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Pathophysiology of myeloma bone disease
Best Practice & Research Clinical Haematology Vol. 20, No. 4, pp. 613–624, 2007 doi:10.1016/j.beha.2007.08.003 available online at http://www.sciencedirect.com
3 Pathophysiology of myeloma bone disease Flavia R. Esteve
MD
Hematology Fellow University of Pittsburgh, Medicine/Hematology-Oncology, 5150 Centre Avenue, Pittsburgh, PA 15213, USA
G. David Roodman *
MD, PhD
Professor of Medicine University of Pittsburgh, Medicine/Hematology-Oncology, VA Pittsburgh Healthcare System R&D (151-U), University Drive C, Pittsburgh, PA 15240, USA
Multiple myeloma is a tumor of terminally differentiated plasma cells that home to and expand in the bone marrow. It is the second most common hematologic malignancy, with approximately 16,000 new cases per year, and accounts for an estimated 11,000 deaths in the USA. It is the most common cancer to metastasize to bone, with up to 90% of patients developing bone lesions. The bone lesions are purely osteolytic in nature, and up to 60% of patients develop a pathologic fracture over the course of their disease. Bone disease is a hallmark of multiple myeloma, and the bone disease differs from other bone metastasis caused by other tumors. Although both myeloma and other osteolytic metastasis induce increased osteoclastic bone resorption, in contrast to other tumors, osteoblast activity in myeloma is either severely decreased or absent. The basis for this severe imbalance between increased osteoclastic bone resorption and decreased bone formation resulting from suppressed osteoblastic activity has been a topic of extensive investigation during the last several years. The clinical consequences of this extensive accelerated and imbalanced bone destruction process include bone pain, pathologic fractures, hypercalcemia and spinal cord compression syndromes, which can be devastating for patients and significantly impact overall quality of life and expected survival. In this chapter, we will discuss the pathophysiology underlying bone disease in myeloma. This results from the uncoupling of bone remodeling and is characterized by markedly increased activity of osteoclasts and profound decreased activity of osteoblasts. In addition, we also review the emerging data on novel targeted therapies aimed at ameliorating myeloma bone disease. Key words: myeloma; metastasis; osteoclasts; bone destruction.
* Corresponding author. Tel.: þ1-412-688-6571; Fax: þ1-412-688-6960. E-mail address: [email protected] 1521-6926/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
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OSTEOLYTIC BONE DESTRUCTION IN MYELOMA BONE DISEASE Normal bone constantly undergoes remodeling, which involves the resorption of bone by osteoclasts and the deposition of new bone by osteoblasts at sites of previous resorption. Osteoclasts arise from precursor cells in the monocyte/macrophage lineage1 that differentiate into inactive osteoclasts. The inactive osteoclasts are then activated, resorb bone, and subsequently undergo apoptosis. Both locally produced cytokines and systemic hormones normally regulate osteoclast formation and activity. In myeloma, many different components interact to increase the bone-resorptive process. These include myeloma cells themselves, bone-marrow stromal cells, and T cells present in the marrow microenvironment. Furthermore, growth factors released by the bone-resorptive process2 also increase the growth of myeloma cells. This creates a vicious cycle, with the bone-resorptive process increasing myelomacell tumor burden, which then results in increased bone resorption. Recent studies have identified several important factors produced by myeloma cells in vivo that have been implicated in the osteolytic bone-resorptive process. These include the receptor activator nuclear factor kB (NF-kB, RANKL), macrophage inflammatory peptide 1a (MIP-1a), interleukin 3 (IL-3) and IL-6.3–6 The RANK/RANKL signaling pathway is a critical component of the bone-remodeling process. RANK is a transmembrane signaling receptor which is a member of the tumor necrosis factor (TNF) receptor superfamily. It is found on the surface of osteoclast precursors.7,8 RANK ligand (RANKL) is expressed as a membrane-bound protein on marrow stromal cells and osteoblasts, and secreted by activated lymphocyte. Its expression is augmented by cytokines that stimulate bone resorption9, such as parathyroid hormone (PTH), 1,25-OH vitamin D3, and prostaglandins.10,11 RANKL binds to RANK receptor on osteoclast precursors and induces osteoclast formation. Rank signals through the NF-kB and JunN terminal kinase pathways and induces increased osteoclastic bone resorption and enhanced osteoclast survival.2 The important role of RANKL in normal osteoclastogenesis has been clearly demonstrated in RANKL or RANK gene knockout mice. These animals lack osteoclasts and as a result develop severe osteopetrosis.12,13 Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and is a member of the TNF receptor superfamily.14 It is produced by osteoblasts, as well as other cell types, and blocks the interactions of RANKL with RANK, thereby limiting osteoclastogenesis. In normal subjects, the ratio RANKL/OPG strongly favors OPG. The importance of OPG has also been shown in studies using knockout mice for the OPG gene; these mice develop severe osteopenia and osteoporosis.13–17 Pearse and co-workers were the first to demonstrate that in bone-marrow biopsies of multiple myeloma (MM) patients RANKL expression was up-regulated while OPG expression was decreased.18 Terpos et al showed that circulating levels of OPG and RANKL correlated with clinical activity of myeloma, severity of bone disease, and poor prognosis.19 Furthermore, murine myeloma models have also determined that inhibition of RANKL can prevent bone destruction in either the SCID-hu model or the T2 MM syngeneic model of myeloma.19,20 These studies revealed that blocking RANKL, either with a soluble form of the RANK receptor or by OPG, minimizes bone destruction and tumor burden. In addition, myeloma cells have been reported to express RANKL, which may further contribute to the bone-destructive process (Figure 1). MIP-1a is a chemokine that is produced by MM cells in 70% of MM patients; it is a potent inducer of human osteoclast formation. MIP-1a can increase osteoclast
Pathophysiology of myeloma bone disease 615
Figure 1. Myeloma cells and T cells secrete RANKL to induce osteoclast (OCL) formation. In addition, multiple myeloma (MM) cells suppress osteoprotegerin (OPG) in the marrow microenvironment. This results in a severe imbalance in the normal RANKL/OPG rates driving osteoclastogenesis.
formation independently of RANKL and can potentiate both RANKL and IL-6 stimulated osteoclast formation.21 Magrangeas et al have shown by gene expression profiling (GEP) that MIP-1a is the gene most highly correlated with bone destruction in myeloma.22 Further, Abe and co-workers have shown that elevated levels of MIP-1a also correlate with an extremely poor prognosis in myeloma.23 In-vivo models of myeloma have demonstrated that MIP-1a can induce osteoclast formation and bone destruction. Blocking MIP-1a expression in myeloma cells injected into severe combined immunodeficient (SCID) mice, or treating the animals with a neutralizing antibody to MIP-1a, results in a decreased tumor burden and bone destruction.24,25 MIP-1a also plays an important role in the homing of myeloma cells to the bone marrow. MIP-1a increases adhesive interactions between myeloma cells and marrow stromal cells by increasing expression of b1 integrins, which takes place through a4b1 or a5b1 integrins and adhesive molecules such as vascular cell adhesion molecule 1 (VCAM-1). This results in production of RANKL, IL-6, vascular endothelial growth factor (VEGF) and TNFa by marrow stromal cells, which further enhances myeloma-cell growth, angiogenesis and bone destruction. Further, Masih-Khan et al reported that the t4:14 translocation results in a constitutive expression of the FGFR3 receptor, which results in high levels of MIP-1a.26 Patients with the t4:14 translocation have a very poor prognosis, which may reflect the increased MIP-1a production in this patient population. IL-3, in addition to RANKL and MIP-1a, is also significantly elevated in bonemarrow plasma of MM patients as compared to that of normal controls.6 IL-3 can induce osteoclast formation in human bone-marrow cultures at levels similar to those
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measured in myeloma patient samples, and osteoclast formation induced by marrow plasma from MM patients could be inhibited by using a blocking antibody to IL-3.6 IL-3 also indirectly influences osteoclastogenesis by enhancing the effects of RANKL and MIP-1a on the growth and development of osteoclasts. It also stimulates myeloma cell growth directly6 and, as discussed later in this chapter, IL-3 has also been shown to inhibit osteoblast formation through a factor produced by macrophages in the marrow microenvironment.27 IL-6 has long been recognized as a proliferative factor for plasma cells, but it is unclear whether IL-6 levels correlate with disease status.28 Levels of IL-6 are elevated in MM patients with osteolytic bone disease, when compared to MM patients without bone disease, as well as in patients with monoclonal gammopathy of unknown significance (MGUS).29 Most studies support the idea that IL-6 is produced by cells in the bone-marrow microenvironment rather than myeloma cells through direct contact with myeloma cells. These cells most likely are osteoclasts and stromal cells, but increased osteoblast production of IL-6 has also been reported in cocultures of human osteoblasts with MM cells.30 Although the precise role of IL-6 in myeloma bone disease is yet to be determined, IL-6 production by osteoclasts can increase tumor burden, leading to enhanced bone destruction as well as acting as an autocrine/ paracrine factor to increase osteoclast formation.31 Adhesive interactions between myeloma and stromal cells play a significant role in the homing of myeloma cells in the bone marrow and augmenting the bone-destructive process. These adhesive interactions result in increased signaling of NF-kB and p38 MAP kinase. The latter is involved in both osteoclastic growth and differentiation as well as induction of RANKL expression by osteoblasts. Blocking p38MAP kinase potently inhibits IL-6 and VEGF production as well as decreased adhesion of myeloma cells to marrow stromal cells.32 Recently, Vanderkerken and co-workers reported that inhibition of p38 MAP kinase in the 5T2 MM murine model of myeloma decreased tumor cell burden, prevented development of bone disease, and increased overall survival in mice having 5T2 cells.33 Therefore, this pathway may be a potential therapeutic target for novel therapies to ameliorate myeloma disease. P62 (sequestosome-1) is a recently described member of the NF-kB pathway. It mediates IL-1-, TNF- and RANKL-mediated NF-kB activation. It is up-regulated in the marrow microenvironment in patients with myeloma. Kurihara and colleagues have examined the role of p62 in myeloma bone disease and found that deleting p62 in marrow stromal cells decreased VCAM-1 expression and the capacity of the cells to support osteoclast formation, to increase myeloma cell growth, and to produce RANKL and TNF-a. They also found that TNF-a could reverse the effects of loss of p62 in co-cultures of MM and stromal cells through increased expression of VCAM-1.34 OSTEOBLASTIC DYSFUNCTION IN MYELOMA Histomorphometric studies of bone lesions from MM have provided important insights into the pathophysiology of myeloma bone disease. They disclosed that the bone-remodeling process was uncoupled, with increased bone resorption due to enhanced osteoclastogenesis and decreased or absent bone formation. In contrast, in either MM patients without bone disease or normal controls, there is balanced bone remodeling with increased osteoblastogenesis and normal bone formation rates.35,36 These histomorphometric studies are also supported by clinical data in
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MM patients with bone disease, which have shown that patients have low levels of bone formation markers, such as alkaline phosphatase and osteocalcin37, and that bone scans underestimated the extent of MM bone disease. Bone scans reflect new bone formation. These data suggest that MM cells suppress osteoblast activity and inhibit bone formation, which plays a critical role in the pathogenesis of myeloma bone disease. In the last few years signaling pathways involved in osteoblastic function have been elucidated, and their perturbations in myeloma have provided a better understanding of the pathophysiology of myeloma bone disease. In addition, these studies have identified several potential therapeutic targets for treating MM bone disease. Runx2/Cbfal transcription factor The formation and differentiation of osteoblasts from mesenchymal cells require the activity and function of the transcription factor Runx2/Cbfal.38–42 Runx2/Cbfaldeficient mice (Runx2) completely lack osteoblasts and bone formation.38–42 Human osteoblast differentiation is associated with increased Runx2/Cbfal activity without a change in Runx2 protein levels, although it has been reported that Runx2/Cbfal over-expression can also impair bone formation. These results indicate that a timedependent expression of Runx2 drives osteoblast differentiation and plays a critical role in this process. Multiple pathways converge to interact with Runx2/Cbfal and regulate osteoblast differentiation, including binding with AP-138,40,42, but Runx2/Cbfal itself is regulated by phosphorylation. The potential role of inhibition of Runx2/Cbfal in MM bone disease has recently begun to be elucidated.43 When MM cells were co-cultured with osteoprogenitor cells, the MM cells inhibited osteoblast differentiation and reduced numbers of both early osteoblast precursors, the colony-forming-unit fibroblast (CFU-F), and the more differentiated precursors, the colony-forming-unit osteoblast (CFU-OB).43 Interestingly, this effect was mediated by blocking Runx2/Cbfal activity in osteoprogenitor cells. In addition, since Runx2/Cbfal stimulated secretion of the RANKL decoy receptor – OPG in osteoprogenitor cells44 – it is possible that inhibition of Runx2/Cbfal activity also increases osteoclastogenesis. The interaction between Runx2/Cbfal and MM cells appears to be mediated by cell–cell interaction between MM cells and osteoprogenitors. This cell–cell interaction is dependent on VLA-4 on MM cells and VCAM-1 on osteoblast precursors, since neutralizing anti-VLA-4 antibodies reduces the inhibitory effect of MM cells on Runx2/Cbfal activity.43 In addition to VLA-4 and VCAM-1, other adhesion molecules also appear to play a role in the inhibition of osteoblasts in MM. NCAM–NCAM interactions between myeloma and stromal/ osteoblastic cells can decrease bone matrix production by osteoblasts and may further contribute to the development of bone lesions in myeloma.45,46 Interleukin 3 IL-3 appears to play a dual role in the bone-destructive process in myeloma. It can stimulate osteoclast formation and therefore bone resorption, and can also indirectly inhibit osteoblast formation. Ehrlich et al demonstrated that treatment of primary mouse or human marrow stromal cells with IL-3 inhibited bone morphogenicprotein-2- (BMP-2)-stimulated osteoblast formation in a dose-dependent manner. Further, marrow plasma from myeloma patients that expressed high IL-3 levels inhibited
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osteoblast differentiation, which was promptly reversed by the addition of anti-IL-3 antibody. Interestingly, IL-3 did not affect the differentiation of osteoblast-like cell lines.47 Inhibition of osteoblast differentiation by IL-3 required the presence of CD45þ cells. On depleting the cell cultures of CD45þ cell, IL-3 did not inhibit osteoblast differentiation. Moreover, the reconstitution of the cultures with CD45þ cells restored the capacity of IL-3 to block osteoblast differentiation. Interleukin 7 IL-7 is one of the several soluble factors that have also been demonstrated to be active in osteoblast inhibition in MM. Giuliani and co-workers reported that IL-7 levels are increased in marrow plasma samples from patients with MM compared to those from normal controls. IL-7 is a very potent inhibitor of osteoblast differentiation, inhibiting differentiation of both early osteoblast precursors CFU-F and more differentiated osteoblast precursors CFU-OB.43 IL-7 can affect osteoblast formation in several ways, including interfering with Runx2 activity.43,48,49 Anti-IL-7 antibody reverses the inhibition of human osteoblast differentiation by myeloma cell lines and primary myeloma cells, as well as the inhibition of bone formation. Therefore, IL-7 appears to be a potential target for future therapies focusing on reversing osteoblast differentiation/suppression in MM. Wnt signaling pathway inhibitors in MM bone disease The Wnt signaling pathway plays an important role in regulation of B-cell and plasmacell motility50 in addition to skeletogenesis. The first link between Wnt signaling and human bone disease came from the observations that inactivating mutations in the Wnt co-receptor, LRP-5, causes the osteoporosis–pseudoglioma syndrome (OPPG).51 Many Wnt effects are mediated by the downstream effector b-catenin, which has a pivotal role in the canonical Wnt pathway. In the absence of Wnt signaling, there is an accumulation of cytoplasmic b-catenin (which normally translocates into the nucleus) where – in conjunction with other transcription factors such as TCf/LEF family members – it promotes the activation of target genes involved in cell-cycle progression, differentiation, and regulation of membrane structure and cell shape.50 As demonstrated by Westendorf and co-workers in murine systems, it appears that Wnt signaling promotes the proliferation, expansion and survival of precursor and immature osteoblast cells.52 BMP-2 and other morphogenic proteins have been reported to induce osteoblast differentiation of murine mesenchymal stem cells by stimulating the Wnt signaling pathway through modulation of Wnt stimulators and inhibitors.52,53 There are several molecules produced by murine osteoblasts which function as soluble inhibitors of canonical Wnt pathway, e.g. Dickkopf (DKK-1), secreted frizzledrelated proteins (sFRP), Wnt inhibitor factor (Wif-1). The importance of DKK-1 in normal skeletal development has been demonstrated by the presence of extra digits in DKK-1 null mice and loss of bony structures in chickens and mice exposed to high levels of DKK-1.54,55 Morvan and colleagues showed that mice lacking a single allele of DKK-1 have a marked increase in bone mass.56 In contrast, transgenic over-expression of DKK-1 caused severe osteopenia.57 Tian and co-workers reported the production of DKK-1 by primary CD138þ MM cells but not by plasma cells from MGUS patients, and also demonstrated that levels of DKK-1 mRNA correlated with focal bone lesions in patients with myeloma.58,59
Pathophysiology of myeloma bone disease 619
In contrast, patients with advanced disease, as well as some human myeloma cell lines, did not express DKK-1, suggesting that such inhibition may mediate bone destruction only in the early phases of disease.58 However, the mechanism by which DKK-1 production by MM cells relates to bone disease is still unclear. Anti-DKK1 antibody neutralized the inhibitory effects of bone-marrow plasma from MM patients on BMP-2-induced alkaline phosphatase expression and osteoblast formation by a murine mesenchymal cell line, but failed to block the inhibitory effects of MM cells on human marrow osteoblast formation.43 Furthermore, only very high levels of DKK-1 seemed capable of inhibiting CFU-F and CFU-OB formation as well as blocking b-catenin signaling in human bone marrow osteoprogenitor cells.43 In addition to osteoblastogenesis inhibition, elevated DKK-1 levels also appear to enhance osteoclastogenesis. Wnt signaling in osteoblasts up-regulates expression of OPG60 and down-regulates the expression of RANKL61, suggesting a possible mechanism by which inhibition of Wnt signaling in osteoblasts would indirectly increase osteoclastogenesis. Taken together, these studies indicate that DKK-1 is a key regulator of bone remodeling in both physiological and pathological conditions, and that blocking this factor may contribute to both stimulation of osteoblastogenesis and inhibition of osteoclasts in myelomatous bones. MM cells may also produce other Wnt inhibitors such as sFRP-3/FRZB. Oshima and colleagues62 reported that myeloma cells suppressed bone formation by secreting sFRP-2. Conditioned media from MM cell lines and primary myeloma blocked in-vitro mineralization and alkaline phosphatase activity induced by BMP-2 treatment of osteoblasts. The authors also found that most MM cells from patients with advanced disease expressed sFRPs, and that if conditioned media were depleted of sFRP the media no longer inhibited osteoblast differentiation. However, as with DKK-1, other studies did not show increased levels of sFRP-2, sFRP-1 and sFRP-4 in myeloma.43 In summary, our understanding of the role of inhibitors of the Wnt signaling pathway in myeloma bone disease is still evolving, and further investigation is required to determine the role of such inhibitors in patients. Other factors Other factors are also involved in the inhibition of mature osteoblastic cells and bone formation. Myeloma cells inhibit osteoblast proliferation18 and up-regulate osteoblast apoptosis. Osteoblasts from patients with advanced myeloma are more prone to undergo apoptosis when compared to myeloma patients without bone lesions.63 In addition to killing osteoblasts, human MM cells also sensitize osteoblastic cells to cell death mediated by recombinant TRAIL (tumor-necrosis-factor-related apoptosis-inducing ligand). TARGETING BONE DISEASE: CURRENT RECOMMENDATIONS AND FUTURE DIRECTIONS Forty-five per cent of patients with myeloma suffer a fracture within the first year of diagnosis, and 65% will have a fracture over the course of their disease. Anti-resorptive therapy with intravenous bisphosphonates (pamidronate, ibandronate, zolendronic acid) has become the cornerstone therapy for multiple myeloma bone disease as well as for other cancer-related bone metastases. By inhibiting osteoclast formation and inducing osteoclast apoptosis, intravenous bisphosphonates have
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reduced the number of SREs (skeletal related events: fractures, hypercalcemia, surgery to bone and radiation to bone) by 50% over the first 9 months of treatment.64 Comparable results were obtained with different bisphosphonates if administered on a monthly schedule. Toxicities of such therapy, albeit uncommon, have been reported. More frequently, renal failure and proteinuria can affect patients. Osteonecrosis of the jaw (ONJ) appears to occur in 4–6% of patients with myeloma treated with intravenous bisphosphonates and can be an extremely morbid condition. This complication may increase with the use of more potent bisphosphonates and prolonged use of these drugs. Neither the mechanism responsible for ONJ nor a cause-and-effect relationship linking bisphosphonate therapy per se with ONJ has been clearly demonstrated to date. Thus, many questions persist about the most appropriate length of treatment with bisphosphonates after the initial year of therapy, and several groups have developed guidelines to address this question.65 Currently, there is an ongoing randomized prospective trial with zolendronic acid in patients with myeloma at standard schedule for 9–12 months, and then the patients are either treated monthly or every 3 months with bisphosphonates to determine the safety, efficacy and toxicity of these drugs given at these two different schedules. Recently, a fully humanized monoclonal antibody to RANKL (Denosumab, Amgen) has been developed. This antibody induces rapid reduction of bone resorption markers in patients, which persist for up to 90 days after therapy.66 This antibody is highly specific for RANKL and does not bind to other members of the TNF-a superfamily. Denosumab is currently in clinical trial in myeloma as well as in other diseases associated with osteoclastic bone destruction. Administration of a single dose of recombinant OPG, which targets the same pathway, has shown a significant decrease in bone resorption markers for up to a month. Because of concerns of developing antiOPG antibodies, this drug has not been brought further into clinical development. Recent discoveries inn the pathogenesis of myeloma bone disease have identified several additional drug targets for treating this process. Inhibition of p38 MAP kinase pathway has a profound effect on osteoclast formation.67,68 A recent phase-II clinical trial using a highly specific MAP kinase inhibitor, SCIO-469 – alone or combined with bortezomib – has just finished, and results are currently pending. Bortezomib, a proteasome antagonist, appears to have excellent effects which are not limited to myeloma but occur also in myeloma bone disease. Several studies have reported that patients on bortezomib have increased serum markers of bone formation, though it remains unclear whether this new bone formation occurs at sites involved with myeloma or at sites that are not involved with disease.69,70 Proteasome-antagonistmediated bone formation has also been shown to occur through the increased production of BMP-2 in preclinical models.71 Additionally, Terpos and colleagues reported that patients receiving bortezomib exhibit increased levels of OPG and decreased levels of RANKL, with a net positive effect on bone formation. Unfortunately, healing of previous lytic lesions has not been observed with bortezomib therapy.72 Supplementary studies are required to assess whether proteasome antagonists, in addition to being anti-myeloma drugs, can also augment bone formation. MIP-1a with its effects on bone resorption may be a key therapeutic target in myeloma bone disease. MIP-1a can either induce osteoclast formation or up-regulate RANKL expression via different chemokine receptors such as CCR1 and CCR5. By blocking these receptors, one should block MIP-1a-mediated osteoclast formation.73 Moreover, MIP-1a can also enhance the growth of MM cells through the CCR1 receptor, as reported by Lentzsch et al.74 Anti-CCR1 agents should be excellent therapeutic agents for clinical trials in the near future.
Pathophysiology of myeloma bone disease 621
Anabolic agents such as PTH may also be potential treatments for myeloma bone disease. Pennisi et al75 reported a 20% increase in bone density and an inhibition of myeloma-cell growth in SCID-rab mice engrafted with human myeloma cells and treated with PTH. However, there is a theoretical concern with PTH treatment. By stimulating osteoblast formation in myeloma bone lesions, there could be increased production of cytokines that increase myeloma tumor burden. Clinical trials with anabolic agents in myeloma are yet to be reported, and their impact on bone formation should be analyzed carefully. Most myeloma patients receive bisphosphonates, which will likely lessen the anabolic effects of PTH.75 Toxic effects of PTH therapy – such as hypercalcemia, nephrolithiasis with renal function compromise – could also pose obstacles in treating myeloma patients with such agents. SUMMARY Recent advances in the pathophysiology underlying multiple myeloma bone disease have identified several novel therapeutic targets such as CCR1, IL-3, IL-7, and Wnt inhibitors. As the understanding of the biology of myeloma bone disease continues to expand, increasing numbers of new potential therapies should emerge. These new agents should be entering clinical trial in the near future and should provide important insights into their utility in ameliorating this devastating complication of myeloma, and in extending survival of patients as well as improving their quality of life. REFERENCES 1. Roodman GD. Cell Biology of the osteoclast. Experimental Hematology 1999; 27: 1229–1241. *2. Roodman GD. Treatment strategies for bone disease. Bone Marrow Transplantation 2007 Aug 6. [Epub ahead of print]. 3. Gunn WG, Conley A, Deininger L et al. A crosstalk between myeloma cells and marrow stromal cells stimulates production of DKK1 and interleukin-6: a potential role in the development of lytic bone disease and tumor progression in multiple myeloma. Stem Cells 2006; 24: 986–991. 4. Giuliani N, Colla S & Rizzoli V. New insight in the mechanism of osteoclast activation and formation in multiple myeloma: focus on the receptor activator NF-Kappa-B ligand (RANKL). Experimental Hematology 2004; 32: 685–691. 5. Choi SJ, Cruz JC, Craig F et al. Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood 2000; 96: 671–675. *6. Lee JW, Chung HY, Ehrlich LA et al. IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells. Blood 2004; 103: 2308–2315. 7. Hsu H, Lacey DL, Dunstan CR et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proceedings of the National Academy of Sciences of the United States of America 1999; 96: 3540–3545. 8. Nakagava N, Kinosaki M, Yamaguchi K et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochemical and Biophysical Research Communications 1998; 253: 395–400. *9. Boyle WJ, Simonet WS & Lacey DL. Osteoclast differentiation and activation. Nature 2003; 423: 337–342. 10. Yasuda H, Shima N, Nakagawa N et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proceedings of the National Academy of Sciences of the United States of America 1998; 95: 3597–3602. 11. Hofbauer LC & Heufelder AE. Osteoprotegerin and its cognate ligand: a new paradigm of osteoclastogenesis. European Journal of Endocrinology 1998; 139: 152–154. 12. Tsukii K, Shima N, Mochizuki S et al. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid
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hormone in the microenvironment of bone. Biochemical and Biophysical Research Communications 1998; 246: 337–341. Dougall WC, Glaccum M, Charrier K et al. RANK is essential for osteoclast and lymph node development. Genes & Development 1999; 13: 2412–2424. Lacey DL, Timms E, Tan HL et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165–176. Simonet WS, Lacey DL, Dunstan CR et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997; 89: 309–319. Bucay N, Sarosi I, Dunstan CR et al. Osteoprotegerin deficient mice develop early onset osteoporosis and arterial calcification. Genes & Development 1998; 12: 1260–1268. Li J, Sarosi I, Yan XQ et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proceedings of the National Academy of Sciences of the United States of America 2000; 97: 1556–1571. Pearse RN, Sordillo EM, Yaccoby S et al. Multiple myeloma disrupts the TRANCE/osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression. Proceedings of the National Academy of Sciences of the United States of America 2001; 98: 11581–11586. Terpos E, Szydlo R, Apperley JF et al. Soluble receptor activator of nuclear factor kappaB ligand- osteoprotegerin ratio predicts survival in multiple myeloma: proposal for a novel prognostic index. Blood 2003; 102: 1064–1069. Yaccoby S, Pearse RN, Johnson CL et al. Myeloma interacts with the bone marrow microenvironment to induce osteoclastogenesis and is dependent on osteoclast activity. British Journal of Haematology 2002; 116: 278–290. Han JH, Choi SJ, Kurihara N et al. Macrophage inflammatory protein-1 alpha is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kappaB ligand. Blood 2001; 97: 3349–3353. Magrangeas F, Nasser V, Avet-Loiseau H et al. Gene expression profiling of multiple myeloma reveals molecular portraits in relation to the pathogenesis of the disease. Blood 2003; 101: 4998–5005. Hashimoto T, Abe M, Oshima T et al. Ability of myeloma cells to secrete macrophage inflammatory protein (MIP)-1 alpha and MIP-1beta correlates with lytic bone lesions in patients with multiple myeloma. British Journal of Haematology 2004; 125: 38–41. Alsina M, Boyce B, Devlin RD et al. Development of an in vivo model of human multiple myeloma bone disease. Blood 1996; 87: 1495–1501. Choi SJ, Oba Y, Gazitt Y et al. Antisense inhibition of macrophage inflammatory protein 1 alpha block bone destruction in a model of myeloma bone disease. The Journal of Clinical Investigation 2001; 108: 1833–1841. Masih-Khan E, Trudel S, Heise C et al. MIP-1a (CCL3) is a downstream target of FGFR3 and RAS-MAPK signaling in multiple myeloma. Blood 2006; 108: 3465–3471. Ehrlich LA, Chung HY, Ghobrial I et al. IL-3 is a potential inhibitor of osteoblast differentiation in multiple myeloma. Blood 2005; 106: 1407–1414. Solary E, Guiguet M, Zeller V et al. Radioimmunoassay for the measurement of serum IL-6 and its correlation with tumour cell mass parameters in multiple myeloma. American Journal of Hematology 1992; 39: 163–171. Sai HI, Apperley JF, Graves M et al. Interleukin -6 is expressed by plasma cells from patients with multiple myeloma and monoclonal gammopathy of unknown significance. British Journal of Haematology 1998; 101: 287–295. Karadag A, Oyajobi BO, Apperley JF et al. Human myeloma cells promote the production of interleukin-6 by primary human osteoblasts. British Journal of Haematology 2000; 108: 383–390. Abe M, Hiura K, Wilde J et al. Osteoclasts enhance myeloma cell growth and survival via cell to cell contact: a vicious cycle between bone destruction and myeloma expansion. Blood 2004; 104: 2484–2491. Nguyen AN, Sttebbins EG, Henson M et al. Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation. Experimental Cell Research 2006; 312: 1909–1923. Vanderkerken K, Medicherla S, Coulton L et al. Inhibition of p38a MAPK reduces tumor burden, prevents the development of myeloma bone disease, and increases survival in the 5T2 and 5T3 murine models of myeloma. Blood 2006; 108: 981a. [abstract 3436].
Pathophysiology of myeloma bone disease 623 34. Kurihara N, Hiruma Y, Hong CS et al. targeting p62 in marrow stromal cells is effective at inhibiting myeloma cell growth. Blood 2006; 108: 155a. [abstract 513]. 35. Bataille R, Chappard D, Marcelli C et al. Mechanisms of bone destruction in multiple myeloma: the importance of an unbalanced process in determining the severity of lytic bone disease. Journal of Clinical Oncology 1989; 7: 1909–1914. 36. Bataille R, Chappard D, Marcelli C et al. Osteoblast stimulation in multiple myeloma lacking bone lytic lesions. British Journal of Haematology 1990; 76: 484–487. 37. Hjorth-Hansen H, Seifert MF, Borset M et al. Marked osteoblastopenia and reduced bone formation in a model of multiple myeloma bone disease in severe combined immunodeficiency mice. Journal of Bone and Mineral Research 1999; 14: 256–263. *38. Kobayashi T & Kronenberg H. Mini review: transcriptional regulation in development of bone. Endocrinology 2005; 146: 1012–1017. 39. Ducy P, Zhang R, Geoffroy V et al. Osf2/Cbfal: a transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747–754. 40. Franceschi RT & Xiao G. Regulation of the osteoblast specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. Journal of Cellular Biochemistry 2003; 88: 446–454. 41. Karsenty G, Ducy P, Starbuck M et al. Cbfal as a regulator of osteoblast differentiation and function. Bone 1999; 25: 107–108. 42. Komori T. Runx2, a multifunctional transcription factor in skeletal development. Journal of Cellular Biochemistry 2002; 87: 1–8. *43. Giuliani N, Colla S, Morandi F et al. Myeloma cells block RUNX2/CBFAL1 activity in human bone marrow osteoblast progenitors and inhibit osteoblast formation and differentiation. Blood 2005; 106: 2472–2483. 44. Thirunavukkarasu K, Halladay DL, Miles RR et al. The osteoblast-specific transcription factor Cbfal contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function. The Journal of Biological Chemistry 2000; 275: 25163–25172. 45. Barille S, Collette M, Bataille R et al. Myeloma cells upregulate interleukin-6 secretion in osteoblastic cells through cell to cell contact but downregulate osteocalcin. Blood 1995; 86: 3151–3159. 46. Ely SA & Knowles DM. Expression of CD56/neural cell adhesion molecule correlates with the presence of lytic bone lesions in multiple myeloma and distinguishes myeloma from monoclonal gammopathy of undetermined significance and lymphomas with plasmacytoid differentiation. The American Journal of Pathology 2002; 160: 1293–1299. 47. Roodman GD. New Potential targets for treating myeloma bone disease. Clinical Cancer Research 2006; 12: 6270s–6273s. 48. Lee SK, Kalinowski JF, Jacquin C et al. Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss. Journal of Bone and Mineral Research 2006; 21: 695–702. 49. Toraldo G, Roggia C, Qian WP et al. IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor kappaB ligand and tumor necrosis factor alpha from T cells. Proceedings of the National Academy of Sciences of the United States of America 2003; 100: 125–130. *50. Qiang Y-W, Endo Y, Rubin JS et al. Wnt signaling in B cell neoplasia. Oncogene 2003; 22: 1536–1545. 51. Gong Y, Slee RB, Fukai N et al. LDL receptor-related protein (LRP5) affects bone accrual and eye development. Cell 2001; 107: 513–523. 52. Westerndorf JJ, Kahler RA & Schroeder TM. Wnt signaling in osteoblasts and bone diseases. Gene 2004; 341: 19–39. 53. Rawadi G, Vayssiere B, Dunn F et al. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. Journal of Bone and Mineral Research 2003; 18: 1842–1853. 54. Mukhopadhyay M, Shtrom S, Rodriguez-Esteban C et al. Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse. Developmental Cell 2001; 1: 423–434. 55. Grotewold L & Ruther U. The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulated programmed cell death. The EMBO Journal 2002; 21: 966–975. 56. Morvan F, Boulukos K, Clement-Lacroix P et al. Deletion of a single allele of the Dkk-1 gene leads to an increase in bone formation and bone mass. Journal of Bone and Mineral Research 2006; 21: 934–954. 57. Li J, Sarosi I, Cattley RC et al. Dkk-1 mediated inhibition of Wnt signaling in bone results in osteopenia. Bone 2006; 39: 754–766.
624 F. R. Esteve and G. D. Roodman *58. Tian E, Zhan F, Walker R et al. The role of Wnt signaling antagonist DKK-1 in the development of osteolytic lesions in multiple myeloma. The New England Journal of Medicine 2003; 349: 2483–2494. 59. Politou MC, Health DJ, Rahemtulla A et al. Serum concentrations of Dickkopf-1 protein are increased in patients with multiple myeloma and reduced after autologous stem cell transplantation. International Journal of Cancer 2006; 119: 1728–1731. 60. Glass DA, Bialek P, Ahn JD et al. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Developmental Cell 2005; 8: 751–764. 61. Spencer GJ, Utting JC, Etheridge SL et al. Wnt signaling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. Journal of Cell Science 2006; 119: 1283–1296. 62. Oshima T, Abe M, Asano J et al. Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor sFRP-2. Blood 2005; 106: 3160–3165. 63. Oyajobi BO & Mundy GR. Receptor activator of NF-kappaB ligand, macrophage inflammatory protein 1-alpha and the proteasome. Cancer 2003; 97: 813–817. *64. Berenson JR, Lichtenstein A, Porter L et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. Myeloma Aredia Study Group. The New England Journal of Medicine 1996; 334: 448–493. 65. Lacy MQ, Dispenzieri A, Gertz MA et al. mayo clinic consensus statement for the use of bisphosphonates in multiple myeloma. Mayo Clinic proceedings 2006; 81: 1047–1053. 66. Body JJ, Facon T, Coleman RE et al. A study of the biological receptor activator of nuclear factor kappaB ligand inhibitor, denosumab, in patients with multiple myeloma or bone metastases from breast cancer. Clinical Cancer Research 2006; 12: 1221–1228. 67. Mbalaviele G, Anderson G, Jones A et al. Inhibition of p38 mitogen-activated protein kinase prevents inflammatory bone destruction. The Journal of Pharmacology and Experimental Therapeutics 2006; 317: 1044–1053. 68. Rossa C, Ehmann K, Liu M et al. MKK3/6-p38 MAPK signaling is required for IL-1 beta and TNF alpha induced RANKL expression in bone marrow stromal cells. Journal of Interferon & Cytokine Research 2006; 26: 719–729. 69. Zangari M, Yaccoby S, Cavallo F et al. Response to bortezomib and activation of osteoblasts in multiple myeloma. Clinical Lymphoma & Myeloma 2006; 7: 109–114. 70. Heider U, Kaiser M, Muller C et al. Bortezomib increases osteoblast activity in myeloma patients irrespective of response to treatment. European Journal of Haematology 2006; 77: 233–238. *71. Garrett IR, Chen D, Gutierrez G et al. Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. The Journal of Clinical Investigation 2003; 111: 1117–1182. 72. Terpos E, Heath D, Rahemtulla A et al. Bortezomib reduces serum dicckopf-1 and RANKL concentrations and normalizes indices of bone remodeling in patients with relapsed multiple myeloma. Blood 2006; 108: 153a. [abstract 506]. 73. Oba Y, Lee JW, Ehrlich LA et al. MIP-1 alpha utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells. Experimental Hematology 2005; 3: 272–278. 74. Lentzsch S, Gries M, Janz M et al. Macrophage inflammatory protein 1-alpha triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma cells. Blood 2003; 101: 3568–3573. 75. Pennisi A, Ling W, Perkins P et al. PTH and Bortezomib suppress growth of primary human myeloma cells through increased bone formation in vivo. Blood 2006; 108: 154a. [abstract 509].
3 Pathophysiology of myeloma bone disease Flavia R. Esteve
MD
Hematology Fellow University of Pittsburgh, Medicine/Hematology-Oncology, 5150 Centre Avenue, Pittsburgh, PA 15213, USA
G. David Roodman *
MD, PhD
Professor of Medicine University of Pittsburgh, Medicine/Hematology-Oncology, VA Pittsburgh Healthcare System R&D (151-U), University Drive C, Pittsburgh, PA 15240, USA
Multiple myeloma is a tumor of terminally differentiated plasma cells that home to and expand in the bone marrow. It is the second most common hematologic malignancy, with approximately 16,000 new cases per year, and accounts for an estimated 11,000 deaths in the USA. It is the most common cancer to metastasize to bone, with up to 90% of patients developing bone lesions. The bone lesions are purely osteolytic in nature, and up to 60% of patients develop a pathologic fracture over the course of their disease. Bone disease is a hallmark of multiple myeloma, and the bone disease differs from other bone metastasis caused by other tumors. Although both myeloma and other osteolytic metastasis induce increased osteoclastic bone resorption, in contrast to other tumors, osteoblast activity in myeloma is either severely decreased or absent. The basis for this severe imbalance between increased osteoclastic bone resorption and decreased bone formation resulting from suppressed osteoblastic activity has been a topic of extensive investigation during the last several years. The clinical consequences of this extensive accelerated and imbalanced bone destruction process include bone pain, pathologic fractures, hypercalcemia and spinal cord compression syndromes, which can be devastating for patients and significantly impact overall quality of life and expected survival. In this chapter, we will discuss the pathophysiology underlying bone disease in myeloma. This results from the uncoupling of bone remodeling and is characterized by markedly increased activity of osteoclasts and profound decreased activity of osteoblasts. In addition, we also review the emerging data on novel targeted therapies aimed at ameliorating myeloma bone disease. Key words: myeloma; metastasis; osteoclasts; bone destruction.
* Corresponding author. Tel.: þ1-412-688-6571; Fax: þ1-412-688-6960. E-mail address: [email protected] 1521-6926/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
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OSTEOLYTIC BONE DESTRUCTION IN MYELOMA BONE DISEASE Normal bone constantly undergoes remodeling, which involves the resorption of bone by osteoclasts and the deposition of new bone by osteoblasts at sites of previous resorption. Osteoclasts arise from precursor cells in the monocyte/macrophage lineage1 that differentiate into inactive osteoclasts. The inactive osteoclasts are then activated, resorb bone, and subsequently undergo apoptosis. Both locally produced cytokines and systemic hormones normally regulate osteoclast formation and activity. In myeloma, many different components interact to increase the bone-resorptive process. These include myeloma cells themselves, bone-marrow stromal cells, and T cells present in the marrow microenvironment. Furthermore, growth factors released by the bone-resorptive process2 also increase the growth of myeloma cells. This creates a vicious cycle, with the bone-resorptive process increasing myelomacell tumor burden, which then results in increased bone resorption. Recent studies have identified several important factors produced by myeloma cells in vivo that have been implicated in the osteolytic bone-resorptive process. These include the receptor activator nuclear factor kB (NF-kB, RANKL), macrophage inflammatory peptide 1a (MIP-1a), interleukin 3 (IL-3) and IL-6.3–6 The RANK/RANKL signaling pathway is a critical component of the bone-remodeling process. RANK is a transmembrane signaling receptor which is a member of the tumor necrosis factor (TNF) receptor superfamily. It is found on the surface of osteoclast precursors.7,8 RANK ligand (RANKL) is expressed as a membrane-bound protein on marrow stromal cells and osteoblasts, and secreted by activated lymphocyte. Its expression is augmented by cytokines that stimulate bone resorption9, such as parathyroid hormone (PTH), 1,25-OH vitamin D3, and prostaglandins.10,11 RANKL binds to RANK receptor on osteoclast precursors and induces osteoclast formation. Rank signals through the NF-kB and JunN terminal kinase pathways and induces increased osteoclastic bone resorption and enhanced osteoclast survival.2 The important role of RANKL in normal osteoclastogenesis has been clearly demonstrated in RANKL or RANK gene knockout mice. These animals lack osteoclasts and as a result develop severe osteopetrosis.12,13 Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and is a member of the TNF receptor superfamily.14 It is produced by osteoblasts, as well as other cell types, and blocks the interactions of RANKL with RANK, thereby limiting osteoclastogenesis. In normal subjects, the ratio RANKL/OPG strongly favors OPG. The importance of OPG has also been shown in studies using knockout mice for the OPG gene; these mice develop severe osteopenia and osteoporosis.13–17 Pearse and co-workers were the first to demonstrate that in bone-marrow biopsies of multiple myeloma (MM) patients RANKL expression was up-regulated while OPG expression was decreased.18 Terpos et al showed that circulating levels of OPG and RANKL correlated with clinical activity of myeloma, severity of bone disease, and poor prognosis.19 Furthermore, murine myeloma models have also determined that inhibition of RANKL can prevent bone destruction in either the SCID-hu model or the T2 MM syngeneic model of myeloma.19,20 These studies revealed that blocking RANKL, either with a soluble form of the RANK receptor or by OPG, minimizes bone destruction and tumor burden. In addition, myeloma cells have been reported to express RANKL, which may further contribute to the bone-destructive process (Figure 1). MIP-1a is a chemokine that is produced by MM cells in 70% of MM patients; it is a potent inducer of human osteoclast formation. MIP-1a can increase osteoclast
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Figure 1. Myeloma cells and T cells secrete RANKL to induce osteoclast (OCL) formation. In addition, multiple myeloma (MM) cells suppress osteoprotegerin (OPG) in the marrow microenvironment. This results in a severe imbalance in the normal RANKL/OPG rates driving osteoclastogenesis.
formation independently of RANKL and can potentiate both RANKL and IL-6 stimulated osteoclast formation.21 Magrangeas et al have shown by gene expression profiling (GEP) that MIP-1a is the gene most highly correlated with bone destruction in myeloma.22 Further, Abe and co-workers have shown that elevated levels of MIP-1a also correlate with an extremely poor prognosis in myeloma.23 In-vivo models of myeloma have demonstrated that MIP-1a can induce osteoclast formation and bone destruction. Blocking MIP-1a expression in myeloma cells injected into severe combined immunodeficient (SCID) mice, or treating the animals with a neutralizing antibody to MIP-1a, results in a decreased tumor burden and bone destruction.24,25 MIP-1a also plays an important role in the homing of myeloma cells to the bone marrow. MIP-1a increases adhesive interactions between myeloma cells and marrow stromal cells by increasing expression of b1 integrins, which takes place through a4b1 or a5b1 integrins and adhesive molecules such as vascular cell adhesion molecule 1 (VCAM-1). This results in production of RANKL, IL-6, vascular endothelial growth factor (VEGF) and TNFa by marrow stromal cells, which further enhances myeloma-cell growth, angiogenesis and bone destruction. Further, Masih-Khan et al reported that the t4:14 translocation results in a constitutive expression of the FGFR3 receptor, which results in high levels of MIP-1a.26 Patients with the t4:14 translocation have a very poor prognosis, which may reflect the increased MIP-1a production in this patient population. IL-3, in addition to RANKL and MIP-1a, is also significantly elevated in bonemarrow plasma of MM patients as compared to that of normal controls.6 IL-3 can induce osteoclast formation in human bone-marrow cultures at levels similar to those
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measured in myeloma patient samples, and osteoclast formation induced by marrow plasma from MM patients could be inhibited by using a blocking antibody to IL-3.6 IL-3 also indirectly influences osteoclastogenesis by enhancing the effects of RANKL and MIP-1a on the growth and development of osteoclasts. It also stimulates myeloma cell growth directly6 and, as discussed later in this chapter, IL-3 has also been shown to inhibit osteoblast formation through a factor produced by macrophages in the marrow microenvironment.27 IL-6 has long been recognized as a proliferative factor for plasma cells, but it is unclear whether IL-6 levels correlate with disease status.28 Levels of IL-6 are elevated in MM patients with osteolytic bone disease, when compared to MM patients without bone disease, as well as in patients with monoclonal gammopathy of unknown significance (MGUS).29 Most studies support the idea that IL-6 is produced by cells in the bone-marrow microenvironment rather than myeloma cells through direct contact with myeloma cells. These cells most likely are osteoclasts and stromal cells, but increased osteoblast production of IL-6 has also been reported in cocultures of human osteoblasts with MM cells.30 Although the precise role of IL-6 in myeloma bone disease is yet to be determined, IL-6 production by osteoclasts can increase tumor burden, leading to enhanced bone destruction as well as acting as an autocrine/ paracrine factor to increase osteoclast formation.31 Adhesive interactions between myeloma and stromal cells play a significant role in the homing of myeloma cells in the bone marrow and augmenting the bone-destructive process. These adhesive interactions result in increased signaling of NF-kB and p38 MAP kinase. The latter is involved in both osteoclastic growth and differentiation as well as induction of RANKL expression by osteoblasts. Blocking p38MAP kinase potently inhibits IL-6 and VEGF production as well as decreased adhesion of myeloma cells to marrow stromal cells.32 Recently, Vanderkerken and co-workers reported that inhibition of p38 MAP kinase in the 5T2 MM murine model of myeloma decreased tumor cell burden, prevented development of bone disease, and increased overall survival in mice having 5T2 cells.33 Therefore, this pathway may be a potential therapeutic target for novel therapies to ameliorate myeloma disease. P62 (sequestosome-1) is a recently described member of the NF-kB pathway. It mediates IL-1-, TNF- and RANKL-mediated NF-kB activation. It is up-regulated in the marrow microenvironment in patients with myeloma. Kurihara and colleagues have examined the role of p62 in myeloma bone disease and found that deleting p62 in marrow stromal cells decreased VCAM-1 expression and the capacity of the cells to support osteoclast formation, to increase myeloma cell growth, and to produce RANKL and TNF-a. They also found that TNF-a could reverse the effects of loss of p62 in co-cultures of MM and stromal cells through increased expression of VCAM-1.34 OSTEOBLASTIC DYSFUNCTION IN MYELOMA Histomorphometric studies of bone lesions from MM have provided important insights into the pathophysiology of myeloma bone disease. They disclosed that the bone-remodeling process was uncoupled, with increased bone resorption due to enhanced osteoclastogenesis and decreased or absent bone formation. In contrast, in either MM patients without bone disease or normal controls, there is balanced bone remodeling with increased osteoblastogenesis and normal bone formation rates.35,36 These histomorphometric studies are also supported by clinical data in
Pathophysiology of myeloma bone disease 617
MM patients with bone disease, which have shown that patients have low levels of bone formation markers, such as alkaline phosphatase and osteocalcin37, and that bone scans underestimated the extent of MM bone disease. Bone scans reflect new bone formation. These data suggest that MM cells suppress osteoblast activity and inhibit bone formation, which plays a critical role in the pathogenesis of myeloma bone disease. In the last few years signaling pathways involved in osteoblastic function have been elucidated, and their perturbations in myeloma have provided a better understanding of the pathophysiology of myeloma bone disease. In addition, these studies have identified several potential therapeutic targets for treating MM bone disease. Runx2/Cbfal transcription factor The formation and differentiation of osteoblasts from mesenchymal cells require the activity and function of the transcription factor Runx2/Cbfal.38–42 Runx2/Cbfaldeficient mice (Runx2) completely lack osteoblasts and bone formation.38–42 Human osteoblast differentiation is associated with increased Runx2/Cbfal activity without a change in Runx2 protein levels, although it has been reported that Runx2/Cbfal over-expression can also impair bone formation. These results indicate that a timedependent expression of Runx2 drives osteoblast differentiation and plays a critical role in this process. Multiple pathways converge to interact with Runx2/Cbfal and regulate osteoblast differentiation, including binding with AP-138,40,42, but Runx2/Cbfal itself is regulated by phosphorylation. The potential role of inhibition of Runx2/Cbfal in MM bone disease has recently begun to be elucidated.43 When MM cells were co-cultured with osteoprogenitor cells, the MM cells inhibited osteoblast differentiation and reduced numbers of both early osteoblast precursors, the colony-forming-unit fibroblast (CFU-F), and the more differentiated precursors, the colony-forming-unit osteoblast (CFU-OB).43 Interestingly, this effect was mediated by blocking Runx2/Cbfal activity in osteoprogenitor cells. In addition, since Runx2/Cbfal stimulated secretion of the RANKL decoy receptor – OPG in osteoprogenitor cells44 – it is possible that inhibition of Runx2/Cbfal activity also increases osteoclastogenesis. The interaction between Runx2/Cbfal and MM cells appears to be mediated by cell–cell interaction between MM cells and osteoprogenitors. This cell–cell interaction is dependent on VLA-4 on MM cells and VCAM-1 on osteoblast precursors, since neutralizing anti-VLA-4 antibodies reduces the inhibitory effect of MM cells on Runx2/Cbfal activity.43 In addition to VLA-4 and VCAM-1, other adhesion molecules also appear to play a role in the inhibition of osteoblasts in MM. NCAM–NCAM interactions between myeloma and stromal/ osteoblastic cells can decrease bone matrix production by osteoblasts and may further contribute to the development of bone lesions in myeloma.45,46 Interleukin 3 IL-3 appears to play a dual role in the bone-destructive process in myeloma. It can stimulate osteoclast formation and therefore bone resorption, and can also indirectly inhibit osteoblast formation. Ehrlich et al demonstrated that treatment of primary mouse or human marrow stromal cells with IL-3 inhibited bone morphogenicprotein-2- (BMP-2)-stimulated osteoblast formation in a dose-dependent manner. Further, marrow plasma from myeloma patients that expressed high IL-3 levels inhibited
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osteoblast differentiation, which was promptly reversed by the addition of anti-IL-3 antibody. Interestingly, IL-3 did not affect the differentiation of osteoblast-like cell lines.47 Inhibition of osteoblast differentiation by IL-3 required the presence of CD45þ cells. On depleting the cell cultures of CD45þ cell, IL-3 did not inhibit osteoblast differentiation. Moreover, the reconstitution of the cultures with CD45þ cells restored the capacity of IL-3 to block osteoblast differentiation. Interleukin 7 IL-7 is one of the several soluble factors that have also been demonstrated to be active in osteoblast inhibition in MM. Giuliani and co-workers reported that IL-7 levels are increased in marrow plasma samples from patients with MM compared to those from normal controls. IL-7 is a very potent inhibitor of osteoblast differentiation, inhibiting differentiation of both early osteoblast precursors CFU-F and more differentiated osteoblast precursors CFU-OB.43 IL-7 can affect osteoblast formation in several ways, including interfering with Runx2 activity.43,48,49 Anti-IL-7 antibody reverses the inhibition of human osteoblast differentiation by myeloma cell lines and primary myeloma cells, as well as the inhibition of bone formation. Therefore, IL-7 appears to be a potential target for future therapies focusing on reversing osteoblast differentiation/suppression in MM. Wnt signaling pathway inhibitors in MM bone disease The Wnt signaling pathway plays an important role in regulation of B-cell and plasmacell motility50 in addition to skeletogenesis. The first link between Wnt signaling and human bone disease came from the observations that inactivating mutations in the Wnt co-receptor, LRP-5, causes the osteoporosis–pseudoglioma syndrome (OPPG).51 Many Wnt effects are mediated by the downstream effector b-catenin, which has a pivotal role in the canonical Wnt pathway. In the absence of Wnt signaling, there is an accumulation of cytoplasmic b-catenin (which normally translocates into the nucleus) where – in conjunction with other transcription factors such as TCf/LEF family members – it promotes the activation of target genes involved in cell-cycle progression, differentiation, and regulation of membrane structure and cell shape.50 As demonstrated by Westendorf and co-workers in murine systems, it appears that Wnt signaling promotes the proliferation, expansion and survival of precursor and immature osteoblast cells.52 BMP-2 and other morphogenic proteins have been reported to induce osteoblast differentiation of murine mesenchymal stem cells by stimulating the Wnt signaling pathway through modulation of Wnt stimulators and inhibitors.52,53 There are several molecules produced by murine osteoblasts which function as soluble inhibitors of canonical Wnt pathway, e.g. Dickkopf (DKK-1), secreted frizzledrelated proteins (sFRP), Wnt inhibitor factor (Wif-1). The importance of DKK-1 in normal skeletal development has been demonstrated by the presence of extra digits in DKK-1 null mice and loss of bony structures in chickens and mice exposed to high levels of DKK-1.54,55 Morvan and colleagues showed that mice lacking a single allele of DKK-1 have a marked increase in bone mass.56 In contrast, transgenic over-expression of DKK-1 caused severe osteopenia.57 Tian and co-workers reported the production of DKK-1 by primary CD138þ MM cells but not by plasma cells from MGUS patients, and also demonstrated that levels of DKK-1 mRNA correlated with focal bone lesions in patients with myeloma.58,59
Pathophysiology of myeloma bone disease 619
In contrast, patients with advanced disease, as well as some human myeloma cell lines, did not express DKK-1, suggesting that such inhibition may mediate bone destruction only in the early phases of disease.58 However, the mechanism by which DKK-1 production by MM cells relates to bone disease is still unclear. Anti-DKK1 antibody neutralized the inhibitory effects of bone-marrow plasma from MM patients on BMP-2-induced alkaline phosphatase expression and osteoblast formation by a murine mesenchymal cell line, but failed to block the inhibitory effects of MM cells on human marrow osteoblast formation.43 Furthermore, only very high levels of DKK-1 seemed capable of inhibiting CFU-F and CFU-OB formation as well as blocking b-catenin signaling in human bone marrow osteoprogenitor cells.43 In addition to osteoblastogenesis inhibition, elevated DKK-1 levels also appear to enhance osteoclastogenesis. Wnt signaling in osteoblasts up-regulates expression of OPG60 and down-regulates the expression of RANKL61, suggesting a possible mechanism by which inhibition of Wnt signaling in osteoblasts would indirectly increase osteoclastogenesis. Taken together, these studies indicate that DKK-1 is a key regulator of bone remodeling in both physiological and pathological conditions, and that blocking this factor may contribute to both stimulation of osteoblastogenesis and inhibition of osteoclasts in myelomatous bones. MM cells may also produce other Wnt inhibitors such as sFRP-3/FRZB. Oshima and colleagues62 reported that myeloma cells suppressed bone formation by secreting sFRP-2. Conditioned media from MM cell lines and primary myeloma blocked in-vitro mineralization and alkaline phosphatase activity induced by BMP-2 treatment of osteoblasts. The authors also found that most MM cells from patients with advanced disease expressed sFRPs, and that if conditioned media were depleted of sFRP the media no longer inhibited osteoblast differentiation. However, as with DKK-1, other studies did not show increased levels of sFRP-2, sFRP-1 and sFRP-4 in myeloma.43 In summary, our understanding of the role of inhibitors of the Wnt signaling pathway in myeloma bone disease is still evolving, and further investigation is required to determine the role of such inhibitors in patients. Other factors Other factors are also involved in the inhibition of mature osteoblastic cells and bone formation. Myeloma cells inhibit osteoblast proliferation18 and up-regulate osteoblast apoptosis. Osteoblasts from patients with advanced myeloma are more prone to undergo apoptosis when compared to myeloma patients without bone lesions.63 In addition to killing osteoblasts, human MM cells also sensitize osteoblastic cells to cell death mediated by recombinant TRAIL (tumor-necrosis-factor-related apoptosis-inducing ligand). TARGETING BONE DISEASE: CURRENT RECOMMENDATIONS AND FUTURE DIRECTIONS Forty-five per cent of patients with myeloma suffer a fracture within the first year of diagnosis, and 65% will have a fracture over the course of their disease. Anti-resorptive therapy with intravenous bisphosphonates (pamidronate, ibandronate, zolendronic acid) has become the cornerstone therapy for multiple myeloma bone disease as well as for other cancer-related bone metastases. By inhibiting osteoclast formation and inducing osteoclast apoptosis, intravenous bisphosphonates have
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reduced the number of SREs (skeletal related events: fractures, hypercalcemia, surgery to bone and radiation to bone) by 50% over the first 9 months of treatment.64 Comparable results were obtained with different bisphosphonates if administered on a monthly schedule. Toxicities of such therapy, albeit uncommon, have been reported. More frequently, renal failure and proteinuria can affect patients. Osteonecrosis of the jaw (ONJ) appears to occur in 4–6% of patients with myeloma treated with intravenous bisphosphonates and can be an extremely morbid condition. This complication may increase with the use of more potent bisphosphonates and prolonged use of these drugs. Neither the mechanism responsible for ONJ nor a cause-and-effect relationship linking bisphosphonate therapy per se with ONJ has been clearly demonstrated to date. Thus, many questions persist about the most appropriate length of treatment with bisphosphonates after the initial year of therapy, and several groups have developed guidelines to address this question.65 Currently, there is an ongoing randomized prospective trial with zolendronic acid in patients with myeloma at standard schedule for 9–12 months, and then the patients are either treated monthly or every 3 months with bisphosphonates to determine the safety, efficacy and toxicity of these drugs given at these two different schedules. Recently, a fully humanized monoclonal antibody to RANKL (Denosumab, Amgen) has been developed. This antibody induces rapid reduction of bone resorption markers in patients, which persist for up to 90 days after therapy.66 This antibody is highly specific for RANKL and does not bind to other members of the TNF-a superfamily. Denosumab is currently in clinical trial in myeloma as well as in other diseases associated with osteoclastic bone destruction. Administration of a single dose of recombinant OPG, which targets the same pathway, has shown a significant decrease in bone resorption markers for up to a month. Because of concerns of developing antiOPG antibodies, this drug has not been brought further into clinical development. Recent discoveries inn the pathogenesis of myeloma bone disease have identified several additional drug targets for treating this process. Inhibition of p38 MAP kinase pathway has a profound effect on osteoclast formation.67,68 A recent phase-II clinical trial using a highly specific MAP kinase inhibitor, SCIO-469 – alone or combined with bortezomib – has just finished, and results are currently pending. Bortezomib, a proteasome antagonist, appears to have excellent effects which are not limited to myeloma but occur also in myeloma bone disease. Several studies have reported that patients on bortezomib have increased serum markers of bone formation, though it remains unclear whether this new bone formation occurs at sites involved with myeloma or at sites that are not involved with disease.69,70 Proteasome-antagonistmediated bone formation has also been shown to occur through the increased production of BMP-2 in preclinical models.71 Additionally, Terpos and colleagues reported that patients receiving bortezomib exhibit increased levels of OPG and decreased levels of RANKL, with a net positive effect on bone formation. Unfortunately, healing of previous lytic lesions has not been observed with bortezomib therapy.72 Supplementary studies are required to assess whether proteasome antagonists, in addition to being anti-myeloma drugs, can also augment bone formation. MIP-1a with its effects on bone resorption may be a key therapeutic target in myeloma bone disease. MIP-1a can either induce osteoclast formation or up-regulate RANKL expression via different chemokine receptors such as CCR1 and CCR5. By blocking these receptors, one should block MIP-1a-mediated osteoclast formation.73 Moreover, MIP-1a can also enhance the growth of MM cells through the CCR1 receptor, as reported by Lentzsch et al.74 Anti-CCR1 agents should be excellent therapeutic agents for clinical trials in the near future.
Pathophysiology of myeloma bone disease 621
Anabolic agents such as PTH may also be potential treatments for myeloma bone disease. Pennisi et al75 reported a 20% increase in bone density and an inhibition of myeloma-cell growth in SCID-rab mice engrafted with human myeloma cells and treated with PTH. However, there is a theoretical concern with PTH treatment. By stimulating osteoblast formation in myeloma bone lesions, there could be increased production of cytokines that increase myeloma tumor burden. Clinical trials with anabolic agents in myeloma are yet to be reported, and their impact on bone formation should be analyzed carefully. Most myeloma patients receive bisphosphonates, which will likely lessen the anabolic effects of PTH.75 Toxic effects of PTH therapy – such as hypercalcemia, nephrolithiasis with renal function compromise – could also pose obstacles in treating myeloma patients with such agents. SUMMARY Recent advances in the pathophysiology underlying multiple myeloma bone disease have identified several novel therapeutic targets such as CCR1, IL-3, IL-7, and Wnt inhibitors. As the understanding of the biology of myeloma bone disease continues to expand, increasing numbers of new potential therapies should emerge. These new agents should be entering clinical trial in the near future and should provide important insights into their utility in ameliorating this devastating complication of myeloma, and in extending survival of patients as well as improving their quality of life. REFERENCES 1. Roodman GD. Cell Biology of the osteoclast. Experimental Hematology 1999; 27: 1229–1241. *2. Roodman GD. Treatment strategies for bone disease. Bone Marrow Transplantation 2007 Aug 6. [Epub ahead of print]. 3. Gunn WG, Conley A, Deininger L et al. A crosstalk between myeloma cells and marrow stromal cells stimulates production of DKK1 and interleukin-6: a potential role in the development of lytic bone disease and tumor progression in multiple myeloma. Stem Cells 2006; 24: 986–991. 4. Giuliani N, Colla S & Rizzoli V. New insight in the mechanism of osteoclast activation and formation in multiple myeloma: focus on the receptor activator NF-Kappa-B ligand (RANKL). Experimental Hematology 2004; 32: 685–691. 5. Choi SJ, Cruz JC, Craig F et al. Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood 2000; 96: 671–675. *6. Lee JW, Chung HY, Ehrlich LA et al. IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells. Blood 2004; 103: 2308–2315. 7. Hsu H, Lacey DL, Dunstan CR et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proceedings of the National Academy of Sciences of the United States of America 1999; 96: 3540–3545. 8. Nakagava N, Kinosaki M, Yamaguchi K et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochemical and Biophysical Research Communications 1998; 253: 395–400. *9. Boyle WJ, Simonet WS & Lacey DL. Osteoclast differentiation and activation. Nature 2003; 423: 337–342. 10. Yasuda H, Shima N, Nakagawa N et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proceedings of the National Academy of Sciences of the United States of America 1998; 95: 3597–3602. 11. Hofbauer LC & Heufelder AE. Osteoprotegerin and its cognate ligand: a new paradigm of osteoclastogenesis. European Journal of Endocrinology 1998; 139: 152–154. 12. Tsukii K, Shima N, Mochizuki S et al. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid
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