tumor burden and bone loss accompanied by increased number of osteoclasts was observed in mice co-inoculated with tumor-challenged MDSCs and 5TGM1 cells compared to the control animals received 5TGM1 cells alone. Finally, treatment of MDSCs from myeloma-challenged mice with Zoledronic acid (ZA), a potent inhibitor of bone resorption, inhibited the number of osteoclasts formed in MDSC cultures and the expansion of MDSCs and bone lesions in mice. Collectively, these data provide in vitro and in vivo evidence that tumor-induced MDSCs exacerbate cancer-associated bone destruction by directly serving as osteoclast precursors.

Bone marrow stromal cells create a permissive microenvironment for myeloma development: a new stromal role for Wnt inhibitor Dkk1.

The rapid progression of multiple myeloma is dependent upon cellular interactions within the bone marrow microenvironment. In vitro studies suggest that bone marrow stromal cells (BMSC) can promote myeloma growth and survival and osteolytic bone disease. However, it is not possible to recreate all cellular aspects of the bone marrow microenvironment in an in vitro system, and the contributions of BMSCs to myeloma pathogenesis in an intact, immune competent, in vivo system are unknown. To investigate this, we used a murine myeloma model that replicates many features of the human disease. Coinoculation of myeloma cells and a BMSC line, isolated from myeloma-permissive mice, into otherwise nonpermissive mice resulted in myeloma development, associated with tumor growth within bone marrow and osteolytic bone disease. In contrast, inoculation of myeloma cells alone did not result in myeloma. BMSCs inoculated alone induced osteoblast suppression, associated with an increase in serum concentrations of the Wnt signaling inhibitor, Dkk1. Dkk1 was highly expressed in BMSCs and in myeloma-permissive bone marrow. Knockdown of Dkk1 expression in BMSCs decreased their ability to promote myeloma and the associated bone disease in mice. Collectively, our results show novel roles of BMSCs and BMSC-derived Dkk1 in the pathogenesis of multiple myeloma in vivo.

Silent information regulator (Sir)T1 inhibits NF-κB signaling to maintain normal skeletal remodeling.

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirtflox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα+/− mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation.

Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo.

The proteasome inhibitor bortezomib has a striking clinical benefit in patients with multiple myeloma. It is unknown whether the bone marrow microenvironment directly contributes to the dramatic response of myeloma cells to proteasome inhibition in vivo. We have used the well‐characterized 5TGM1 murine model of myeloma to investigate myeloma growth within bone and response to the proteasome inhibitor bortezomib in vivo. Myeloma cells freshly isolated from the bone marrow of myeloma‐bearing mice were found to have an increase in proteasome activity and an enhanced response to in vitro proteasome inhibition, as compared with pre‐inoculation myeloma cells. Treatment of myeloma‐bearing mice with bortezomib resulted in a greater reduction in tumor burden when the myeloma cells were located within the bone marrow when compared with extra‐osseous sites. Our results demonstrate that myeloma cells exhibit an increase in proteasome activity and an enhanced response to bortezomib treatment when located within the bone marrow microenvironment in vivo. Am. J. Hematol., 2009.

A murine model of myeloma that allows genetic manipulation of the host microenvironment

SUMMARY Multiple myeloma, and the associated osteolytic bone disease, is highly dependent upon cellular interactions within the bone marrow microenvironment. A major limitation of existing myeloma models is the requirement for a specific host strain of mouse, preventing molecular examination of the bone marrow microenvironment. The aim of the current study was to develop a model of myeloma in which the host microenvironment could be modified genetically. The Radl 5T murine model of myeloma is well characterized and closely mimics human myeloma. In the current study, we demonstrate 5T myeloma establishment in recombination activating gene 2 (RAG-2)-deficient mice, which have improper B- and T-cell development. Importantly, these mice can be easily bred with genetically modified mice to generate double knockout mice, allowing manipulation of the host microenvironment at a molecular level. Inoculation of 5TGM1 myeloma cells into RAG-2−/− mice resulted in myeloma development, which was associated with tumor growth within bone and an osteolytic bone disease, as assessed by microcomputed tomography (microCT), histology and histomorphometry. Myeloma-bearing RAG-2−/− mice displayed many features that were similar to both human myeloma and the original Radl 5T model. To demonstrate the use of this model, we have examined the effect of host-derived matrix metalloproteinase 9 (MMP-9) in the development of myeloma in vivo. Inoculation of 5TGM1 myeloma cells into mice that are deficient in RAG-2 and MMP-9 resulted in a reduction in both tumor burden and osteolytic bone disease when compared with RAG-2-deficient wild-type myeloma-bearing mice. The establishment of myeloma in RAG-2−/− mice permits molecular examination of the host contribution to myeloma pathogenesis in vivo.

Diet-induced obesity promotes a myeloma-like condition in vivo.

Multiple myeloma remains a fatal haematological malignancy associated with clonal plasma cell expansion within the bone marrow, osteolytic bone disease, anaemia and renal failure. In almost all cases, myeloma is preceded by the nonmalignant plasma cell disorder monoclonal gammopathy of undetermined significance (MGUS), characterized by an increase in monoclonal immunoglobulin secretion, <10% plasma cells within the bone marrow and a lack of lytic bone lesions. The mechanisms underlying the pathogenesis of MGUS and progression to myeloma are complex with a major role attributed to the host microenvironment. A loss of host-derived adiponectin is known to promote myeloma development in vivo, both in murine models and in patients with MGUS.1 Adiponectin is inversely linked to obesity, with increasing epidemiological evidence supporting an association between obesity and MGUS or myeloma.2, 3, 4, 5, 6, 7 In the present study, we have combined a well-characterized murine model of myeloma with diet-induced obesity and a genetic model of obesity to determine the effect of increased obesity on myeloma development in vivo.

Tumour-derived alkaline phosphatase regulates tumour growth, epithelial plasticity and disease-free survival in metastatic prostate cancer

Background:Recent evidence suggests that bone-related parameters are the main prognostic factors for overall survival in advanced prostate cancer (PCa), with elevated circulating levels of alkaline phosphatase (ALP) thought to reflect the dysregulated bone formation accompanying distant metastases. We have identified that PCa cells express ALPL, the gene that encodes for tissue nonspecific ALP, and hypothesised that tumour-derived ALPL may contribute to disease progression.Methods:Functional effects of ALPL inhibition were investigated in metastatic PCa cell lines. ALPL gene expression was analysed from published PCa data sets, and correlated with disease-free survival and metastasis.Results:ALPL expression was increased in PCa cells from metastatic sites. A reduction in tumour-derived ALPL expression or ALP activity increased cell death, mesenchymal-to-epithelial transition and reduced migration. Alkaline phosphatase activity was decreased by the EMT repressor Snail. In men with PCa, tumour-derived ALPL correlated with EMT markers, and high ALPL expression was associated with a significant reduction in disease-free survival.Conclusions:Our studies reveal the function of tumour-derived ALPL in regulating cell death and epithelial plasticity, and demonstrate a strong association between ALPL expression in PCa cells and metastasis or disease-free survival, thus identifying tumour-derived ALPL as a major contributor to the pathogenesis of PCa progression.

Preclinical animal models of multiple myeloma

Multiple myeloma is an incurable plasma-cell malignancy characterized by osteolytic bone disease and immunosuppression. Murine models of multiple myeloma and myeloma bone disease are critical tools for an improved understanding of the pathogenesis of the disease and the development of novel therapeutic strategies. This review will cover commonly used immunocompetent and xenograft models of myeloma, describing the advantages and disadvantages of each model system. In addition, this review provides detailed protocols for establishing systemic and local models of myeloma using both murine and human myeloma cell lines.