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 ...

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Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)-hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144-52)

The accurate measurement of tissue mineral density, ρm, in specimens of unequal size or quantities of bone mineral using polychromatic μCT systems is important, since studies often compare samples with a range of sizes and bone densities. We assessed the influence of object size on μCT measurements of ρm using (1) hydroxyapatite rods (HA), (2) precision-manufactured aluminum foams (AL) simulating trabecular bone structure, and (3) bovine cortical bone cubes (BCt). Two beam-hardening correction (BHC) algorithms, determined using a 200 and 1200 mg/cm3 HA wedge phantom, were used to calculate ρm of the HA and BCt. The 200 mg/cm3 and an aluminum BHC algorithm were used to calculate the linear attenuation coefficients of the AL foams. Equivalent ρm measurements of 500, 1000, and 1500 mg HA/cm3 rods decreased (r2> 0.96, p< 0.05 for all) as HA rod diameter increased in the 200 mg/cm3 BHC data. Errors averaged 8.2% across these samples and reached as high as 29.5%. Regression analyses suggested no size effects in the 1200 mg/cm3 BHC data but differences between successive sizes still reached as high as 13%. The linear attenuation coefficients of the AL foams increased up to approximately 6% with increasing volume fractions (r2> 0.81, p< 0.05 for all) but the strength of the size-related error was also BHC dependent. Equivalent ρm values were inversely correlated with BCt cube size (r2> 0.92, p< 0.05). Use of the 1200 mg/cm3 BHC ameliorated the size-related artifact compared to the 200 mg/cm3 BHC but errors with this BHC were still significant and ranged between 5% and 12%. These results demonstrate that object size, structure, and BHC algorithm can influence μCT measurements of ρm. Measurements of ρm of specimens of unequal size or quantities of bone mineral must be interpreted with caution unless appropriate steps are taken to minimize these potential artifacts.