LYMPHOID NEOPLASIA
Pharmaceutical inhibition of glycogen synthetase kinase-3
reduces multiple
myeloma–induced bone disease in a novel murine plasmacytoma xenograft model
*W. Grady Gunn,1 *Ulf Krause,2 Narae Lee,1 and Carl A. Gregory2
1Center for Gene Therapy, and Department of Medicine, Tulane University Health Sciences Center, New Orleans, LA; and 2Institute for Regenerative Medicine
at Scott and White Hospital, Texas A&M Health Science Center, Temple, TX
Multiple myeloma (MM) is amalignancy of
plasma cells that accumulate in the bone
marrow. MM is incurable with approxi-
mately 100 000 patients currently in the
United States and 20 000 new cases diag-
nosed yearly. Themalignancy causes dis-
placement of hematopoiesis and forma-
tion of osteolytic bone lesions also known
as myeloma bone disease (MBD). At diag-
nosis, 79% of patients suffer from MBD
associatedwith severe pain and increased
mortality. Wnt inhibitors secreted by MM
cells inhibit osteogenesis and promote
osteoclastogenesis, therefore rapid tar-
geting of Wnt inhibitors is necessary to
prevent potentially irreversible effects on
the stroma, which could lead to incurable
MBD. Inhibition of glycogen synthetase
kinase-3
(GSK3
) causes accelerated
Wnt signaling and enhanced osteogen-
esis in mesenchymal stem/progenitor
cells, irrespective of the extracellular con-
centration of Wnt inhibitors. Our primary
goal of this studywas to evaluate aGSK3

inhibitor (6-bromoindirubin-3
-oximeBIO)
for amelioration of bone destruction in a
murine model of MBD. When measured
using histomorphometry, peritumoral BIO
administration improved bone quality at
the bone-tumor interface and, surpris-
ingly, increased histologically apparent
tumor necrosis. Furthermore, in vitro
assays demonstrated a proapoptotic ef-
fect on numerous MM cell lines. These
preliminary data suggest that pharma-
ceutical GSK3
inhibition may improve
bone quality in myeloma and other
malignant bone diseases. (Blood. 2011;
117(5):1641-1651)
Introduction
Multiple myeloma (MM) is a malignancy of plasma cells (CD138/
CD38 B cells) that accumulate in the bone marrow. MM is to date
incurable, with approximately 100 000 patients currently in the
United States and 20 000 new cases diagnosed nationally each
year. The aggregate median survival for MM is 4 years.1 The
malignant cells reside primarily in the bone marrow, resulting in
displacement of hematopoiesis, production of very high levels of
monoclonal immunoglobulin, and formation of osteolytic bone
lesions (OLs) also known as myeloma bone disease (MBD). MBD
is one of the major challenges in MM therapy. At diagnosis, 79% of
patients suffer from OLs, osteoporosis, or bone fractures.2 These
occurrences not only reduce quality of life for patients, but they are
also associated with approximately 20% increased mortality.3 OLs
are formed by MM cells through a change in the cytokine milieu of
bone marrow, which causes intensified osteoclastogenesis and
inhibits differentiation of mesenchymal stem cells/marrow stromal
cells (MSCs), presumptive source of new mature osteoblasts.4-7 For
years, the treatment of OLs has focused on the inhibition of
osteoclastogenesis by administration of bisphosphonates, but even
when osteoclast activity is controlled and successful chemotherapy
is achieved, no osteoblastic repair occurs,8 and skeletal events
continue to occur in approximately 40% of patients,9 suggesting
that MM cells have the capacity to irreversibly disrupt the anabolic
axis of bone formation. Indeed, there is an increasing body of
literature demonstrating that MM cells secrete factors that cause
lingering effects on osteoprogenitor cells such as MSCs. For
instance, MM cells secrete factors that inhibit osteogenic differen-
tiation of MSCs such as canonical Wnt inhibitors,4,6,10,11 which in
turn cause the release of numerous prosurvival cytokines, such as
interleukin-6 (IL-6), from the undifferentiated MSCs.5,12 As well as
inhibiting osteogenesis and enhancing stromal support of MM by
MSCs, Wnt inhibitors have also been reported to shift the ratio of
osteoblastic receptor activator of NF-B ligand (RANKL) and
osteoprotegerin (OPG) secretion in favor of osteoclastogenesis.7
The MM-derived factors seem to have lasting effects on MSCs,
even when examined ex vivo in the absence of MM cells,13-15
therefore rapid targeting of Wnt inhibitors is necessary to prevent
potentially irreversible effects on the stroma that could lead to
intractable MBD.
In the canonical Wnt signaling pathway, secreted Wnt glycopro-
teins bind to the transmembrane receptor frizzled (Frz) and the
coreceptor lipoprotein-related protein 5 and protein 6 on the surface
of the target cell. Activation of receptor Frz recruits the cytoplasmic
bridging molecule, disheveled, so as to inhibit the action of
glycogen synthetase kinase-3
(GSK3
). Inhibition of GSK3

decreases phosphorylation of
-catenin, preventing its degradation
by the ubiquitin-mediated pathway. The stabilized
-catenin acts
on the nucleus by activating T-cell factor/lymphoid enhancer
factor–mediated transcription of target genes that elicits a variety of
effects including induction of differentiation and in some cases,
proliferation. Canonical Wnt signaling is tightly regulated by a
combination of positive induction through the binding of the Wnt
ligand and negative regulation through numerous mechanisms by
at least 4 classes of the following secreted Wnt inhibitors: the
Submitted September 21, 2010; accepted November 18, 2010. Prepublished
online as Blood First Edition paper, December 1, 2010; DOI 10.1182/blood-
2010-09-308171.
*W.G.G. and U.K. are equal contributing authors.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by The American Society of Hematology
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VOLUME 117, NUMBER 5

dickkopf (Dkk) inhibitors, sclerostin, soluble Frz receptors, and
Wnt inhibitory factor (reviewed in Kawano and Kypta16 and
Gregory et al17). To date, immunosequestration of Dickkopf-1
(Dkk-1) has been reported to alleviate MBD in animal models.18,19
Dkk-1 acts by blocking the binding of the Wnt ligand to the
lipoprotein-related protein receptor, which results in its internaliza-
tion and degradation.20,21
Given that effective inhibition of GSK3
is necessary for
effective canonical Wnt signaling and such signals are necessary
for osteogenic differentiation,5,22-27 GSK3
inhibitors are likely
candidates for osteoinductive therapy in MM. Because this class of
agent acts in the cytoplasm, and the Wnt inhibitors are secreted
factors reliant on membrane bound receptors, the therapy would be
predicted to function without hindrance in the presence of very
high concentrations of Wnt inhibitor. Naturally, there are concerns
involving the potential for Wnt modulators to accelerate the
expansion of the MM cells,28 but if this effect does occur, it is
expected it will be counteracted by antimitotic therapy, or that the
putative increase in MM proliferation will be negated by the
reduced number of OLs available to serve as a niche for the
malignant cells.29 Of additional note are recent studies document-
ing the antimitotic effect of GSK3
inhibitors on malignant
lymphoid cells.30,31
We therefore examined the potential osteoprotective capacity of
GSK3
inhibition in a xenograft model of MBD. We chose
6-bromoindirubin-3-oxime (BIO), a specific inhibitor of GSK3
,
and relative of the cyclin-dependent kinase (CDK) inhibitor
indirubin-3-monooxime.32 In our recently published studies, BIO
has been shown to improve in vitro osteogenic differentiation by
human MSCs,27 block the osteoinhibitory action of Dkk-15 and
modestly inhibit the proliferation of cultured osteosarcoma cell
lines.33 Our primary goal was to examine whether BIO could block
the effects of Wnt inhibitors during bone destruction caused by
MBD. For this reason, our measurements focused on histomorpho-
metric parameters at the bone-tumor interface. We demonstrate that
systemic injections of BIO improved the percentage of bone
volume/tissue volume (BV/TV) of the proximal tibial heads of
wild-type (WT) mice. Furthermore, in a novel model of MBD,
peritumoral BIO administration slowed bone degradation and
increased tumor necrosis when the Dkk-1 expressing MM cell line
INA612,34 is engrafted into severe combined immunodeficiency
(SCID) beige mice. We further demonstrate that, contrary to
predictions of the established dogma, administration of a GSK3

inhibitor did not increase morbidity or tumor burden and caused
apoptosis of MM cell lines in vitro. We therefore suggest that
GSK3
inhibition should be explored further as a potential adjunct
therapy for malignant diseases that destroy bone.
Methods
Additional details on methods can be reviewed in supplemental methods
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article).
Inhibitor
BIO ((2Z,3E)-6-bromoindirubin-3-oxime) was acquired commercially
(GSK3 inhibitor IX; Calbiochem). The 1-mg aliquots were diluted in
dimethylsulfoxide (DMSO) to 2.8mM, then diluted in sterile phosphate-
buffered saline to 20M. Aliquots were stored at 80°C.
Tissue culture
We expanded RPMI 8226 and INA6 MM cells, kindly provided by John
Shaughnessy (University of Arkansas for Medical Sciences, Little Rock,
AR), in medium consisting of RPMI 1640, 10% fetal bovine serum, 10%
MarrowMax supplement, 100M pyruvate, 100 units/mL penicillin G, and
100 g/mL streptomycin (Invitrogen).
Animal care and use
All animal experiments were performed in accordance with animal use
protocols approved by the Tulane University Animal Care and Use
committee. For initial experiments involving systemic administration of
BIO, male C57BL/6 WT mice at 1 month of age were acquired from The
Jackson Laboratory. Mice were caged in groups of 3 to 4 animals and
allowed to access standard mouse chow and water ad libitum.
For establishment of the MBD model, male and female C57BL6/Prkdc
scid mice (20 per group) were purchased from The Jackson Laboratory and
housed 2 to 4 animals per cage under specific pathogen–free conditions.
They were allowed access to sterile standard mouse chow and water ad
libitum.
Micro computer-aided tomography and conventional
x-ray imaging
A Skyscan 1174 specimen imager was used for micro computer-aided
tomography (CT) imaging. A 25-m filtered beam set to 48 kV was used
with a resolution of approximately 6 m. Conventional x-rays were
achieved using a Faxitron specimen imager.
Alkaline phosphatase measurement
Alkaline phosphatase (ALP) levels were measured as described previously5
from serum using a kinetic assay based on the conversion of p-nitrophenol
phosphate to nitrophenolate.
ELISA
The pyridinoline (PYD) and tartrate-resistant acid phosphatase (TRAP) 5b
enzyme-linked immunosorbent assays (ELISAs) were obtained from Quidel.
The
-catenin and Dkk-1 ELISAs were obtained from R&D Systems.
Where necessary, values were normalized to cell number using hemacyto-
metric analyses.
Histology
Mice were killed, and leg bones were dissected and placed immediately in
10% (vol/vol) buffered formalin, and stored at 4°C from 24 hours to 3 days.
Decalcification was done using 0.5M ethylenediaminetetraacetic acid in
phosphate-buffered saline until x-ray scans showed loss of bone opacity.
Processing, embedding, and sectioning were performed at the Tulane
Histology Core Facility. Sections were stained with hematoxylin and eosin.
Statistics
In vivo statistical tests were performed with groups of 4 mice or more using
standard analysis of variance (ANOVA) or 2-tailed Student t test. In vitro
assays of cell viability and cell-cycle data were analyzed using ANOVA
after arcsine transformation of proportions calculated from sample sets
consisting of 3 to 9 replicates. Values were calculated using R Version
2.10.1 software (http://www.R-project.org). Statistical significance was
defined at a value P  .05.
Results
Effect of systemic BIO administration on the bone quality of
proximal tibial heads of WT mice
To examine whether BIO could influence bone homeostasis in
vivo, 1-month-old male C57BL/6 WT mice (n  10) received a
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VOLUME 117, NUMBER 5

100-L intraperitoneal injection of 2M BIO every 48 hours for
6 weeks. Controls received the vehicle dimethylsulfoxide
(DMSO). No toxicity was apparent by gross inspection, and
body masses were comparable among groups. In some in-
stances, the gut and lung tissue were histologically surveyed for
signs of neoplasia but none were detected (data not shown).
After 6 weeks, the mice were euthanized and the tibias were
explanted. Fixed, dried bones were subjected to micro computer-
aided tomography (CT). The length, surface area, and volume
of the tibias were not affected by BIO treatment, and remained
constant with variation not exceeding 15%. For microanalysis,
region of interest (ROI) was defined as the proximal tibial head
and diaphysis constituting 10% of the longitudinal length of the
bone (Figure 1A-B). Binary representations of the transverse
sections (15 m each) were generated from the scans, and
BV/TV values were calculated for each 3-dimensional ROI.
Trabecular parameters were significantly affected by BIO
treatment with increased trabecular number (Tb N.) and a
corresponding decrease in trabecular spacing (Tb Sp., P  .005;
Figure 1C-D). Furthermore, the BIO-treated mice had, on
average, an 18% (P  .005) increase in BV/TV (Figure 1F).
Because BIO has the capacity to up-regulate osteogenic markers
such as ALP levels and OPG secretion in some cell types,
including stromal cells,27 we analyzed the serum of the test
mice. ALP levels were raised in the BIO-treated animals by an
average of 30% (P  .005), confirming that the inhibitor had a
systemic effect on ALP expression (Figure 1E). Serum RANKL
and OPG levels were subject to a high degree of variation, but
the ratio of serum RANKL to OPG did not significantly differ
from controls (data not shown). Given the modest effect of BIO
on the bone, it is possible that these blood markers do not
significantly change against a background of high variation.
Figure 1. Effect of systemic BIO administration compared with vehicle (DMSO) on murine proximal tibial bone parameters as measured by CT. (A) Mean volume of
the ROIs analyzed to confirm comparability between groups. (B) The 3-dimensional CT rendering (right) of a control proximal tibia with ROI (left). The 3-dimensional
reconstruction of ROIs from binary images (right). (C) Mean Tb N. (D) Mean Tb Sp. (E) Relative serum ALP activity. (F) Mean percentage of BV/TV. Statistics analyzed by
Student t test for n  10 mice per group; experiment was performed twice.
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Because intraperitoneally administered BIO had a systemic
osteogenic effect in WT, healthy mice, it was decided to test BIO
in a model of MM-induced MBD.
Establishing the model of MM-induced bone disease
To establish an OL model to present a measurable bone phenotype,
we first administered the human RPMI 8226 MM cell line into the
tibial medullary canal of nonlethally irradiated SCID mice. This
method was used to facilitate osteogenic-relevant plasmacytoma
formation. Human antibody fragments secreted by RPMI 8226
cells, were used to track tumor formation because there was a good
correlation between soluble levels and cell number (R  0.91).
Tumor engraftment, as defined by presence of a palpable tumor
(supplemental Figure 1A-B, available on the Blood Web site; see
the Supplemental Materials link at the top of the online article) or
detection of systemic human chains, remained below 50%. Of the
mice with engrafted tumors, histologically detectable bone involve-
ment was rare and mild (supplemental Figure 1C), and detection of
systemic bone resorption markers TRAP 5b and type I collagen–
derived PYD cross-linked products was sporadic.
To improve engraftment and initiate bone involvement, an
alternative Dkk-1 expressing cell line (INA6) was used. INA6 cells
are described as a plasmacytoma-forming plasma cell line estab-
lished from a patient 80 years of age with plasma cell leukemia.
The cells express several plasma cell antigens such as  light chain,
CD75w, and CD138 while being negative for common pan-B cell
markers such as CD19 and CD20.12 Although the rate of tumor
development varied, with animals developing palpable tumors
between 1 and 4 weeks after injection, tumor induction efficiency
was 97%. Tumors were localized at the injection site as a solid
plasmacytoma (Figure 2A). To standardize treatment strategies and
data analyses, a simple staging system was used to categorize
tumors based on cross-section (Figure 2A). Established tumors
increased in size rapidly, with accompanying lysis of adjacent bone
that could be detected radiologically and histologically (Figure 2B).
The 3-dimensional CT scans demonstrated formation of focal
lesions in the cortical bone characteristic of MM (Figure 2C
arrowhead), which was more apparent in axial images. Additional
weakening of the bone tissue was evident, such as invasion of the
metaphyses and disruption of the growth plate when examined
histologically (Figure 2D-E). Further examination confirmed colo-
nization of the tibial marrow cavity and surrounding muscle tissue
with myeloma cells, as well as extensive cortical degradation
(Figure 2B,D).
Upon explant, engrafted bones were qualitatively more fragile
than the contralateral counterparts with severe tumors causing
complete ablation of red marrow from the engrafted bone
(Figure 2F bottom). Exudates from the medullary canals of mice
with tumors contained an abundance of human CD138 cells
(Figure 2F top). A panel of serum markers of bone resorption was
used for following tumor progression from blood samples. Dkk-1,4
PYD cross-links derived from collagen I degradation and TRAP 5b
was elevated in sera from tumor bearing mice (Figure 3A,C), but
the levels did not correlate with tumor size, suggesting that the
angiogenesis in the plasmacytomas might proceed at a different
rate than tumor expansion. There was a weak correlation with
TRAP and PYD levels and the severity of osteolysis, but unfortu-
nately, the serum markers alone were not sufficient to accurately
measure the bone disease nor the size of tumor in this model. Like
many naturally occurring MM cases, circulating cells could not be
detected in the blood by costaining for CD138 and CD38 followed
by flow cytometry, and only under the rarest of circumstances could
tumors or lytic lesions be found in distal locations after necropsy.
These data therefore suggested that a reproducible model of
localized MM-induced bone disease with minimal complications
from secondary sites had been developed.
Effect of peritumoral-administered BIO in experimentally
induced MBD mice
For examination of the effects of BIO on MM-induced bone
resorption, mice were randomly divided into treated and untreated
groups. Administration of BIO did not begin until tumors devel-
oped. Dose and frequency were based on the in vitro studies by
Gunn et al5 and Krause et al27 with a peritumoral route to best
maintain relevant local concentrations. Although lesions were not
apparent in whole body x-ray scans taken at earlier stages,
osteolytic lesions and localized bone resorption were detected after
approximatelyday 17 of the experiment. The severity of the bone
resorption from radiologic data was scored in blinded fashion by an
institutional veterinarian according to the degree of osteopenia,
periosteal reaction, and bony regeneration. BIO-treated animals
had predominantly mild and moderate bone resorption, whereas
animals receiving only vehicle had exclusively moderate to severe
resorption. Surprisingly, there was no strong relationship between
tumor size and radiologic or histologic appearance of bone
resorption. Mice that reached endpoint criteria as defined by the
animal use protocol were euthanized, and hind limbs were submit-
ted for histopathology and bone histomorphometry. Gross inspec-
tion suggested that knee joints treated with BIO appeared to be in
better condition than those that received vehicle with improved
protection of the subchondral bone and trabecular architecture
(Figure 4A). This was supported by static histomorphometry,
where statistically significant improvements in bone area, trabecu-
lar thickness, bone perimeter, and bone fraction were evident in the
BIO-treated specimens for both tibias and femurs compared with
DMSO controls (Figure 4B-C). Of note is the observation that all
histomorphometric measurements that did not show statistically
significant improvement followed a trend in favor of BIO treat-
ment. Similarly, systemic indicators of bone destruction, TRAP 5b
and PYD, were not changed in a statistically significant manner by
BIO treatment, but in general, BIO-treated mice had lower levels of
these factors (Figure 5A).
Given the function of GSK3
in the canonical Wnt signaling
pathway is to phosphorylate, and thus destabilize
-catenin,
inhibition of GSK3
is predicted to increase soluble
-catenin
levels in the tumor and surrounding tissue. To examine the
bioavailability of BIO, the tumor and associated stromal cells
were extracted from hind limbs and soluble extracts were
prepared. The amount of DNA in the digests was used to
normalize samples for cell number, and soluble
-catenin was
measured by ELISA. Extracts from DMSO-treated animals had
a remarkably constant level of soluble
-catenin (Figure 5B),
whereas the range of
-catenin values for BIO-treated extracts
was higher but variable. Irrespective of the variation, soluble

-catenin levels were significantly higher in BIO-treated ex-
tracts, demonstrating that it was having the expected biochemi-
cal effect on the tumor and surrounding tissue. Because

-catenin up-regulation is sometimes associated with tumor
initiation and expansion, and BIO appears to increase
-catenin
levels, there is understandable concern that BIO could promote
the malignancy. Nevertheless, BIO caused no increase in tumor
morbidity when analyzed by the Kaplan and Meier method
(P  .62, n  20). End point morbidity was defined as a
palpable tumor of 1.0 cm in diameter. Tumor growth was similar
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VOLUME 117, NUMBER 5

in both treated and untreated groups with 60% versus 40%
animals remaining in the study at day 10, 50% versus 30% at
day 20 and 10% versus 5% surviving until day 30 after treat-
ment, respectively. Although reducing tumor burden was not a
primary goal of our study, tumor sections were scored for
cross-sectional area and area of tumor necrosis. Contrary to
expectations, it became apparent that BIO administration substan-
tially increased appearance of necrotic tumor tissue and as a
result, reduced viable tumor area in sections from BIO-treated
mice (Figure 5C).
The effect of BIO on viability of cultured INA6 WT cells and
cells derived from control tumors
Given that indirubin derivatives have additional inhibitory
effects on CDK1 and CDK235 and BIO itself has been shown to
cause growth inhibition, cell-cycle arrest, and apoptosis in
numerous malignant cell lines,33,36-38 including those of lym-
phoid origin,30,31 we decided to examine the effect of BIO on
INA6 viability in more detail in vitro. INA6 sublines were
established from tumors explanted from control animals by
enzymatic dissociation and culture. The process of in vivo
passage selected for those MM cells with greater proliferative
potential and adaption to the in vivo microenvironment. The
INA6 subline cells were therefore deemed to be a more relevant
test line than INA6 cells expanded from parental stocks. One
line in particular, designated INA6-10.13, had an in vitro
proliferative rate substantially higher than the parental strain.
This characteristic could be explained at least in part by a loss of
IL-6 dependency in the subline because murine IL-6 does not
stimulate the human IL-6 receptor.12
Figure 2. Major characteristics of the INA6 model of
MBD. (A) X-ray imaging of the various arbitrary stages
(stage 1-4) of plasmacytoma formation. (B) Gross (left),
x-ray (center), and histologic (right) appearance of a high
grade plasmacytoma and associated bone destruction.
The histology shows the fibula with the tumor (T), bone
marrow (BM), and cortical bone (B) labeled. (C) The
3-dimensional CT renderings of contralateral (unaf-
fected left) and engrafted (affected middle) proximal
tibias showing focal OLs penetrating deep into the corti-
ces (arrowheads). Axial reconstructions at levels A and B
of the affected tibia are presented (right panels) demon-
strating the depth of the lesions. (D-E) Histologic analysis
of contralateral (D) and engrafted (E) knee joints demon-
strating infiltration and disruption of the growth plate
architecture. (F) Staining of bone marrow preparations
from engrafted long bones with human-specific anti–
CD138 antibody (top), demonstrating frequent CD138
cells. Nuclei are counterstained with 4,6-diamidino-2-
phenylindole. Slides were examined using a Nikon Eclipse
800 upright microscope fitted with a Nikon DXM1200F
digital camera. (F) Gross appearance of engrafted and
contralateral tibias (bottom), demonstrating displacement
of hematopoiesis. Representative images of n  10 mice
per group; experiment was performed twice.
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To test whether the INA6 derivatives were more resistant to
BIO and whether BIO had a general effect on MM cell survival, we
incubated INA6-WT cells, INA6-10.13 cells, and other IL-6–
independent cell lines in the presence of 0 to 1600nM BIO. After
72 hours, BIO reduced viable cell yield in all cell lines tested in a
dose-dependent manner (supplemental Figure 2 and Figure 6A-B).
Guided by previous literature32,35 describing additional affinity for
BIO on cyclins, we measured cell-cycle parameters of the BIO-
treated cells. We expected cell-cycle rate, as defined by the
proportion of cells in S-phase, to be reduced by BIO, accounting
for the profound reduction of viable MM cells, but we found that
the effect on S-phase was marginal. Surprisingly, the most apparent
effect of BIO treatment was cell-cycle blockade at the G2M
checkpoint, with subsequent loss of the G2 peak to apoptosis, which
could be detected as a sub-G1 peak on the flow cytometric plots
(Figure 7). To further examine this observation, INA6-WT and
INA6-10.13 cells were incubated in increasing concentrations of
BIO for 18 hours and examined for early induction of apoptosis by
measurement of cleaved caspase-3 fragments. Both INA6-WT and
IL-6–independent subclone INA6-10.13 cells showed a dose-
dependent accumulation of cleaved caspase-3 fragments
(Figure 6C).
To mimic the putative protective effect by MSCs on MM cells
that is presumed to occur in vivo, cultures were supplemented with
10% MSC conditioned medium, a rich source of IL-6 and other
gp130 signaling cytokines.5 Although the addition of MSC condi-
tioned medium could rescue MM cells from BIO at lower doses,
this could be overcome by higher concentrations of BIO (1000nM).
This observation correlates with the finding that in numerous
endogenously IL-6–independent cell lines higher doses of BIO are
needed for a reduction in cell viability (supplemental Figure 2).
Therefore, it appears that BIO reduces MM cell expansion rather
than stimulating it. This probably occurs through the promiscuity
of the molecule in inhibiting a range of CDKs, a variety of other
pathways, and of course, GSK3
.
Discussion
Formation of OLs is a major contributor to morbidity associated
with MM, occurring throughout the skeleton and causing severe
bone pain and pathologic fractures. Until recently, OLs were
assumed to be the result of a cytokine-enriched microenviron-
ment generated by the MM resulting in osteoclastogenesis and
local bone resorption. Therefore, the treatment of OLs has
focused on the inhibition of osteoclastogenesis, such as by
administration of bisphosphonates.1 However, when the tumor
load is controlled, and the MM cells are absent from the OLs,
they seldom heal,8 suggesting that the anabolic axis of bone
formation had also been irreversibly altered. The first study to
Figure 3. Serum markers of bone disease in the INA6
model of myeloma. Serum levels of human Dkk-1 (A),
PYD (B), and TRAP (C) in engrafted and nonengrafted
(controls) mice. Range (left) and mean with SD (right) are
presented. Error bar represents SD for n  5 samples per
group. **P  .01, ***P  .005 with Student t test. Experi-
ment was performed twice.
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suggest a mechanism behind the apparent interference of bone
repair by MM was from Tian et al4 who observed that secretion
of an inhibitor of the canonical Wnt signaling pathway, Dkk-1,
by MM cells strongly correlated with the severity of OLs. Given
that Wnt signaling has been demonstrated necessary for the
differentiation of osteoprogenitors to osteoblasts, the presence
of high levels of Wnt inhibitors seemed to explain the deficit in
bone repair. Subsequently, it has been shown that exposure to
MM cells in vivo and in vitro irreversibly conditions osteopro-
genitors to be deficient in osteogenic differentiation, as well as
up-regulating some key cytokines that support MM cell sur-
vival.5,13-15 The preliminary evidence therefore suggests that
early inhibition of the action of Wnt inhibitors could (1) reduce
the formation of OLs by enhancing osteogenic signals and (2)
reduce the number of undifferentiated stromal cells that provide
support for MM cell expansion and survival. In this study, we
have attempted to target the activity of Wnt inhibitors that are
secreted by many MM tumors by GSK3
inhibition.
To enhance Wnt signaling in vivo, we chose the GSK3

inhibitor BIO, a nucleotide analog related to indirubin-3-
monooxime (IO). Preparations containing IO have been used
throughout history for the treatment of leukemic malignancies
and continue to be the subject of antimitotic research mainly
because of the inhibitory activity of IO on CDKs.32 BIO had
been modified to substantially improve its fidelity for GSK3
,
but some cross-reactivity with CDKs has been reported. Initial
experiments were designed to examine short-term safety of BIO
when administered systemically at large doses, and whether
steady state bone turnover could be perturbed. We calculated
dose based on the IC50 of BIO (2 M/L) and the LD50 of the
parent molecule, indirubin39 (400 mg/kg), which was the only
toxicity data available at the time. We administered a total of 60
nmoles of BIO, which was well below the published toxicity.
The mice received intraperitoneal injections of the inhibitor well
with no detectable complications over the treatment period.
After 6 weeks, the mice were euthanized, and bones were
analyzed. We hypothesized that GSK3
inhibition, through
BIO, would increase Wnt signaling in bone marrow MSCs, and
thus increase the proportion of MSCs with a greater propensity
for osteogenesis. This finding was supported by our previous
Figure 4. Histomorphometric scoring of hind limb
bones adjacent to tumors in mice treated with a
peritumoral dose of BIO or vehicle (DMSO). (A) Ex-
ample of sections used for the histomorphometric scor-
ing, demonstrating tibial and femoral destruction in un-
treated but INA6-engrafted knees. Note destruction of the
bone at the articular surfaces of the knee (arrowed) and
disruption of the trabecular structures that is less appar-
ent in the BIO-treated animals (asterisk). (B) Static
histomorphometric assays of peritumoral bone tissue in
INA6 bearing mice locally treated with BIO or with vehicle
(DMSO). The bone area (B. Area), marrow star volume
(MSV), trabecular thickness (Tb. Th.), trabecular star
volume (TSV), bone perimeter (B. Perim), and percent-
age of bone/total tissue (frac
100) are shown. Bars
represent mean ( SEM) for n  4-5 samples. *P  .05
with Student t test. Where values for P were borderline
significant, they are given on the plot. Experiment was
performed twice.
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observations that BIO accelerates osteogenesis by human MSCs
in vitro.27 Superficial histologic examination of the long bones
and vertebrae demonstrated that although no gross abnormali-
ties were evident, it was difficult to discern whether BIO had a
positive effect on bone quality. Micro-CT analysis was therefore
performed to measure the morphometric parameters of the bone
tissue. Upon analysis of the proximal tibial head and diaphysis,
it was apparent that the BIO had improved trabecular parame-
ters, increasing the Tb N. and reducing Tb Sp. In these
experiments, the effects of BIO seemed to be confined to the
trabecular bone without detectable changes in cortical bone.
Nevertheless, given the much lower rate of turnover in cortical
relative to trabecular bone, it is feasible that cortical parameters
could also be improved by extended BIO treatment. These data
suggest a role for GSK3
inhibition for osteoinductive therapy
and agree with previous animal studies24,26 and a human study
exploring the effects of chronic lithium use and fracture
frequency.40 It is noteworthy that although our results do not
unequivocally prove a direct effect of BIO on osteoblast
activity, these data described in this study and in related studies
strongly suggest that osteoblasts and osteoprogenitors are
indeed the target of BIO.
The main goal of this study was to examine the effect of
direct GSK3
inhibition on the formation and repair of OLs in
MBD by direct histomorphometric evaluation of the bone-tumor
interface. Given that Wnt inhibition (through Dkk-1 and soluble
Frz-related protein) has been implicated in the formation and
persistence of such lesions4-7,10,11 and immunosequestration of
Dkk-1 as well as peritumoral Wnt3a administration can reduce
MBD in vivo,19,34,41 direct modulation of Wnt signaling through
GSK3
inhibition appeared feasible and potentially useful for
clinical application. Several excellent in vivo MM assays have
been described, but most recapitulate the systemic elements of
the disease, resulting in randomly distributed foci. These models
effectively reproduce MM pathology, but may have limited
utility for specifically examining localized MBD.42-44 A model
involving the local implantation of MM cells and a human bone
substrate would be particularly suitable, but the procedure
requires primary human biomaterials45 that are in short supply
for most investigators. Recently, direct implantation models,
similar to the procedure described in this study have been used
to examine the antitumoral effects of tumor necrosis factor-
related apoptosis-inducing ligand involving RPMI 8226 and
KMS-11 human MM cell lines.46 The model described in this
study produces a solitary plasmacytoma with highly aggressive
bone resorption qualities. Although systemic effects are negli-
gible, serum levels of bone resorption markers could be used to
follow bone involvement at the single site (Figure 3), making
this model extremely convenient for direct study of MBD in a
well-defined system. It therefore contrasts with the INA6-based
model of disseminated MM disease originally reported by
Burger et al.12
Upon detection of INA6-plasmacytomas, peritumoral BIO
injections were performed. The dosage corresponded with in
vitro studies5 that defined the effective concentration to be
approximately 280nM. Because the pharmacokinetics of BIO is
currently uncharacterized and bioavailability could not be
guaranteed, peritumoral injection was deemed the most reliable
way to maintain appropriate local concentrations. Upon conclu-
sion of the study, affected limbs were assigned a code and
analyzed by histomorphometry, and pathology was outsourced
to Premier Laboratories (Boulder, CO). In all samples, bone
involvement was substantial, and localized to the bone-tumor
interface. However, in agreement with the systemic injection
Figure 5. Pathology and biochemical markers in tumor-bearing mice treated with a peritumoral dose of BIO or vehicle (DMSO). (A) Serum PYD and TRAP 5b in
response to peritumoral BIO administration. Despite values for P  .05, BIO-treated mice have generally lower levels of circulating resorption markers. (B) ELISA detection of
soluble
-catenin in tissue lysates generated from the injection sites of treated and untreated animals. BIO treatment increases
-catenin levels, *P  .05. (C) Pathologic
scoring on a 5-point linear scale of bone-tumor interface. Tumor necrosis and tumor cells refer to the sectional area containing dead or live tumor cells, respectively. Error bar
represents SEM for n  4-5 samples, *P  .05. Experiment was performed twice.
1648 GUNN et al BLOOD, 3 FEBRUARY 2011

VOLUME 117, NUMBER 5

studies, histomorphometric measurements demonstrated that
BIO significantly improved bone quality tibias and femurs
(Figure 4). Of note is the observation that contralateral limbs
were also affected by BIO administration, although they did not
harbor detectable levels of MM cells (data not shown). The
results therefore support a potential osteoprotective role for
GSK3
inhibitors in MM and probably other malignant diseases
of the skeleton. Furthermore, the apparent systemic effect of
BIO suggests that testing in disseminated and immune-
competent models of MM lines is a necessary course of further
study.
One understandable argument against the use of GSK3

inhibitors arises from the observation that Wnt signaling can
increase MM cell proliferation in early stages of tumorigenesis
(eg,28,47-49). However, we found that BIO administration did not
adversely affect tumor morbidity. In contrast, we found that BIO
increased the necrotic component of the plasmacytoma and as a
result, reduced the area of viable tumor observed on pathology
slides. Furthermore we could partially reproduce the findings of a
previous study30 by demonstrating that BIO could reduce MM cell
viability and cell-cycle turnover and thus slow MM cell expansion
in vitro. These observations therefore suggest that a less specific
inhibitor, with affinity for CDKs as well as GSK3
may indeed be
the agent of choice for treatment of severe OLs. Nevertheless, very
specific inhibition of GSK3
by thiadiazolidinone has been
reported to inhibit in vitro MM cell expansion and cause apoptosis
through a mechanism involving the proapoptotic protein Fas
ligand.31 Furthermore, fortification of bone tissue by increased Wnt
signaling results in displacement and regression of modeled MM
due to enhanced osteoblast differentiation without affecting prolif-
eration or differentiation of preosteoclasts.29,41,50 Therefore, BIO is
likely to act through several additive or synergizing mechanisms
that target MM cells and the surrounding microenvironment.
From the data presented in this study, adjuvant osteoprotective
therapy in the form of GSK3
inhibition represents a feasible
strategy for the adjunct treatment of MM. Contrary to conventional
Figure 6. Effect of BIO on viability of INA6 MM cells in vitro. Viability of INA6-WT (A) and INA6-10.13 (B) cells exposed to increasing concentrations of BIO (0-1600nM) for
72 hours with or without addition of 10% MSC-conditioned medium as determined by trypan blue exclusion. Mean ( SD) for n  6-9 samples is shown. ANOVA was
performed after arcsine transformation of proportions. Asterisk indicates cut-off dose of BIO causing statistically significant reduction of viability (*P  .05). There were 2 to
3 sets of independent triplicates analyzed. (C) Western blot for cleaved caspase-3 showing induction of apoptosis in INA6-WT and INA6-10.13 cells by BIO after 18 hours
exposure. Note the initial rescuing effect of MSC-conditioned medium, which is overcome by higher concentrations of BIO (1000nM). Membranes were cut and exposed with
different sensitivity using an automated imaging system. To ensure comparability, INA6-WT or INA6-10.13 cells were run on the same gel, respectively, and lanes were spliced
together for presentation purposes. Experiment was performed twice independently.
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dogma, GSK3
inhibition did not increase tumor load, but rather
induced tumor necrosis. With improved knowledge of its pharma-
cokinetics and safety profile in humans, this inhibitor could
represent a valuable supportive clinical tool for treatment of MBD,
and in general, bone destruction occurring through other malignant
diseases.
Acknowledgments
We thank Tara G. Ooms, DVM, DACLAM, Department of
Comparative Medicine, Tulane University, New Orleans, LA, for
help with the histology scoring and Dina Gaupp, Department of
Medicine, Center for Gene Therapy, Tulane University Health
Sciences Center, for excellent help with the histology. W.G.G.
thanks Tina Kilts, National Institute of Dental and Craniofacial
Research, National Institutes of Health, Bethesda, MD, for training
and the development and optimization of the injection techniques.
This work was supported in part by National Institutes of Health
grants DK071780, and R020152, by the Louisiana Gene Therapy
Research Consortium, and by Scott and White Hospital, Texas
A&M Health Science Center (to C.A.G.)
Authorship
Contribution: W.G.G., U.K., and C.A.G. designed and performed
research, analyzed and interpreted the data and wrote the manu-
script; and N.L. performed research.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Carl A. Gregory PhD, Institute for Regenera-
tive Medicine, Texas A&M Health Sciences Center, 5701 Airport
Rd, Module C, Temple, TX 76502; e-mail: cgregory@medicine.
tamhsc.edu.
Figure 7. Effect of BIO on cell cycle of INA6MMcells in vitro. Cell-cycle analysis of INA6-WT and INA6-10.13 cells exposed to various concentrations of BIO for 72 hours. At
higher concentrations, a significant sub-G1 peak is evident (arrow), demonstrating dead cell debris. Note the loss of the G2 population. Experiment was performed twice
independently.
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