Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/ß-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

Evaluating results from the multiple myeloma patient subset treated with denosumab or zoledronic acid in a randomized phase 3 trial.

In a phase 3 trial of denosumab vs zoledronic acid in patients (n=1776) with bone metastases and solid tumors or multiple myeloma, denosumab was superior to zoledronic acid for the primary end point of prevention of skeletal-related events. There was no difference in overall survival between the two groups; however, an ad hoc overall survival analysis in the multiple myeloma subset of patients (n=180) favored zoledronic acid (hazard ratio (HR) 2.26; 95% confidence interval (CI) 1.13-4.50; P=0.014). In the present analysis, we found imbalances between the groups with respect to baseline risk characteristics. HRs with two-sided 95% CIs were estimated using the Cox model. After adjustment in a covariate analysis, the CI crossed unity (HR 1.86; 95% CI 0.90-3.84; P=0.0954). Furthermore, we found a higher rate of early withdrawals for the reasons of lost to follow-up and withdrawal of consent in the zoledronic acid group; after accounting for these, the HR was 1.31 (95% CI 0.80-2.15; P=0.278). In conclusion, the survival results in multiple myeloma patients in this trial were confounded and will eventually be resolved by an ongoing phase 3 trial.

Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Pathogenesis of myeloma bone disease.

Bone disease in multiple myeloma (MM) is characterized by lytic bone lesions, which can cause severe bone pain, pathologic fractures and hypercalcemia. However, the lytic bone disease in MM differs from that in other cancer patients who have lytic bone metastases. Although increased osteoclastic bone destruction is involved in MM and other tumors involving bone, in contrast to other tumors, once the MM tumor burden exceeds 50% in a local area, osteoblast activity is either suppressed or absent. The basis for this severe imbalance between increased osteoclastic bone resorption and decreased bone formation has been a topic of intensive investigation over the last several years and will be reviewed in this article.

In vivo impact of a 4 bp deletion mutation in the DLX3 gene on bone development.

Distal-less 3 (DLX3) gene mutations are etiologic for Tricho-Dento-Osseous syndrome. To investigate the in vivo impact of mutant DLX3 on bone development, we established transgenic (TG) mice expressing the c.571_574delGGGG DLX-3 gene mutation (MT-DLX3) driven by a mouse 2.3 Col1A1 promoter. Microcomputed tomographic analyses demonstrated markedly increased trabecular bone volume and bone mineral density in femora from TG mice. In ex vivo experiments, TG mice showed enhanced differentiation of bone marrow stromal cells to osteoblasts and increased expression levels of bone formation markers. However, TG mice did not show enhanced dynamic bone formation rates in in vivo fluorochrome double labeling experiments. Osteoclastic differentiation capacities of bone marrow monocytes were reduced in TG mice in the presence of osteoclastogenic factors and the numbers of TRAP(+) osteoclasts on distal metaphyseal trabecular bone surfaces were significantly decreased. TRACP 5b and CTX serum levels were significantly decreased in TG mice, while IFN-gamma levels were significantly increased. These data demonstrate that increased levels of IFN-gamma decrease osteoclast bone resorption activities, contributing to the enhanced trabecular bone volume and mineral density in these TG mice. These data suggest a novel role for this DLX-3 mutation in osteoclast differentiation and bone resorption.

Involvement of hepcidin in the anemia of multiple myeloma.

PURPOSE: Hepcidin is a liver-produced peptide implicated in the anemia of inflammation. Because interleukin (IL)-6 is a potent inducer of hepcidin expression and its levels are elevated in multiple myeloma, we studied the role of hepcidin in the anemia of multiple myeloma. EXPERIMENTAL DESIGN: Urinary hepcidin and serum levels of IL-6, ferritin, C-reactive protein, tumor necrosis factor-alpha, and IL-1 beta were studied in newly diagnosed myeloma patients. In vitro hepcidin induction assay was assessed by real-time PCR assay. RESULTS: Pretreatment urinary hepcidin levels in 44 patients with stage III multiple myeloma were 3-fold greater than normal controls. In the subset of multiple myeloma patients without renal insufficiency (n = 27), a marked inverse correlation was seen between hemoglobin at diagnosis and urinary hepcidin level (P = 0.014) strongly supporting a causal relationship between up-regulated hepcidin expression and anemia. The urinary hepcidin also significantly (P < 0.05) correlated with serum ferritin and C-reactive protein, whereas its correlation with serum IL-6 levels was of borderline significance (P = 0.06). Sera from 14 multiple myeloma patients, with known elevated urinary hepcidin, significantly induced hepcidin mRNA in the Hep3B cells, whereas normal sera had no effect. For 10 patients, the ability of anti-IL-6 and anti-IL-6 receptor antibodies to prevent the serum-induced hepcidin RNA was tested. In 6 of these patients, hepcidin induction was abrogated by the anti-IL-6 antibodies, but in the other 4 patients, the neutralizing antibodies had no effect. CONCLUSIONS: These results indicate hepcidin is up-regulated in multiple myeloma patients by both IL-6-dependent and IL-6-independent mechanisms and may play a role in the anemia of multiple myeloma.

Treatment strategies for bone disease.

Multiple myeloma is characterized by extensive bone destruction with little or no new bone formation. A multiplicity of factors including receptor activator NF-kappaB (RANKL), macrophage inflammatory protein-1alpha, interleukin-3 and interleukin-6 can induce osteoclast formation in myeloma and drive the bone destructive process. Furthermore, factors are also produced either in the microenvironment or by myeloma cells themselves, which inhibit osteoblast differentiation and new bone formation. The combination of increased osteoclast formation with little or no bone repair in response to the previous bone destruction explains the severity of the bone disease in myeloma. Studies of the pathophysiology of myeloma bone disease have identified several novel therapeutic targets. These include antibodies to RANKL, chemokine receptor antagonists, which block the effects of chemokines on osteoclast differentiation and proteasome antagonists, which can affect both RANKL production and osteoprotegerin levels as well as inhibit osteoclast and enhance osteoblast differentiation. In addition, many of the new biologic agents being used for the treatment of patients with myeloma also further inhibit the bone destructive process. New therapies that can target both the tumor as well as the severe bone disease should be on the horizon to treat this devastating complication of myeloma.

Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease.

We recently identified macrophage inflammatory protein 1-alpha (MIP-1alpha) as a factor produced by multiple myeloma (MM) cells that may be responsible for the bone destruction in MM (1). To investigate the role of MIP-1alpha in MM bone disease in vivo, the human MM-derived cell line ARH was stably transfected with an antisense construct to MIP-1alpha (AS-ARH) and tested for its capacity to induce MM bone disease in SCID mice. Human MIP-1alpha levels in marrow plasma from AS-ARH mice were markedly decreased compared with controls treated with ARH cells transfected with empty vector (EV-ARH). Mice treated with AS-ARH cells lived longer than controls and, unlike the controls, they showed no radiologically identifiable lytic lesions. Histomorphometric analysis demonstrated that osteoclasts (OCLs) per square millimeter of bone and OCLs per millimeter of bone surface of AS-ARH mice were significantly less than in EV-ARH mice, and the percentage of tumors per total bone area was also significantly decreased. AS-ARH cells demonstrated decreased adherence to marrow stromal cells, due to reduced expression of the alpha(5)beta(1) integrin and diminished homing capacity and survival. These data support an important role for MIP-1alpha in cell homing, survival, and bone destruction in MM.

Paget's disease of bone: a disease of the osteoclast.

Paget's disease is a chronic focal disease of the skeleton that affects up to 2-3% of the population over the age of 60 years. There is a genetic predisposition for Paget's disease, with one predisposition locus identified on chromosome 18q-21-22. Osteoclasts and osteoclast precursors from Paget's patients are abnormal and appear hyperresponsive to 1,25(OH)2D3 and RANK ligand and contain paramyxoviral transcripts (Fig. 1). The basis for the abnormalities detected in Paget's disease and the role that the paramyxoviruses may play in this disease are still unclear.

Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment.

Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-kappaB (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors.

Studies in Paget's disease and their relevance to oncology.

Paget's disease and bone metastases in cancer patients share many common properties. Both are characterized by a localized increase in osteoclast (OCL) formation leading to bone resorption. In both Paget's disease and bone metastases the increased OCL formation and the increased osteoclastogenic nature of the bone microenvironment are mediated by common factors, namely interleukin (IL)-6 and RANK ligand (RANKL). Available data suggest that in the case of Paget's disease there is increased RANKL and IL-6 production, and IL-6 enhances the responsivity of the OCL precursors to RANKL, contributing to the elevated numbers of OCLs. In patients with multiple myeloma, 95% to 100% of whom develop bone lesions, both IL-6 and RANKL levels are increased. Bisphosphonates bind locally to the surfaces of the bone undergoing osteoclastic resorption to inhibit this process. Paget's disease has in the past and will continue in the future to provide a model to test the efficacy of bisphosphonates in inhibiting bone resorption. Paget's disease provides an ideal model in which to investigate the efficacy of the new third-generation bisphosphonates in the treatment of bone metastases as well as nonmalignant bone disease.

Myeloma bone disease.

Bone destruction is a hallmark of myeloma, with 70% to 80% of patients manifesting bone involvement. Destruction is mediated through normal osteoclasts (OCLs), which respond to local osteoclast-activating factors (OAFs) produced by myeloma cells or by other cells in the local microenvironment. OAFs implicated in myeloma bone disease include tumor necrosis factor-beta (TNFbeta), RANK ligand (RANKL), interleukin-1 (IL-1), parathyroid hormone-related protein (PTHrP), hepatocyte growth factor (HGH), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha), and macrophage inflammatory protein-1-alpha (MIP-1alpha). To date, the leading candidates for OAFs are MIP-1alpha and RANKL. Adhesive interactions between marrow stromal cells and myeloma cells induce marrow stromal cells to secrete IL-6, a potent myeloma growth/survival factor that may contribute to the bone disease. Evaluation of myeloma bone disease includes plain radiographs, and newer methods, such as magnetic resonance imaging (MRI), positron emission tomography (PET) scans, technetium-99m-sestamibi (Mibi) scanning, and dual-energy x-ray absorptiometry (DEXA) scanning, may provide more complete information. In addition, biochemical markers of bone resorption are being evaluated, although the limited availability of these assays and lack of extensive testing in patients make their routine use premature. Treatment of myeloma bone disease includes radiation therapy, vertebroplasty, surgery, and bisphosphonates. New developments on the pathogenesis and treatment of myeloma bone disease present great opportunities to combat bone disease.

Biology of osteoclast activation in cancer.

Bone is a frequent site of cancer metastasis. Bone metastases can result in bone destruction or new bone formation. Bone destruction is mediated by factors produced or induced by tumor cells that stimulate formation and activation of osteoclasts, the normal bone-resorbing cells. Local bone destruction also occurs in patients with osteoblastic metastases and may precede or occur simultaneously with increased bone formation. Several factors, including interleukin (IL)-1, IL-6, receptor activator of NF-kappaB (RANK) ligand, parathyroid hormone-related protein (PTHrP), and macrophage inflammatory protein-1-alpha (MIP-1alpha), have been implicated as factors that enhance osteoclast formation and bone destruction in patients with neoplasia. PTHrP seems to be the major factor produced by breast cancer cells that induces osteoclast formation through upregulation of RANK ligand. Enhanced RANK ligand expression also plays an important role in bone destruction in patients with myeloma. RANK ligand can act to enhance the effects of other factors produced by myeloma cells or in response to myeloma cells, such as MIP-1alpha and/or IL-6, to induce osteoclast formation. Understanding of the molecular mechanisms responsible for osteoclast activation in osteolytic metastases should lead to development of novel therapeutic approaches for this highly morbid and potentially fatal complication of cancer.

Osteoclasts formed by measles virus-infected osteoclast precursors from hCD46 transgenic mice express characteristics of pagetic osteoclasts.

Pagetic osteoclasts (OCLs) are abnormal in size and contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus (MV). However, the role that MV infection plays in Paget's disease is unknown, because no animal model of Paget's disease is available. Therefore, we targeted a cellular MV receptor, human CD46 (hCD46), to cells in the OCL lineage in transgenic mice using the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. In vitro infection of OCL precursors from hCD46 transgenic mice with MV significantly increased OCL formation in bone marrow cultures. The numbers of TRAP-positive mononuclear cells and CFU-GM, the earliest identifiable OCL precursor, were also significantly increased. MV-infected OCLs formed from hCD46 marrow were increased in size, contained markedly increased numbers of nuclei, and had increased bone-resorbing capacity per OCL compared with OCLs formed from marrow of nontransgenic littermates. Furthermore, IL-6 and 24-hydroxylase messenger RNA expression levels were increased in MV-infected hCD46 transgenic mouse bone marrow cultures. Treatment of MV-infected hCD46 marrow cultures with a neutralizing antibody to IL-6 blocked the increased OCL formation seen in these cultures. These data demonstrate that MV infection of OCL precursors results in OCLs that have many features of pagetic OCLs, that the enhanced OCL formation is in part mediated by increased IL-6 expression induced by MV infection, and suggest that the hCD46 transgenic mouse may be a useful model for examining the effects of MV infection on OCL formation in vivo.

Prognostic factors and response to fludarabine therapy in patients with Waldenström macroglobulinemia: results of United States intergroup trial (Southwest Oncology Group S9003).

Current information on Waldenström macroglobulinemia (WM) is based on retrospective or single-institution studies of patients requiring therapy. Between 1992 and 1998, 231 patients with WM were enrolled in a prospective observational multicenter clinical trial. Of these, 182 patients with symptomatic or progressive disease were treated with 4 to 8 cycles of therapy with a purine nucleoside analogue, fludarabine (FAMP; 30 mg/m(2) of body-surface area daily for 5 days every 28 days). A serum beta2-microglobulin (beta2M) level below 3 mg/L and a hemoglobin level of at least 120 g/L (12 g/dL) at presentation predicted a lower likelihood of requiring therapy. The overall rate of response to FAMP therapy was 36% (95% confidence interval, 29%-44%), with 2% complete remissions. Patients who were 70 years old or older had a substantially lower likelihood of response (odds ratio, 0.34; P =.004) than younger patients. On multivariate analysis, a serum beta2M level of 3 mg/L or higher, hemoglobin level below 120 g/L, and serum IgM level below 40 g/L [4 g/dL] were significant adverse prognostic factors for survival. We developed a simple staging system for WM by using these variables and identified 4 distinct subsets of patients with estimated 5-year overall survival rates of 87%, 64%, 53%, and 22%, and 5-year progression-free survival rates of 83%, 55%, 33%, and 12%. Prognosis in WM is highly variable and serum beta2M was the dominant predictor of a need for therapy and of survival. FAMP has activity against WM. Our staging system may provide guidance for a risk-based approach to the treatment of WM.

Macrophage inflammatory protein-1alpha is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kappaB ligand.

A complementary DNA expression library derived from marrow samples from myeloma patients was recently screened and human macrophage inflammatory protein-1alpha (hMIP-1alpha) was identified as an osteoclastogenic factor expressed in these samples. hMIP-1alpha enhanced osteoclast (OCL) formation in human marrow cultures and by highly purified OCL precursors in a dose-dependent manner (5-200 pg/mL). Furthermore, hMIP-1alpha enhanced OCL formation induced by human interleukin-6 (IL-6), which is produced by marrow stromal cells when they interact with myeloma cells. hMIP-1alpha also enhanced OCL formation induced by parathyroid hormone-related protein (PTHrP) and receptor activator of nuclear factor kappaB ligand (RANKL), factors also implicated in myeloma bone disease. Time-course studies revealed that the hMIP-1alpha acted during the last 2 weeks of the 3-week culture period. Reverse transcription-polymerase chain reaction analysis showed that the chemokine receptors for hMIP-1alpha (CCR1 and CCR5) were expressed by human bone marrow and highly purified early OCL precursors. Furthermore, hMIP-1alpha did not increase expression of RANKL. These data demonstrate that hMIP-1alpha is an osteoclastogenic factor that appears to act directly on human OCL progenitors and acts at the later stages of OCL differentiation. These data further suggest that in patients with myeloma, MIP-1alpha produced by myeloma cells, in combination with RANKL and IL-6 that are produced by marrow stromal cells in response to myeloma cells, enhances OCL formation through their combined effects on OCL precursors. (Blood. 2001;97:3349-3353)

ADAM8: a novel osteoclast stimulating factor.

We used polymerase chain reaction (PCR)-selective complementary DNA (cDNA) subtraction hybridization with an immortalized murine osteoclast (OCL) precursor cell line to identify genes that are highly expressed in OCLs compared with OCL precursors and which may be involved in the OCL differentiation process. ADAM8 was one of the 50 genes identified. ADAM (a disintegrin and metalloproteinase) peptides are membrane-bound proteins that can act as cell-to-cell and cell-to-matrix adhesion molecules, degrade the extracellular matrix, and play a role in tissue morphogenesis. Addition of antisense (AS) S-oligonucleotides for ADAM8 (1-10 nM) to mouse bone marrow cultures treated with 10(-9) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibited OCL formation compared with treatment with the control S-oligonucleotide. Furthermore, conditioned media from 293 cells transiently transfected with a secretable form of the ADAM8 cDNA increased OCL formation in a dose-dependent manner. In addition, treatment of OCLs with soluble ADAM8 conditioned media significantly increased pit formation per dentin slice compared with control OCLs. Time course studies indicated that ADAM8 increased OCL formation only when it was present during days 4-7 of the 7-day culture period. Structural analysis, using truncated constructs of ADAM8, showed that the cysteine-rich/disintegrin domain was responsible for its OCL stimulatory activity. Western blot analysis confirmed that the soluble form of ADAM8 is present in normal marrow cultures. These data suggest that ADAM8 plays an important role in OCL formation and acts primarily at the later stages of OCL differentiation.

Ibandronate decreases bone disease development and osteoclast stimulatory activity in an in vivo model of human myeloma.

The benefits of bisphosphonate therapy for multiple myeloma bone disease have been clearly documented. However, the effects of bisphosphonates on the osteoclast stimulatory activity (OSA) that is present in the marrow of patients with multiple myeloma, even before the bone disease is detectable, are unknown. Therefore, we examined the effects of ibandronate (IB) treatment prior to the development of bone disease in a murine model of human myeloma. Sublethally irradiated severe combined immunodeficient (SCID) mice were transplanted with ARH-77 cells on day 0. These ARH-77 mice were treated daily with subcutaneous injections of IB started before or at different times after tumor injection as follows: group 1 was started on day -7; group 2 on day 0; group 3 on day +7; group 4 on day +14 after IB administration; and group 5 (control) received no IB. Mice were sacrificed after they developed paraplegia. The onset of paraplegia was delayed in group 1 vs all other groups (mean day 27 vs day 32; p = 0.0098). The number of lytic lesions and the bone surface area of resorption (mm(2)) were significantly decreased in groups 1, 2, and 3, which were treated early with IB, when compared with groups 4 and 5 (p = 0.003 and 0.002, respectively). OSA, as measured by the capacity of bone marrow plasma from ARH-77 mice to induce osteoclast (OCL) formation in human bone marrow cultures, was decreased proportionally to the length of IB treatment. Group 1 had the lowest OSA compared with the other groups (p = 0.003). However, all mice eventually developed paraplegia, and at time of sacrifice, tumor burden was not grossly different among the groups. Interestingly, macroscopic abdominal tumors were more frequent in mice treated with IB. These data demonstrate that early treatment of ARH-77 mice with IB prior to development of myeloma bone disease decreases OSA and possibly retards the development of lytic lesions, but not eventual tumor burden.