What hope is there for myeloma bone disease? 2011
Myeloma bone disease causes severe morbidity in patients with myeloma characteristically including
pain, loss of mobility and disfigurement. The pathological features of myeloma bone disease are
osteoporosis, focal lytic lesions often leading to pathological fractures, vertebral collapse and
hypercalcaemia (Figure 1). Neurological sequelae secondary to bone disease are common caused by
compression of nerves by damaged and displaced bone and most dramatically include spinal cord
compression which often presents as a neuro-surgical emergency occurring in up to 5% of patients
with myeloma (Figure 2) (Kyle, Gertz et al. 2003). Indeed, consequences of osteolytic bone disease
are often the presenting features of myeloma. Approximately 67% of patients with myeloma present
with bone pain and up to 90% of patients with myeloma exhibit features of myeloma bone disease
at some stage of the disease course (Coleman 1997; Kariyawasan, Hughes et al. 2007).
Figure 1 – Typical features of myeloma bone disease including from left to right – vertebral wedge
fracture, ‘pepper-pot’ skull, pathological fracture arising from a lytic lesion of the humerus, large
lytic lesion of the proximal tibia and lytic lesion causing extensive destruction of the mandible.
Last month we reviewed the exciting news from the MRC Myeloma IX trial demonstrating that not
only is zolendronic acid superior to clodronate in the prevention of skeletal events events (27% vs
35%) but it is also associated with a survival advantage. Even after a relatively short median followup of 3.7 years, patients receiving zoledronic acid survive 5.5 months longer than those receiving
clodronate. Progression free survival is also increased by 2 months (19.5 months vs 17.5 months)
(Morgan, Davies et al. 2010). However, bisphosphonates are only partially successful in the
prevention of skeletal events. New lesions appear and crucially, existing lesions are not repaired.
There is therefore an urgent need for additional agents and strategies. So what else is under
development?
Figure 2 – MRI images of spinal cord compression involving the cervical spine (left) and the
thoracic spine (right).
Pathophysiology of myeloma bone disease
A brief review of the pathophysiology of myeloma bone disease provides the rationale for new
therapeutic targets and strategies. Myeloma bone disease is caused by disruption of physiological
bone remodelling specifically the uncoupling of the normal balance between bone resorption
mediated by osteoclasts and bone formation mediated by osteoblasts resulting in net osteolysis
(Mundy 1974; Bataille, Chappard et al. 1991; Roodman 1992; Bataille, Chappard et al. 1995; Bataille,
Chappard et al. 1996; Croucher and Apperley 1998). Over thirty years ago, in studies of myeloma,
Mundy et al. (1974) suggested that increased osteoclastogenesis was mediated by secreted
osteoclast activating factors (OAFs). Histological and biochemical analysis has confirmed increased
bone resorption associated with tumour infiltration (Taube, Beneton et al. 1992). Initially, the
identity of the OAFs was uncertain. Since then many factors have been confirmed to have
osteoclastogenic effects including interleukin 1β (Il-1β) (Cozzolino, Torcia et al. 1989; Kawano,
Yamamoto et al. 1989; Yamamoto, Kawano et al. 1989), interleukin-6 (IL-6) (Lowik, van der Pluijm et
al. 1989; Ishimi, Miyaura et al. 1990), interleukin-11 (Il-11) (Paul 1990), tumour necrosis factor-α
(TNF-α) (Lichtenstein, Berenson et al. 1989), tumour necrosis factor-β (TNF-β) (Garrett, Durie et al.
1987; Bataille, Klein et al. 1989), macrophage inflammatory protein 1 α (MIP 1- α) (Wolpe, Davatelis
et al. 1988), and hepatocyte growth factor (HGF) (Kukita, Nomiyama et al. 1997). It has been
proposed that many of these osteoclast activating factors mediate their effects via the receptor
activator nuclear factor kappa B ligand/osteoprotogerin (RANKL/OPG) system, which acts as a final
common pathway (Simonet, Lacey et al. 1997; Lacey, Timms et al. 1998; Hsu, Lacey et al. 1999; Choi,
Cruz et al. 2000).
Anti resorptive strategies
Given the above, the rationale for anti osteoclastic agents is clear. In addition to bisphosphonates,
recombinant OPG and OPG mimetics, and anti RANK-L constructs, are under development (Body,
Greipp et al. 2003; Body, Facon et al. 2006; Heath, Vanderkerken et al. 2007). An OPG mimetic has
shown some anti resorptive effects in the 5T2MM murine model of myeloma, and no adverse effects
in a preliminary phase 1 trial (Body, Greipp et al. 2003). More attention, however, has focused on
the anti RANK-L monoclonal antibody, denusomab (Amgen). In 2006, a head to head comparison
between pamidronate and denusomab demonstrated prompt and substantial reduction in urinary
and serum N-telopeptide, a marker of bone resorption in both pamidronate and denusomab treated
patients. However, the reduction in urinary and serum N-telopeptide was sustained for 84 days in
the denusomab treated patients whilst the reductions were not sustained in the pamidronate
treated patients.. The authors concluded that denusomab compared favourably given the lack of
adverse effects and a sustained reduction in bone turnover for at least 84 days following a single s.c.
dose (Body, Facon et al. 2006). However, apparent benefits, indicated by markers of bone
resorption, are of limited significance until they are backed up by clear evidence of a reduction in
hard clinical endpoints eg actual skeletal events. Hard clinical advantage is yet to be demonstrated
for denusomab or other anti-resorptive strategies.
Bone formation is also inhibited in myeloma hence the rationale for bone anabolic strategies
Given the limited benefit of anti-resorptive strategies, attention has switched to bone anabolic
strategies. In addition to increased recruitment and activity of osteoclasts, it has become apparent
that osteoblast activity is also inhibited. Histomorphometric evidence suggests that early in the
disease process there is an increase in the recruitment of both osteoclasts and osteoblasts. There
are well documented reports of myeloma bone disease with an osteosclerotic phenotype (Roberts,
Rinaudo et al. 1974; McCluggage, Jones et al. 1995; Mulleman, Gaxatte et al. 2004). However,
typically as disease progresses osteoblast function is inhibited, osteoclast activity is stimulated
resulting in net osteolysis (Bataille, Chappard et al. 1991). However, the molecular mechanism
responsible for the inhibition of osteoblast function is not yet accounted for. Following biochemical
analysis of markers of bone metabolism, Abildgaard et al. suggested that an unknown factor in
myeloma was responsible for inhibition of later stage osteoblast differentiation and function leading
to the uncoupling of osteoblast and osteoclast activity and net bone lysis (Abildgaard, Glerup et al.
2000).
Targeting Dkk-1 and other inhibitors of Wnt signalling
Evidence is now accumulating to suggest that the Wnt/β-catenin signalling pathway inhibitor,
dickkopf (Dkk-1) contributes to osteolytic disease in myeloma via the inhibition of osteoblast differ
entiation and therefore, may be if not the only osteoblast inhibitor active in myeloma bone disease,
one of the critical factors driving osteolytic bone disease. The first important discovery to suggest
this was made following gene array analysis of genes upregulated in myeloma (Tian 2003) .
These authors screened patients with multiple myeloma and radiological evidence of lytic lesions
and compared them with patients with myeloma and no radiological evidence of lytic lesions. Other
control groups included patients with other lymphoid malignancies eg Waldenström
macroglobulinaemia, a condition related to myeloma but which does not lead to lytic disease,
patients with monoclonal gammopathy of undetermined significance (MGUS), a precursor condition
of myeloma and healthy controls. They identified overexpression of four genes in patients with
myeloma and lytic lesions. One of these genes codes for Dkk-1. Furthermore, expression of the DKK1 gene increased from a basal level in healthy controls, to patients with MGUS, to patients with
myeloma and at least one lytic lesion and was highest in patients with myeloma with more than one
lytic lesion. Bone marrow plasma levels of Dkk-1 were measured by enzyme linked immunosorbent
assay (ELISA) in all patients and a similar trend was seen. The authors proceeded to examine the
effects of recombinant Dkk-1, in the presence of bone morphogenic protein-2 on C2C12 osteoblast
precursors and inhibition of osteoblast differentiation with the addition of anti-Dkk-1. They
confirmed that the addition of Dkk-1 inhibited osteoblast differentiation and that this inhibition was
reversed using an anti-Dkk-1 antibody. The correlation between serum Dkk-1 levels and osteolytic
bone disease has been subsequently confirmed by other groups (Politou, Heath et al. 2006).
A.
Lytic
lesions
Naive
b.
Naive
5T2MM 5T2MM
+ veh
5T2MM
5T2MM++anti
AntiDkk1
-Dkk-1
5T2MM + veh
5T2MM + anti Dkk1
B.
Naive
C. i. trabecular volume - tibiae
p<0.05
p=0.09
p<0.001
%
mm
p<0.001
ii. trabecular thickness - tibiae
iii. trabecular number - tibiae
p <0.05
iv. cortical volume - tibiae
p=0.062
P <0.01
P <0.01
1
3
mm 3
mm
mm-1
0.8
0.6
0.4
0.2
0
naïve
5T2MM + veh
5T2MM + anti
Dkk1
Figure 3 – Treatment with anti Dkk-1prevents myeloma induced reductions in trabecular and
cortical bone volume in the 5T2MM murine model of myeloma (Heath et al 2009).
Since then a number of in vivo studies have demonstrated the effectiveness of targeting Dkk-1 in
various murine models of myeloma (Yaccoby, Ling et al. 2006; Edwards, Edwards et al. 2007). In
particular, our group has shown a reduction in osteolytic lesions, increases in bone volume and bone
formation rate using an anti-Dkk-1 antibody (BHQ880, Novartis) in the 5T2MM murine model of
myeloma (Figure 3) (Heath, Chantry et al. 2009). A humanised version of this anti-body is currently
the subject of a large, multi-centered phase 2 trial in patients with myeloma and results are eagerly
anticipated.
There is also increasing evidence to suggest that other inhibitors of Wnt/β-catenin signalling may
play a role in the reduction of osteoblast numbers and inhibition of osteoblast function in myeloma
bone disease and as such constitute potential therapeutic targets. For example, the role of sFRP-2
has been investigated (Oshima, Abe et al. 2005). These authors took conditioned media from the
human derived myeloma cell lines RPMI8226 and U266 and primary myeloma cells and observed the
effect on bone morphogenic protein induced alkaline phosphatase activity in osteoblasts precursors
and in vitro mineralization. They found reduced alkaline phosphatase activity and mineralization.
These cell lines and most of the primary myeloma cells used expressed sFRP-2 but not sFRP-1, sFRP-3
or Dkk-1.
Dkk-1 is also not the only Wnt component found to be upregulated in gene micro-array analyses of
myeloma (De Vos, Couderc et al. 2001; Zhan, Hardin et al. 2002; Davies, Dring et al. 2003). De Vos et
al. (2001) detected up-regulation of the gene encoding the Wnt inhibitor sFRP3/FRZB. Davies et al.
(2003) demonstrated that FRZB was the most up-regulated gene during the progression from
monoclonal gammopathy of uncertain significance to myeloma. Zhan et al (2002) demonstrated
down regulation of Wnt 10B and up regulation of Wnt 5A and sFRP-3 (aka FRZB). These findings
therefore suggest that there a number of different components and inhibitors of the Wnt/β-catenin
signalling pathway implicated in the pathogenesis of myeloma bone disease.
Other osteoblast inhibitors active in myeloma bone disease
Interleukin-7 (Il-7)
There is also evidence to suggest other molecules and pathways play a role in osteoblast inhibition in
myeloma bone disease. Investigators have reviewed the expression of seven of the known inhibitors
of osteoblast differentiation, some of which act upon the Wnt signalling pathway (Dkk-1, sFRP-1,
sFRP-2, sFRP-3, sFRP-4) and some which act on other pathways (gremlin, noggin, Il-7) in myeloma
cell lines (Giuliani, Colla et al. 2005). They began by confirming the reduction of markers of
osteoblast differentiation and nodule formation in the colony forming assay (colony forming unit –
osteoblast, CFU-OB) when osteoblast precursors were co-cultured with myeloma cell lines. The
expression and activity of the osteoblast differentiation transcription factor Runx2/Cbfa1 mRNA
measured by RT-PCR was also reduced. They also noted that the inhibitory effect was significantly
more marked with increased cell to cell contact reiterating the role of the very late antigen 4 (VLA-4)
integrin system. They then co-cultured human bone marrow derived osteoblast precursors (Human
BM PreOBs) with Il-7 and Dkk-1 and found a significant inhibitory effect with Il-7 but contrary to
previous findings, not with Dkk-1. They therefore suggest that Il-7 is a key inhibitor of osteoblast
differentiation and inhibitior of Runx2/Cbfa and furthermore may also influence osteoclast numbers
via the reduction of OPG production in differentiated osteoblasts. This inhibiton was blocked by the
addition of Il-7 antibodies but not by the addition of Dkk-1 antibody. Il-7 is secreted by T-cells and is
known to stimulate osteoclast activation via up regulation of RANKL (Giuliani, Colla et al. 2002) but
its’ effects on osteoblasts were previously unknown. The authors suggest that Il-7 probably does not
act via Wnt signalling demonstrating the likely contribution of other pathways on osteoblast
differentiation.
Hepatocyte growth factor
Hepatocyte growth factor (HGF) has also been implicated (Grano, Galimi et al. 1996). These authors
identified HGF production by osteoclasts and expression of the HGF receptor by both osteoclasts and
osteoblasts. These data suggested HGF mediated autocrine regulation of osteoclasts and paracrine
regulation of osteoblasts and that HGF therefore acted as a coupling factor between osteoclast and
osteoblast activity. Subsequently, a number of studies noted expression of HGF by myeloma cell
lines and purified primary myeloma cells (Borset, Hjorth-Hansen et al. 1996; Seidel, Borset et al.
1998; Standal, Abildgaard et al. 2007). Standal et al. have recently demonstrated that HGF inhibits
BMP induced expression of alkaline phosphatase in human mesenchymal stem cells and the murine
osteoblast pre-cursor cell line C2C12.
Interleukin-3 (Il-3)
Similarly, recent studies have suggested that Il-3 plays a dual role in the pathogenesis of myeloma
bone disease (Lee, Chung et al. 2004). Investigators demonstrated that Il-3 induced
osteoclastogenesis and increased myeloma cell growth. More recently, the same group confirmed
that Il-3 was significantly increased in the bone marrow plasma of patients with myeloma compared
to healthy controls and that Il-3 was also able to inhibit basal and BMP induced osteoblast
differentiation (Ehrlich, Chung et al. 2005).
TGF-β signalling also regulates osteoblast differentiation
Given the importance of osteoblast inhibition in the pathogenesis of myeloma bone disease,
attention has also focussed on other pathways that may regulate osteoblastogenesis. Transforming
growth factor beta (TGF-β) signalling is one such pathway (Geiser, Zeng et al. 1998). The TGF-β
superfamily of cytokines, comprising TGF-βs, the bone morphogenic proteins (BMPs) and the
activin/inhibin system, is a highly conserved system performing a wide variety of biological functions
ranging from regulation of embryogenesis to regulation of reproduction and widespread tissue
homeostasis (Guo and Wang 2009).
Within the context of myeloma, investigators have recently suggested that TGF-β released from
bone mineral matrix as a result of increased, myeloma driven osteoclastic resorption, inhibits
osteoblast differentiation (Yata, Abe et al. 2008). Recent studies inhibiting the TGF-beta type I
receptor in mice has demonstrated anabolic and anti-catabolic effects on bone mediated by
increased osteoblast differentiation and bone formation and reducing osteoclast differentiation and
resorption (Mohammad, Chen et al. 2009).
Targeting the TGF-β ligand, activin-A prevents myeloma bone disease
Furthermore, recent studies have identified activin-A as an important regulator of bone phenotype.
Activins are members of the transforming growth factor beta (TGF-β) superfamily closely related to
their natural antagonists, the inhibins. Inhibins are comprised of a common α sub-unit coupled to
one of two β sub-units leading to inhibin-A (αβA) or inhibin-B (αβB). Activins are homo- or
heterodimers of the β subunits. The most commonly occurring β subunits are βA and βB leading to
the homodimers activin-A (βAβA), activin-B (βBβB) and the heterodimer activin-AB (βAβB). Activins
and inhibins were first identified as important regulators of pituitary follicle stimulating hormone
release (Schwartz and Channing 1977; Ling, Ying et al. 1986; Vale, Rivier et al. 1986; Woodruff and
Mather 1995). Subsequent studies have demonstrated that activins are expressed in diverse tissues
and play important roles in embryology, wound healing and tissue homeostasis (Feijen, Goumans et
al. 1994; Tuuri, Eramaa et al. 1994; Munz, Smola et al. 1999; Chen, Lui et al. 2002; Zhang, Deng et al.
2005).
A.
Naive
5T2MM + veh
B. i. Osteolytic lesion count, tibiae
60
p <0.05
5T2MM + ActRIIA
ii. Osteolytic lesion area, tibiae
50
p <0.05
2000
40
pixcels 2
no of lesions
p <0.05
2500
p <0.05
30
1500
1000
20
500
10
0
0
naïve
5T2MM + veh
5T2MM + ActRIIA
naïve
5T2MM + veh
5T2MM + ActRIIA
Figure 4 - Treatment with ActRIIA.muFc reduces the number and area of osteolytic lesions in the
tibiae of mice bearing 5T2MM cells as shown by analysis of 3d μCT reconstructions (Chantry et al
2010).
Recently, a number of investigators have identified the activin/inhibin system as an important
regulator of bone formation (Eijken, Swagemakers et al. 2007). Specifically, activin-A has been
shown to be abundantly expressed in bone (Ogawa, Schmidt et al. 1992). Conversely, neither activinB nor activin-AB has been detected in bone. Conflicting evidence exists concerning the role of
activin-A in bone. It has been reported to both inhibit and stimulate osteoblastogenesis in vitro, and
to promote osteoclast formation in vitro (Ikenoue, Jingushi et al. 1999; Fuller, Bayley et al. 2000;
Gaddy-Kurten, Coker et al. 2002; Eijken, Swagemakers et al. 2007). In vivo, direct administration of
activin-A increases bone mineral density (Oue, Kanatani et al. 1994), whereas, over-expression of
inhibin-A, which blocks activin-A and other TGF-β family members, increases bone mass (Bonewald
and Mundy 1990; Perrien, Akel et al. 2007). Other investigators have recently demonstrated that
activin-A signalling can be blocked in vivo with a soluble ActRIIA.muFc fusion protein leading to
increased bone mass and strength (Pearsall, Canalis et al. 2008). ACE-011, a soluble form of the
extra-cellular domain of the human activin type II receptor, fused to a human IgG-Fc fragment, has
also been shown to increase bone formation and decrease bone resorption markers in healthy, post
menopausal women (Ruckle J 2007). Recently, two studies have targeted activin-A in murine models
of myeloma demonstrating substantial protection against Osteolytic disease (Chantry, Heath et al.
2010; Vallet, Mukherjee et al. 2010). Our group has recently targeted activin-A using a soluble decoy
receptor in the 5T2MM and 5T33MM murine models of myeloma demonstrating not only
prevention of osteolytic bone disease but also a survival advantage (Figure 4)(Chantry, Heath et al.
2010). Given these promising results, targeting activin-A signalling may well be beneficial in
myeloma bone disease.
Dual anti-resorptive and bone anabolic strategies offer the greatest promise for those who suffer
with myeloma bone disease
As the overall prognosis for patients with myeloma improves, especially with the arrival of novel
therapies, improved treatment of myeloma bone disease remains a clinical priority. Considering the
pathophysiology of myeloma bone disease, targeting both increased bone resorption and decreased
bone formation appears to offer not only eminently sensible, but the strategy most likely to offer
most hope. Anti resorptive therapies are well established but the addition of bone anabolic agents,
as yet clinically unproven may represent a vital step forward.
Andy Chantry
Clinician Scientist
Mellanby Centre for Bone Research
University of Sheffield
References
Abildgaard, N., H. Glerup, et al. (2000). "Biochemical markers of bone metabolism reflect
osteoclastic and osteoblastic activity in multiple myeloma." Eur J Haematol 64(2): 121-129.
Bataille, R., D. Chappard, et al. (1995). "Excessive bone resorption in human plasmacytomas: direct
induction by tumour cells in vivo." Br J Haematol 90(3): 721-724.
Bataille, R., D. Chappard, et al. (1996). "Quantifiable excess of bone resorption in monoclonal
gammopathy is an early symptom of malignancy: a prospective study of 87 bone biopsies."
Blood 87(11): 4762-4769.
Bataille, R., D. Chappard, et al. (1991). "Recruitment of new osteoblasts and osteoclasts is the
earliest critical event in the pathogenesis of human multiple myeloma." J Clin Invest 88(1):
62-66.
Bataille, R., B. Klein, et al. (1989). "Spontaneous secretion of tumor necrosis factor-beta by human
myeloma cell lines." Cancer 63(5): 877-880.
Body, J. J., T. Facon, et al. (2006). "A study of the biological receptor activator of nuclear factorkappaB ligand inhibitor, denosumab, in patients with multiple myeloma or bone metastases
from breast cancer." Clin Cancer Res 12(4): 1221-1228.
Body, J. J., P. Greipp, et al. (2003). "A phase I study of AMGN-0007, a recombinant osteoprotegerin
construct, in patients with multiple myeloma or breast carcinoma related bone metastases."
Cancer 97(3 Suppl): 887-892.
Bonewald, L. F. and G. R. Mundy (1990). "Role of transforming growth factor-beta in bone
remodeling." Clin Orthop Relat Res(250): 261-276.
Borset, M., H. Hjorth-Hansen, et al. (1996). "Hepatocyte growth factor and its receptor c-met in
multiple myeloma." Blood 88(10): 3998-4004.
Chantry, A. D., D. Heath, et al. (2010). "Inhibiting activin-A signaling stimulates bone formation and
prevents cancer-induced bone destruction in vivo." J Bone Miner Res 25(12): 2357-2370.
Chen, Y. G., H. M. Lui, et al. (2002). "Regulation of cell proliferation, apoptosis, and carcinogenesis by
activin." Exp Biol Med (Maywood) 227(2): 75-87.
Choi, S. J., J. C. Cruz, et al. (2000). "Macrophage inflammatory protein 1-alpha is a potential
osteoclast stimulatory factor in multiple myeloma." Blood 96(2): 671-675.
Coleman, R. E. (1997). "Skeletal complications of malignancy." Cancer 80(8 Suppl): 1588-1594.
Cozzolino, F., M. Torcia, et al. (1989). "Production of interleukin-1 by bone marrow myeloma cells."
Blood 74(1): 380-387.
Croucher, P. I. and J. F. Apperley (1998). "Bone disease in multiple myeloma." Br J Haematol 103(4):
902-910.
Davies, F. E., A. M. Dring, et al. (2003). "Insights into the multistep transformation of MGUS to
myeloma using microarray expression analysis." Blood 102(13): 4504-4511.
De Vos, J., G. Couderc, et al. (2001). "Identifying intercellular signaling genes expressed in malignant
plasma cells by using complementary DNA arrays." Blood 98(3): 771-780.
Edwards, C. M., J. R. Edwards, et al. (2007). "Increasing Wnt signaling in the bone marrow
microenvironment inhibits the development of myeloma bone disease and reduces tumor
burden in bone in vivo." Blood.
Ehrlich, L. A., H. Y. Chung, et al. (2005). "IL-3 is a potential inhibitor of osteoblast differentiation in
multiple myeloma." Blood 106(4): 1407-1414.
Eijken, M., S. Swagemakers, et al. (2007). "The activin A-follistatin system: potent regulator of
human extracellular matrix mineralization." Faseb J 21(11): 2949-2960.
Feijen, A., M. J. Goumans, et al. (1994). "Expression of activin subunits, activin receptors and
follistatin in postimplantation mouse embryos suggests specific developmental functions for
different activins." Development 120(12): 3621-3637.
Fuller, K., K. E. Bayley, et al. (2000). "Activin A is an essential cofactor for osteoclast induction."
Biochem Biophys Res Commun 268(1): 2-7.
Gaddy-Kurten, D., J. K. Coker, et al. (2002). "Inhibin suppresses and activin stimulates
osteoblastogenesis and osteoclastogenesis in murine bone marrow cultures." Endocrinology
143(1): 74-83.
Garrett, I. R., B. G. Durie, et al. (1987). "Production of lymphotoxin, a bone-resorbing cytokine, by
cultured human myeloma cells." N Engl J Med 317(9): 526-532.
Geiser, A. G., Q. Q. Zeng, et al. (1998). "Decreased bone mass and bone elasticity in mice lacking the
transforming growth factor-beta1 gene." Bone 23(2): 87-93.
Giuliani, N., S. Colla, et al. (2005). "Myeloma cells block RUNX2/CBFA1 activity in human bone
marrow osteoblast progenitors and inhibit osteoblast formation and differentiation." Blood
106(7): 2472-2483.
Giuliani, N., S. Colla, et al. (2002). "Human myeloma cells stimulate the receptor activator of nuclear
factor-kappa B ligand (RANKL) in T lymphocytes: a potential role in multiple myeloma bone
disease." Blood 100(13): 4615-4621.
Grano, M., F. Galimi, et al. (1996). "Hepatocyte growth factor is a coupling factor for osteoclasts and
osteoblasts in vitro." Proc Natl Acad Sci U S A 93(15): 7644-7648.
Guo, X. and X. F. Wang (2009). "Signaling cross-talk between TGF-beta/BMP and other pathways."
Cell Res 19(1): 71-88.
Heath, D. J., A. D. Chantry, et al. (2009). "Inhibiting Dickkopf-1 (Dkk1) removes suppression of bone
formation and prevents the development of osteolytic bone disease in multiple myeloma." J
Bone Miner Res 24(3): 425-436.
Heath, D. J., K. Vanderkerken, et al. (2007). "An osteoprotegerin-like peptidomimetic inhibits
osteoclastic bone resorption and osteolytic bone disease in myeloma." Cancer Res 67(1):
202-208.
Hsu, H., D. L. Lacey, et al. (1999). "Tumor necrosis factor receptor family member RANK mediates
osteoclast differentiation and activation induced by osteoprotegerin ligand." Proc Natl Acad
Sci U S A 96(7): 3540-3545.
Ikenoue, T., S. Jingushi, et al. (1999). "Inhibitory effects of activin-A on osteoblast differentiation
during cultures of fetal rat calvarial cells." J Cell Biochem 75(2): 206-214.
Ishimi, Y., C. Miyaura, et al. (1990). "IL-6 is produced by osteoblasts and induces bone resorption." J
Immunol 145(10): 3297-3303.
Kariyawasan, C. C., D. A. Hughes, et al. (2007). "Multiple myeloma: causes and consequences of
delay in diagnosis." QJM 100(10): 635-640.
Kawano, M., I. Yamamoto, et al. (1989). "Interleukin-1 beta rather than lymphotoxin as the major
bone resorbing activity in human multiple myeloma." Blood 73(6): 1646-1649.
Kukita, T., H. Nomiyama, et al. (1997). "Macrophage inflammatory protein-1 alpha (LD78) expressed
in human bone marrow: its role in regulation of hematopoiesis and osteoclast recruitment."
Lab Invest 76(3): 399-406.
Kyle, R. A., M. A. Gertz, et al. (2003). "Review of 1027 patients with newly diagnosed multiple
myeloma." Mayo Clin Proc 78(1): 21-33.
Lacey, D. L., E. Timms, et al. (1998). "Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast
Differentiation and Activation." Cell 93(2): 165.
Lee, J. W., H. Y. Chung, et al. (2004). "IL-3 expression by myeloma cells increases both osteoclast
formation and growth of myeloma cells." Blood 103(6): 2308-2315.
Lichtenstein, A., J. Berenson, et al. (1989). "Production of cytokines by bone marrow cells obtained
from patients with multiple myeloma." Blood 74(4): 1266-1273.
Ling, N., S. Y. Ying, et al. (1986). "Pituitary FSH is released by a heterodimer of the beta-subunits
from the two forms of inhibin." Nature 321(6072): 779-782.
Lowik, C. W., G. van der Pluijm, et al. (1989). "Parathyroid hormone (PTH) and PTH-like protein (PLP)
stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in
osteoclastogenesis." Biochem Biophys Res Commun 162(3): 1546-1552.
McCluggage, W. G., F. G. Jones, et al. (1995). "Sclerosing IgA multiple myeloma." Acta Haematol
94(2): 98-101.
Mohammad, K. S., C. G. Chen, et al. (2009). "Pharmacologic inhibition of the TGF-beta type I receptor
kinase has anabolic and anti-catabolic effects on bone." PLoS One 4(4): e5275.
Morgan, G. J., F. E. Davies, et al. (2010). "First-line treatment with zoledronic acid as compared with
clodronic acid in multiple myeloma (MRC Myeloma IX): a randomised controlled trial."
Lancet 376(9757): 1989-1999.
Mulleman, D., C. Gaxatte, et al. (2004). "Multiple myeloma presenting with widespread
osteosclerotic lesions." Joint Bone Spine 71(1): 79-83.
Mundy, G. R., Raisz, L.G., Cooper, R.A., Schecter, G.P., Salmon, S.E. (1974). "Evidence for the
secretion of an osteoclast stimulating factor in myeloma." N Eng J Med, 291, 1041-1046.
Munz, B., H. Smola, et al. (1999). "Overexpression of activin A in the skin of transgenic mice reveals
new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair."
EMBO J 18(19): 5205-5215.
Ogawa, Y., D. K. Schmidt, et al. (1992). "Bovine bone activin enhances bone morphogenetic proteininduced ectopic bone formation." J Biol Chem 267(20): 14233-14237.
Oshima, T., M. Abe, et al. (2005). "Myeloma cells suppress bone formation by secreting a soluble
Wnt inhibitor, sFRP-2." Blood 106(9): 3160-3165.
Oue, Y., H. Kanatani, et al. (1994). "Effect of local injection of activin A on bone formation in
newborn rats." Bone 15(3): 361-366.
Paul, S. R., Bennett, F., Calvetti, J.A., Kelleher, K., Wood, C.R., O'Hara, Jr, R.M., Leary, A.C., Sibley, B.,
Clark, S.C. and Williams, D.A. (1990). "Molecular cloning of a cDNA encoding interleukin 11, a
stromal cell-derived lymphopoietic and haematopoietic cytokine." Proceedings of the
National Academy of Sciences of the United States of America, 98, 11581-11586.
Pearsall, R. S., E. Canalis, et al. (2008). "A soluble activin type IIA receptor induces bone formation
and improves skeletal integrity." Proc Natl Acad Sci U S A 105(19): 7082-7087.
Perrien, D. S., N. S. Akel, et al. (2007). "Inhibin A is an endocrine stimulator of bone mass and
strength." Endocrinology 148(4): 1654-1665.
Politou, M. C., D. J. Heath, et al. (2006). "Serum concentrations of Dickkopf-1 protein are increased in
patients with multiple myeloma and reduced after autologous stem cell transplantation." Int
J Cancer 119(7): 1728-1731.
Roberts, M., P. A. Rinaudo, et al. (1974). "Solitary sclerosing plasma-cell myeloma of the spine. Case
report." J Neurosurg 40(1): 125-129.
Roodman, G. D. (1992). "Interleukin-6: an osteotropic factor?" J Bone Miner Res 7(5): 475-478.
Ruckle J, J. M., Kramer W, Kumar R, Underwood K, Pearsall S, Pearsall A, Seehra J, Condon C,
Sherman ML (2007). "A single dose of ACE-011 is associated with increases in bone
formation and decreases in bone resorption markers in healthy, postmenopausal women
Abstract ASBMR 2007."
Schwartz, N. B. and C. P. Channing (1977). "Evidence for ovarian "inhibin": suppression of the
secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of
porcine follicular fluid." Proc Natl Acad Sci U S A 74(12): 5721-5724.
Seidel, C., M. Borset, et al. (1998). "Elevated serum concentrations of hepatocyte growth factor in
patients with multiple myeloma. The Nordic Myeloma Study Group." Blood 91(3): 806-812.
Simonet, W. S., D. L. Lacey, et al. (1997). "Osteoprotegerin: A Novel Secreted Protein Involved in the
Regulation of Bone Density." Cell 89(2): 309.
Standal, T., N. Abildgaard, et al. (2007). "HGF inhibits BMP-induced osteoblastogenesis: possible
implications for the bone disease of multiple myeloma." Blood 109(7): 3024-3030.
Taube, T., M. N. Beneton, et al. (1992). "Abnormal bone remodelling in patients with myelomatosis
and normal biochemical indices of bone resorption." Eur J Haematol 49(4): 192-198.
Tian, E. Z., Fenghuang; Walker, Ronald; Rasmussen, Erik; Ma, Yupo; Barlogie, Bart; Shaughnessy,
John D. Jr. (2003). "The Role of the Wnt-Signaling Antagonist DKK1 in the Development of
Osteolytic Lesions in Multiple Myeloma." The New England Journal of Medicine 349 No.26(N
Engl J Med 2003;349: 2483-94.): 2483-2494.
Tuuri, T., M. Eramaa, et al. (1994). "The tissue distribution of activin beta A- and beta B-subunit and
follistatin messenger ribonucleic acids suggests multiple sites of action for the activinfollistatin system during human development." J Clin Endocrinol Metab 78(6): 1521-1524.
Vale, W., J. Rivier, et al. (1986). "Purification and characterization of an FSH releasing protein from
porcine ovarian follicular fluid." Nature 321(6072): 776-779.
Vallet, S., S. Mukherjee, et al. (2010). "Activin A promotes multiple myeloma-induced osteolysis and
is a promising target for myeloma bone disease." Proc Natl Acad Sci U S A.
Wolpe, S. D., G. Davatelis, et al. (1988). "Macrophages secrete a novel heparin-binding protein with
inflammatory and neutrophil chemokinetic properties." J Exp Med 167(2): 570-581.
Woodruff, T. K. and J. P. Mather (1995). "Inhibin, activin and the female reproductive axis." Annu
Rev Physiol 57: 219-244.
Yaccoby, S., W. Ling, et al. (2006). "Antibody-based inhibition of DKK1 suppresses tumor- induced
bone resorption and multiple myeloma growth in- vivo." Blood: blood-2006-2009-047712.
Yamamoto, I., M. Kawano, et al. (1989). "Production of interleukin 1 beta, a potent bone resorbing
cytokine, by cultured human myeloma cells." Cancer Res 49(15): 4242-4246.
Yata, K., M. Abe, et al. (2008). "[Mechanisms for formation of myeloma bone disease]." Clin Calcium
18(4): 438-446.
Zhan, F., J. Hardin, et al. (2002). "Global gene expression profiling of multiple myeloma, monoclonal
gammopathy of undetermined significance, and normal bone marrow plasma cells." Blood
99(5): 1745-1757.
Zhang, L., M. Deng, et al. (2005). "MEKK1 transduces activin signals in keratinocytes to induce actin
stress fiber formation and migration." Mol Cell Biol 25(1): 60-65.