Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). IntroductionA cardinal clinical feature of multiple myeloma (MM) is the presence of osteolytic bone lesions. Myeloma cells disrupt the delicate balance between bone formation and bone resorption. 1,2 Various clinical observations 3 and experimental studies 4,5 have linked the level of MM bone disease with disease burden. Increased osteoclastic activity and its molecular basis have long been considered a primary pathogenic event in MM bone disease. However, a molecular basis for the well recognized lack of osteoblast (OB) function, specifically DKK-1, in the MM bone disease has only recently been described. 6,7 Canonical Wnt pathway plays an important role in controlling proliferation, differentiation, and survival of OB. [8][9][10][11] Previous studies have reported high expression levels of the canonical Wnt inhibitor DKK1 and osteolytic bone lesions in various tumor types including breast, 12,13 neuroblastoma, 14 esophageal, and lung cancer, 15 and conversely enhanced OB activity and osteoblastic bone lesions associated with decreased DKK1 levels in prostate and colon cancers. [16][17][18] In MM, high serum DKK1 levels were correlated with focal bone lesions. 19 The DKK1 produced by MM cells can inhibit the differentiation of OB precursor cells 19 and bone formation in vitro 20 through a DKK1-mediated attenuation of Wnt3a-induced stabilization of ␤-catenin. 21 These findings confirm DKK1 as an important regulator of bone formation in the bone microenvironment. The importance of DKK1 secretion in diseases associated with bone destruction is reinforced by a recent study showing that DKK1 mediates the bone destructive effects of rheumatoid arthritis and that a neutralizing antibody to DKK1 could inhibit the bone destructive process in that disease. 22 There is also emerging evidence that the cellular bone compartment affects MM cell growth and progression. This is supported by the observation that osteoclasts can support long-term survival and proliferation of primary MM cells, 23,24 and OB may impede MM cell growth. 7,25 Thus, targeting these cellular elements may also favorably affect disease control. Therefore, we have evaluated DKK1 as a therapeutic target in MM in the context of the bone marrow (BM) microenvironment, analyzing the effect of a human DKK1 neutralizing antibody (BHQ880). We show that this clinically applicable antibody increases OB function and number and also has anti-MM effect when evaluated in the presence of the BM milieu. Methods ReagentsBHQ880 is a phage-derived DKK1 neutralizing human immunoglobulin G1 (IgG1) antibody (provided by Novartis, Cambridge, MA). BHQ880 has a high affinity for and can neutralize both human DKK1 and murine DKK1. IgG1 isotype antibody was used as control. CellsBone marrow mononuclear cells (BMMNCs) and primary MM cells were isolated using Ficoll-Hypaque density gradient sedimentation from BM Submitted November 25, 2008; accepted April 20, 2009. Prepublished online ...

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distalless homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.osteoblasts | osteoclasts | tumor niche

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110␦ signaling in multiple myeloma ( IntroductionThe bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM) by promoting cell proliferation, survival, migration, and drug resistance. [1][2][3][4] The PI3K/AKT pathway mediates growth and drug resistance in MM cells and also plays a significant role in autophagy. 5,6 PI3K is activated via upstream tyrosine kinase-associated receptors for growth factors, cytokines, antigens, and costimulatory molecules. It in turn activates AKT, which mediates cell proliferation, cell cycle, apoptosis, and autophagy. 7 Class IA PI3K consists of 5 isoforms of regulatory subunits (p85␣, p50␣, p55␣, p85␤, and p55␥), which interact with class IA isoforms. Class IA PI3K is composed of p110␣, -␤, and -␦ isoforms. 8 Among the 8 distinct mammalian isoforms of PI3K, class I PI3Ks are responsible for Akt activation. Importantly, p110␦ is expressed in many cancers, including colon and bladder carcinoma, glioblastoma, and acute myeloid leukemia blasts. 9,10 In the current study, we demonstrate high expression of p110␦ in patient MM cells. Previous studies have shown that CAL-101, a potent and selective p110␦ inhibitor, has broad antitumor activity against cancer cells of hematologic origin. 11,12 Moreover, inhibition of p110␦ induces cleavage of caspases and LC3, consistent with apoptotic and autophagic cell death, respectively. Here we show that p110␦ blockade with CAL-101, a potent and selective p110␦ inhibitor, inhibits MM cell growth even in the presence of interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or bone marrow stromal cells (BMSCs), associated with decreased phosphorylation of AKT and P70S6k. We also confirmed inhibition of human MM cell growth triggered by p110␦ inhibition in our xenograft mouse models of human MM. These studies therefore show that small molecule inhibitors of p110␦ trigger significant anti-MM cytotoxicity both in vitro and in vivo, providing the framework for their clinical evaluation to improve patient outcome in MM. Methods Materialsp110␦ inhibitor CAL-101 and IC488743 were provided by Calistoga Pharmaceuticals. CAL-101 was dissolved in dimethyl sulphoxide at 10mM and stored at Ϫ20°C for in vitro study. IC488743 was dissolved in 1% carboxyl methylcellulose/0.5% Tween 80 and stored at 4°C for in vivo study. Recombinant human p110␣, ␤, ␥, and ␦ were reconstituted with sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. Bortezomib was provided by Millennium Pharmaceuticals. 3-Methyladenine was purchased from Sigma-Aldrich. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Cell culture Dex 1460BLOOD, 2 SEPTEMBER 2010 ⅐ VOLUME 116, NUMBER 9For personal use only. on March 28, 2019. by guest www.bloodjournal.org From Germany). LB human MM ce...

IntroductionMultiple myeloma (MM) is a B-cell malignancy characterized by proliferation of monoclonal plasma cells in the bone marrow (BM). Despite the recent emergence of novel therapies including bortezomib, 1,2 thalidomide, 3,4 and lenalidomide, 5 it remains incurable due to the development of drug resistance. [5][6][7] Among the factors that lead to this resistance are defects in apoptotic signaling pathways and overexpression of the multidrug resistance protein (MRP) pumps that enhance drug efflux. 8 In addition, the BM microenvironment confers drug resistance in MM via (1) secretion of cytokines such as interleukin 6 (IL-6) and insulin-like growth factor 1 (IGF-1), which mediate survival signals in MM cells, 9-11 as well as (2) direct interaction with MM cells, which results in cell adhesion-mediated drug resistance. 12,13 Despite recent progress, MM remains incurable, and new therapeutic agents with novel mechanism of actions are urgently needed.JS-K (O 2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a diazeniumdiolate class of prodrug designed to release nitric oxide (NO • ) when metabolized by glutathione S-transferases (GSTs; Figure 1A). 14 GSTs are enzymes that catalyze the conjugation of xenobiotics with glutathione (GSH), thereby facilitating their subsequent efflux through MRP pumps. 15 GSTs are frequently overexpressed in a broad spectrum of tumors. 16,17 In the context of conventional chemotherapy, this provides tumor cells with a selective survival advantage over normal cells by enhancing drug efflux and thus decreasing therapeutic efficacy. In contrast, since JS-K uniquely requires GST for its optimal activity, it can potentially turn GST overexpression to the tumor's disadvantage by generating relatively high intracellular concentrations of cytotoxic NO • , specifically within tumor cells. Importantly, JS-K has recently been shown to inhibit tumor growth in both in vitro and in vivo models of human prostate cancer and human leukemia. 14 Importantly, GSTs are overexpressed in 10% to 70% of patients with MM at diagnosis, and in 30% of patients at relapse. 8 In addition, in our recent study comparing gene expression profiles of patient MM cells with normal plasma cells from a genetically identical twin, we observed that GST was overexpressed by 7-fold in MM cells. 18 Furthermore, in our high-resolution genomic and expression profiling of primary tumor cells from 67 patients with MM and plasma cells from 12 healthy donors, 19 33% and 39% of the MM cells overexpressed GSTP1 and GSTM1, respectively, when compared with plasma The online version of this manuscript contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 10, 2018. by guest www.bloodjournal.org From cell controls. To date, however, the biological effects of JS-K on MM cells ha...

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Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G 2 -M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.The cell surface proteoglycan CD138 (syndecan-1) is an integral membrane protein acting as a receptor for the extracellular matrix. Within the normal human hematopoetic compartment, CD138 is expressed on differentiated plasma cells and is a primary diagnostic marker of multiple myeloma (MM; ref. 1). The large extracellular domain of CD138 binds via its heparin sulfate chains to soluble extracellular molecules, including the growth factors epidermal growth factor, fibroblast growth factor, and hepatocyte growth factor, and to insoluble extracellular molecules, such as collagen and fibronectin (2, 3). CD138 also mediates cell-cell adhesion through interactions with heparinbinding molecules. Studies of plasma cell differentiation show that CD138 is a differentiation antigen (4) and a coreceptor for MM growth factors (5).Several monoclonal antibodies (mAb; i.e.,

Osteolytic bone disease in multiple myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising response rates in relapsed and newly diagnosed MM, and bortezomib has recently been reported to inhibit OCLs. We here investigated the effect of lenalidomide on OCL formation and osteoclastogenesis in comparison with bortezomib. Both drugs decreased aVb3-integrin, tartrate-resistant acid phosphatase-positive cells and bone resorption on dentin disks. In addition, both agents decreased receptor activator of nuclear factor-jB ligand (RANKL) secretion of bone marrow stromal cells (BMSCs) derived from MM patients. We identified PU.1 and pERK as major targets of lenalidomide, and nuclear factor of activated T cells of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was observed. We demonstrated a significant decrease of growth and survival factors including macrophage inflammatory protein-a, B-cell activating factor and a proliferation-inducing ligand. Importantly, in serum from MM patients treated with lenalidomide, the essential bone-remodeling factor RANKL, as well as the RANKL/OPG ratio, were significantly reduced, whereas osteoprotegerin (OPG) was increased. We conclude that both agents specifically target key factors in osteoclastogenesis, and could directly affect the MM-OCL-BMSCs activation loop in osteolytic bone disease.