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OPEN
ORIGINAL ARTICLE
Notch pathway inhibition controls myeloma bone disease in the
murine MOPC315.BM model
R Schwarzer
1,9
, N Nickel
1,9
, J Godau
1,9
, BM Willie
2
, GN Duda
2
, R Schwarzer
3
, B Cirovic
4
, A Leutz
4
, R Manz
5
, B Bogen
6,7
,BDo¨ rken
1,4
and F Jundt
1,8
Despite evidence that deregulated Notch signalling is a master regulator of multiple myeloma (MM) pathogenesis, its contribution
to myeloma bone disease remains to be resolved. Notch promotes survival of human MM cells and triggers human osteoclast
activity in vitro. Here, we show that inhibition of Notch through the g-secretase inhibitor XII (GSI XII) induces apoptosis of murine
MOPC315.BM myeloma cells with high Notch activity. GSI XII impairs murine osteoclast differentiation of receptor activator of NF-kB
ligand (RANKL)-stimulated RAW264.7 cells in vitro. In the murine MOPC315.BM myeloma model GSI XII has potent anti-MM activity
and reduces osteolytic lesions as evidenced by diminished myeloma-specific monoclonal immunoglobulin (Ig)-A serum levels and
quantitative assessment of bone structure changes via high-resolution microcomputed tomography scans. Thus, we suggest that
Notch inhibition through GSI XII controls myeloma bone disease mainly by targeting Notch in MM cells and possibly in osteoclasts
in their microenvironment. We conclude that Notch inhibition is a valid therapeutic strategy in MM.
Blood Cancer Journal (2014) 4, e217; doi:10.1038/bcj.2014.37; published online 13 June 2014
INTRODUCTION
Impressive improvement in the treatment of multiple myeloma
(MM) has occurred during the past decade, as evidenced by an
increase in the number of progression-free cases and overall
survival rates of patients through the use of target-specific agents.
However, there is still a need for the identification of novel
substances, as in most cases MM remains an incurable disease.
1
High-dose therapy with the alkylating agent melphalan in
combination with autologous stem cell transplantation is used
as the standard therapy in patients, who are eligible for the
procedure.
1
In addition, novel agents such as the proteasome
inhibitor bortezomib are successfully employed to further increase
remission durations.
1
Nevertheless, still most MM patients suffer
from relapses even after many years due to intrinsic or acquired
drug resistance.
1
Recently, we showed that Notch signalling is a survival factor in
Hodgkin lymphoma, anaplastic large-cell lymphoma and particu-
larly in MM.
2–6
The evolutionarily conserved Notch signalling
pathway mediates cell–cell communication and regulates cell
growth, cell death, differentiation programs and self-renewing
processes in a context-dependent manner.
7,8
Its deregulation is
involved in developmental syndromes and cancer.
7,9
In order to
transmit a signal, Notch receptors undergo a series of proteolytic
cleavages after binding their cognate ligands of the Delta-like and
Jagged family.
7,10
Thereby the g-secretase membrane protease
complex cleaves the membrane-bound form of Notch. As a result,
the intracellular domain of Notch receptors (NIC) translocates
into the nucleus and takes part in the Notch transcriptional
complex, which in addition includes scaffold proteins of the
mastermind-like family and the DNA-binding factor RBP-Jk.
g-secretase inhibitors block the cleavage of the Notch receptors
and inhibit release of NIC.
7,8
It has been clearly established that Notch is a master regulator
of MM pathogenesis providing a rationale for evaluating anti-
Notch approaches in MM.
11
Houde et al.
12
showed that Notch
activation can be triggered by overexpressed Jagged2 in the
premalignant condition monoclonal gammopathy of
undetermined significance. Overexpression of Jagged2 can be
triggered either by promoter hypomethylation,
12
through
aberrant expression of the ubiquitin ligase Skeletrophin
13
or by
loss of SMRT/NCoR2 corepressor, which results in abnormal
acetylation of the Jagged2 promoter.
14
In MM, Notch confers
resistance to apoptotic stimuli and protects against
chemotherapy-induced toxicity.
15,16
Nefedova et al.
15
described
that Notch signalling is involved in de novo drug resistance
triggered by bone marrow stroma. Most recently, it has been
shown that Notch activation contributes to bortezomib resistance
in MM.
17
In addition, Notch might accelerate MM progression by
promoting cancer stem cell self-renewal.
18,19
Our own data
showed that activated Notch signalling promotes proliferation
and survival of MM cells.
3
The Notch1 receptor and both its
cognate ligands, Jagged1 and Jagged2, are highly expressed in
primary and cultured MM cells.
3
In addition, activated Notch
signalling is involved in the interactions between MM cells and
their microenvironment.
5
We have demonstrated that Notch
inhibition by g-secretase inhibitors might be a promising
treatment option in MM, as these inhibitors control proliferation
in cultured MM cells
3
and suppress Notch-dependent osteoclast
1
Department of Hematology, Oncology and Tumor Immunology, Campus Virchow-Klinikum, Charite
´—Universita
¨tsmedizin Berlin, Berlin, Germany;
2
Julius Wolff Institute and
Berlin-Brandenburg Center for Regenerative Therapies, Charite
´—Universita
¨tsmedizin Berlin, Berlin, Germany;
3
Institute of Biology and Molecular Biophysics, Humboldt University
Berlin, Berlin, Germany;
4
Max Delbru¨ ck Center for Molecular Medicine, Berlin, Germany;
5
Institute for Systemic Inflammation Research (ISEF), University of Lu¨ beck, Lu¨ beck,
Germany;
6
Centre for Immune Regulation, Institute of Immunology, Oslo University Hospital, Oslo, Norway;
7
Jebsen Centre for Research on Influenza Vaccines, University of Oslo,
Oslo, Norway and
8
Department of Internal Medicine II, University Hospital Wu¨rzburg, University of Wu¨ rzburg, Wu¨ rzburg, Germany. Correspondence: Professor F Jundt,
Department of Internal Medicine II, University Hospital Wu¨ rzburg, University of Wu¨ rzburg Medizinische Klinik und Poliklinik II, Oberdu¨ rrbacher Strasse 6, Wu¨ rzburg 97080,
Germany.
E-mail: jundt_f@ukw.de
9
These authors contributed equally to this work.
Received 23 April 2014; accepted 30 April 2014
Citation: Blood Cancer Journal (2014) 4, e217; doi:10.1038/bcj.2014.37
&
2014 Macmillan Publishers Limited All rights reserved 2044-5385/14
www.nature.com/bcj
activation in vitro.
5
In conclusion, we provided evidence for Notch
activation and deregulation in late-MM stages.
In this study, we used the g-secretase inhibitor XII (GSI XII) for
Notch inhibition. It has been demonstrated that GSI XII controls
the Notch pathway in human lymphoma cells,
6,20,21
and enhances
cytotoxic effects of bortezomib reducing proteasome activity in
MM.
20
To confirm its Notch-specific activity in MM, GSI XII has
been compared with other selective compounds such as the
g-secretase inhibitor DAPT and SAHM1, the dominant-negative
fragment of the Notch co-activator mastermind-like 1, which
selectively blocks the Notch transcriptional complex.
21
These
analyses revealed that GSI XII has similar anti-Notch effects as
DAPT or SAHM1, but even more effectively inhibits MM growth
and induces apoptosis, possibly due to concomitant proteasome
inhibition.
21
To test whether Notch inhibition through GSI XII affects
myeloma bone disease, we used a recently described
murine MOPC315.BM model,
22
which recapitulates the main
characteristics of human MM with particular respect to
myeloma-specific monoclonal IgA serum levels and abundant
osteolytic lesions.
MATERIALS AND METHODS
Mice
Six-week-old female BALB/c mice were obtained from Charles River
(Sulzfeld, Germany). All experiments were approved by the local
committee of the Landesamt fu¨ r Gesundheit und Soziales Berlin
(Berlin, Germany).
Cell culture and reagents
Human MM cell lines (NCI-H929 and OPM-2) were used (DSMZ,
Braunschweig, Germany). MOPC315 cells were obtained from ATCC
(Manassas, VA, USA) as an in vitro-adapted cell line and were repeatedly
injected subcutaneously into BALB/c mice, resulting in the MOPC315.4 cell
line.
23
The MOPC315.4 cell line was injected intravenously into BALB/c
mice. After nine in vivo/in vitro cycles, a cell line was generated that had a
tropism for bone marrow and was therefore named as MOPC315.BM.
22
MOPC315.BM cells produce an IgA myeloma protein, M315, which can be
measured by enzyme-linked immunosorbent assay. MOPC315.BM cells
were incubated 24–48 h with increasing doses of GSI XII, which was
obtained from Calbiochem (San Diego, CA, USA). The RAW264.7 murine
monocyte/macrophage cell line was cultured in Dulbecco’s modified
Eagle’s medium and induced to differentiate into bone resorbing
osteoclasts by 10 ng/ml receptor activator of NF-kB ligand (RANKL; 462-
TEC-010, R&D Systems, Wiesbaden, Germany) in minimal essential medium
alpha as described.
24
Immunoblotting
Whole-cell extracts were prepared and immunoblotting was performed as
described.
2
Nuclear extracts were prepared using the Nuclear Extract Kit
(Active Motif, Carlsbad, CA, USA). Blots were incubated with monoclonal
rabbit anti-Notch1 antibodies (cat# 1935-1, Epitomics, Burlingame, CA,
USA), anti-cleaved Notch1 NIC (Val1744; cat# 4147; Cell Signaling
Technologies, Frankfurt, Germany), anti-poly-(ADP-ribose) polymerase
(PARP, cat# 9532, Cell Signaling Technologies), anti-cleaved PARP
(Asp214; cat# 9541, Cell Signaling Technologies) or rabbit monoclonal
anti-tubulin antibodies (cat# 2125; Cell Signaling Technologies). Detection
was performed using Pico or Dura chemiluminescence reagents (Perbio
Science, Bonn, Germany).
RNA preparation and quantitative reverse transcription-PCR
analysis
RNA preparation and complementary DNA synthesis were performed as
described.
4
Reverse transcription-PCR analysis was performed as described
using primers and probes for murine HEY1, RANKL, NFATc1 and TRAP5.
4
As
an internal control murine hypoxanthin-guanin-phosphoribosyltransferase
was amplified. Primer sequences are available upon request.
Viability assay and assessment of apoptosis
Viability of cells was determined by CellTiter-Glo Luminescent Cell Viability
Assay (Promega, Mannheim, Germany). Each treatment was done in three
independent replicates. Luminescence was recorded; average values were
calculated and normalized to the respective dimethyl sulphoxide-treated
sample. Amount of apoptotic cells was determined using the human
AnnexinV-FITC Kit (Bender Medsystems, Vienna, Austria).
TRAP staining
After cultivation of 0.75 10
5
RAW264.7 per 12-well for 72 h, cells were
washed with phosphate-buffered saline (PBS) and fixed in 4% PBS-buffered
formaldehyde for 10 min at room temperature (RT). Cells were shown to be
tartrate-resistant acid phosphatase (TRAP)-positive by staining using the
Acid Phosphatase Leukocyte Kit (Sigma-Aldrich, Seelze, Germany) with an
adapted protocol. Staining solution was prepared with 0.2 M tartrate and
using half as much GBC (40-amino-2,30-dimethylazobenzene) solution as
described in manufacturer’s protocol. After adequate incubation with
staining solution, cells were once washed with water and stored in PBS/4%
formaldehyde for further analysis.
Differential interference contrast (DIC) microscopy
After labelling, TRAP-positive cells were subjected to DIC microscopy.
Images were collected using an inverted Olympus IX-81 microscope
(Olympus, Tokyo, Japan) equipped with a cooled monochrome CCD
camera. All images that were used for the analysis are shown in
Supplementary Figure S1. The cells were imaged using a 20 UPlanFL
air objective (numerical aperture 0.4) with a typical exposure time of 10 ms.
Cell image analysis
DIC images were analysed using the CellProfiler image analysis software
(version 2.0) using a self-provided pipeline (Supplementary Figures S2A–C).
25
Briefly, nuclei were identified by Hoechst 33258 (Sigma-Aldrich) staining and
subsequently, based on propagation from the nuclei, cell segmentation was
performed on inverted DIC images after automated editing of image
properties, such as image intensity and contrast. Then, the normalized
staining intensity of individual cells was assessed from the original, non-
processed images and saved in a spreadsheet for further analysis.
Enzyme-linked immunosorbent assay
Murine blood samples were obtained from cheeks. Blood samples were
allowed to clot for 2 h at RT before centrifugation for 10 min at 2000 g. Sera
were collected and stored at 20 1C. The M315 myeloma protein, which is
2,4-dinitrophenol specific, was detected by enzyme-linked immunosorbent
assay. Ninety-six-well plates were coated with 1 mg/ml dinitrophenol-
conjugated bovine serum albumin and incubated at 41C overnight.
Unspecific binding sites were blocked for 30 min at RT using PBS/5%
bovine serum albumin followed by three washing steps with PBS/0.1%
Tween. Serum samples were diluted 1:500–1:5000 in PBS containing 0.1 or
5% bovine serum albumin. M315 standard protein was serially diluted
within a range of 10–2560 ng/ml. Plates were incubated for 2 h at 37 1C
with serum samples and standard protein. After plates were washed three
times with PBS/0.1% Tween, 1 mg/ml biotinylated rat anti-mouse IgA (clone
C10-1, BD Pharmingen, Heidelberg, Germany) was added for 1 h at RT.
Plates were again washed three times and incubated with streptavidin-
conjugated alkaline phosphatase (1:3000; Roche, Mannheim, Germany) for
1 h at RT. After three washes, phosphatase substrate (Roche) was added at
1 mg/ml in substrate buffer and absorbance was measured at 415 nm. Sera
were collected from vehicle-treated and GSI XII-treated mice at time points
indicated (Figure 2a) or every 2–8 days (Figure 3a).
High-resolution mCT scans
For trabecular and cortical bone structural analysis, microcomputed
tomography (mCT) at an isotropic voxel size of 10.5 mm (vivaCT 40, Scanco
Medical, Bru¨ ttisellen, Switzerland; 55 kVp, 145 mA, 600 ms integration time,
no frame averaging) was performed on dissected mice tibias to assess
bone. Guidelines for assessment of bone microstructure in rodents were
applied for evaluation.
26
For each tibia, a trabecular and cortical bone
volume of interest was defined. The total tibia length was measured using
digital calipers on freshly isolated bones. The trabecular volume of interest
included secondary spongiosa in the proximal metaphysis, starting 700 mm
below the growth plate and extending distally 5% of the tibial length.
Notch in myeloma bone disease
R Schwarzer et al
2
Blood Cancer Journal &2014 Macmillan Publishers Limited
The trabecular bone volume of interest excluded the cortical shell.
Thresholds of 456 mg (14 days GXI XII treatment) and 447 mg (36 days of
GSI XII treatment) hydroxylapatite/cm
3
were used to segment proximal
trabecular bone from water and soft tissue. Trabecular (Tb.) bone outcome
parameters included: bone volume fraction (BV/TV, mm
3
/mm
3
), trabecular
thickness (Tb.Th, mm), average number of trabeculae per unit length (Tb.N,
1/mm), trabecular separation (Tb.Sp, mm) and trabecular volumetric bone
mineral density (mg hydroxylapatite/cm
3
). The second analysed volume of
interest included only cortical bone at the proximal metaphysis, excluding
trabecular bone and the marrow cavity. A 563-mg hydroxylapatite/cm
3
threshold (36 days GXI XII treatment) was used to segment cortical bone
from water and soft tissue. The following parameters were used for
analysis of cortical (Ct.) bone: cortical bone area ¼cortical volume/(number
of slices slice thickness) (Ct.Ar, mm
2
), cortical thickness (Ct.Th, mm) and
total cross-sectional area inside the periosteal envelope (Tt.Ar, mm
2
). Tt.Ar
was contoured to only include the metaphyseal cortical bone and porosity
within the bone, with the medullary canal (including trabecular bone and
bone marrow) excluded from the analysis. Also, cortical porosity
area ¼cortical porosity volume/(number of slices slice thickness)
(Ct.Po.Ar, mm
2
) and cortical volumetric bone mineral density were
measured. All quantitative analyses were performed with the system’s
software (Scanco Eval 6.5-1, Scanco Medical, Bru¨ttisellen, Switzerland).
MM model and drug treatment
MOPC315.BM cells were cultured in RPMI with 10% fetal calf serum in vitro
(37 1C, 5% CO
2
) and harvested for injection into tail veins. 5 10
5
MOPC315.BM cells were injected per mouse. The mice were killed on days
indicated or when end points were reached. Mice were treated with
10 mg/kg GSI XII intraperitoneally either for 14 days (Figure 3, dissolved in
PBS/cremophor/peanut oil, PCO) or for 36 days (Figure 4, dissolved in
dimethyl sulphoxide). Quantification of bone structure changes was
performed after 14 days and after 36 days of treatment, respectively.
Statistics
Statistical analysis was performed with Prism 5 software (GraphPad, La
Jolla, CA, USA). One- or two-tailed t-tests were used as appropriate to
analyse statistical significance. All data are shown as mean±s.d. Logrank
test was used to compare serum increases over time between vehicle-
treated and GSI XII-treated mice as shown in a Kaplan–Meier plot. P-values
o0.05 were considered as statistically significant.
RESULTS
Notch inhibition reduces viability and induces apoptosis in
MOPC315.BM cells with deregulated Notch activity in vitro
Our recent data showed aberrant Notch activation in cultured and
primary human MM cells.
3,5
In contrast, freshly isolated mature
CD19
þ
B cells and CD19
þ
B cells, which we differentiated to
CD38
þ
plasmablastic cells in vitro, were almost completely devoid
of Notch expression.
3,5
To test whether the murine MM cell line
MOPC315.BM has activated Notch signalling, we first confirmed
expression of the full-length transmembrane-bound form of the
Notch receptors (Notch1 and Notch2) and their ligands (Jagged1
and Jagged2) by immunoblotting. MOPC315.BM cells express
both Notch receptors and ligands (Figure 1a). Human NCI-H929
and OPM-2 cultured MM cells served as positive controls
(Figure 1a). To further demonstrate activated Notch signalling,
we used nuclear extracts and verified abundant expression of the
intracellular form of Notch1 (N1IC; Figure 1b). Therefore, we
confirmed activation of the Notch pathway in MOPC315.BM cells.
In addition, N1IC could be specifically downregulated through the
g-secretase inhibitor GSI XII (Figure 1b). Concomitantly, messenger
RNA expression of the Notch target gene HEY1 was suppressed
after GSI XII treatment in MOPC315.BM cells (Figure 1c). These data
indicate that activated Notch signalling was blocked in
MOPC315.BM cells by use of GSI XII.
To analyse functional consequences of Notch inhibition for
tumour cell biology, we performed viability assays, Annexin
V-staining and immunoblotting for PARP cleavage. GSI XII reduced
cell viability (Figure 1d) and induced apoptosis (Figures 1e and f)
in MOPC315.BM cells in a dose-dependent manner.
Notch inhibition impairs osteoclast differentiation of RANKL-
stimulated RAW264.7 cells
Recently, we provided evidence in vitro that Notch inhibition has
no effect on osteoblasts and their progenitors and blocks human
osteoclast activity by downregulation of TRAP5, a marker for
osteoclast differentiation and activity.
5,27
To analyse whether GSI
XII impairs murine osteoclast differentiation, we used the murine
monocyte/macrophage cell line RAW264.7.
24
RAW264.7 cells were
efficiently induced to differentiate into osteoclasts within 72 h
12 μM15μM
DMSO GSI XII
2-ΔΔCt
HEY1
MOPC315.BM
OPM-2
NCI-H929
Jagged1
Notch2
Jagged2
Notch1
α-tubulin
N1IC
PARP
DMSO
7.5 μM
10 μM
GSI
DMSO GSI XII
relative viability
0.2
0.4
0.6
0.8
1.0
1.2
0
0.2
0.4
0.6
0.8
1.0
1.2
0
1.4
cleaved
PARP
α-tubulin
DMSO
12 μM
15 μM
GSI
DMSO
PI
AnnexinV
1%
10 μM GSI
PI
AnnexinV
46%
7%
12 μM 15 μM
1%
Figure 1. Notch inhibition controls viability and induces apoptosis in
MOPC315.BM cells with activated Notch signalling. (a) Immunoblot-
ting of Jagged1, Jagged2, Notch1 and Notch2 in MOPC315.BM cells
and in the human MM cell lines NCI-H929 and OPM-2. Staining for
a-tubulin served as control for equal loading. (b) Immunoblotting of
the intracellular form of Notch1 (N1IC), and PARP (loading control) in
nuclear extracts of MOPC315.BM cells after GSI XII treatment.
(c) Quantitative reverse transcription-PCR analysis of HEY1 messenger
RNA expression in MOPC315.BM cells after GSI XII treatment.
(d) Viability of MOPC315.BM cells after GSI XII treatment. Numbers of
viable cells are given relative to dimethyl sulphoxide (DMSO)-treated
samples. (e) Immunoblotting of PARP cleavage in whole-cell extracts of
MOPC315.BM cells treated with different concentrations of GSI XII.
(f) AnnexinV/propidium iodide (PI) staining of DMSO and GSI XII-
treated cells. Percentages of early (FITC þPI ) and late apoptotic or
necrotic (F ITC þPI þ) cells as determined by AnnexinV/PI staining.
Notch in myeloma bone disease
R Schwarzer et al
3
&2014 Macmillan Publishers Limited Blood Cancer Journal
through stimulation with 10 ng/ml RANKL.
24
RANKL is known to
selectively induce NFATc1 expression during differentiation of
RAW264.7 cells into TRAP þosteoclasts.
28,29
Reverse transcription-
PCR analysis revealed that in RANKL-induced osteoclasts GSI XII
diminished messenger RNA expression of the Notch target gene
HES1 and in parallel of the transcription factor NFATc1, a master
regulator of osteoclast differentiation (Figures 2a and b;
Supplementary Figure S3A). Concomitantly, messenger RNA
expression of the osteoclast-specific gene TRAP5 was down-
regulated both on the messenger RNA (Figure 2b, right panel;
Supplementary Figure S3B) and on the protein level (Figure 2c).
TRAP5 is a known target gene of NFATc1.
28
Both NFATc1 and
TRAP5 were regulated in a dose-dependent manner by GSI XII
(Supplementary Figures S3A and B). We further performed DIC
microscopy, to quantify differences in the staining intensity of
TRAP þand TRAP þþ osteoclasts after Notch inhibition (Figures
2d and e; Supplementary Figure 4). Our data showed that GSI XII
treatment almost completely abolished RANKL-induced osteoclast
differentiation in vitro.
Notch inhibition reduces myeloma-specific paraprotein levels in
the MOPC315.BM model
Next, GSI XII efficacy was examined in the MOPC315.BM mouse
model.
22
To that end, we designed two different experiments. The
first experimental setup was designed to evaluate GSI XII
treatment in mice, which had already established MM cell
growth. Mice were inoculated with MOPC315.BM cells and
randomized into vehicle-treated mice and GSI XII-treated mice
once myeloma-specific monoclonal IgA (M315) serum levels
DMSO GSI DMSO GSI
- RANKL + RANKL
2−ΔΔCt
HES1
0
5
10
15
20
DMSO GSI DMSO GSI
- RANKL + RANKL
2-ΔΔCt
NFATc1
0
50
100
150
200
250
DMSO GSI DMSO GSI
- RANKL + RANKL
2-ΔΔCtCt
TRAP5
0
0.08
0.16
0.24
0.32
0.40
0.48
intensit
y
units
TRAP staining
TRAP+
TRAP++
100 μm
- RANKL + DMSO
100 μm
+ RANKL + GSI
100 μm
+ RANKL + DMSO
TRAP staining
DMSO GSI
- RANKL + RANKL
TRAP+
***p<0.0001
DMSO GSI
- RANKL + RANKL
TRAP++
**p=0.0011
-RANKL
+ RANKL + DMSO
+ RANKL + GSI
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0
5
10
15
20
25
30
35
100
80
60
40
20
0
events (%)
events (%)
60
40
20
0
events (%)
Figure 2. Notch inhibition impairs differentiation of RAW264.7 cells into osteoclasts in vitro (aand b) Treatment of the murine monocyte/
macrophage cell line RAW264.7 without ( ) and with ( þ) 10 ng/ml RANKL for stimulation of osteoclast differentiation. Quantitative reverse
transcription-PCR analysis of HES1, NFATc1 and TRAP5 in RAW264.7 cells treated with 10 mMGSI XII. Dimethyl sulphoxide (DMSO) was used as
solvent control. (c) Osteoclast differentiation visualized by TRAP staining. RAW264.7 cells differentiated into TRAP-positive osteoclasts after
72 h of RANKL-stimulation (middle). Treatment of RAW264.7 cells with 10 mMGSI XII inhibited osteoclast differentiation and RANKL-induced
RAW264.7 cells remain TRAP negative (right). RAW.264.7 cells treated with DMSO as solvent control (left). (d) Differential interference contrast
microscopy was used to quantify differences in staining intensity of TRAP þand TRAP þþ RANKL-stimulated osteoclasts. Cells were treated
with GSI XII or DMSO as solvent control. (e) Percentages of TRAP þand TRAP þþ cells. Two-tailed t-test was used for statistical analysis.
P-values as indicated.
Notch in myeloma bone disease
R Schwarzer et al
4
Blood Cancer Journal &2014 Macmillan Publishers Limited
approximated 10–30 mg/ml at two consecutive time points
(Figure 3a). Threshold corresponded to the lowest detectable
IgA levels. GSI XII was then administered at a dose of 10 mg/kg for
14 days (Figure 3a). GSI XII-treated mice showed a strong
reduction in M315 levels as compared with vehicle-treated mice
over time (Figure 3b). In addition, progression-free survival of GSI
XII-treated mice was significantly increased (Figure 3c). Progres-
sion was determined when M315 levels exceeded 150 mg/ml.
If bone structure changes were already apparent, it would be
difficult to detect changes in bone mass and volume during a
treatment period of only 14 days. Therefore, we designed a
second experiment (Figure 4a) and focused our analysis on IgA
levels (Figure 4b) and bone structure changes employing mCT
scans (Figures 4c and d; Tables 1 and 2). MOPC315.BM cells were
transplanted at day 0 and mice were randomized into two groups
(vehicle- and GSI XII-treated mice) at the same day. GSI XII was
administered at a dose of 10 mg/kg for 36 days (Figure 4a). GSI XII
treatment reduced tumour burden as evidenced by diminished
M315 levels at day of death (Figure 4b). We conclude that GSI XII
reduces MM development and progression but is not able to
completely cure treated mice. In GSI-treated groups still a few
mice develop MM, however, with lower tumour load and slower
increase of IgA levels over time as compared with control animals.
Notch inhibition diminishes osteolytic lesions in the MOPC315.BM
mice with GSI XII
We recently demonstrated osteolytic lesions in the MOPC315.BM
model by use of mCT analysis, TRAP staining for osteoclasts in the
bone marrow and measurement of serum Ca2 þlevels in BALB/c
mice.
22
To determine quantitative bone structural changes
between vehicle-treated and GSI XII-treated mice, we performed
mCT analysis and used recently published guidelines for
assessment of bone microstructure in rodents.
26
Representative
data of proximal tibiae of mice are shown (Figures 4c and d;
Tables 1 and 2). Transversal sections (Figure 4c) and three-
dimensional reconstruction (Figure 4d) of tibiae revealed
increased cortical porosity (Ct.Po.Ar; Figure 4e; Table 1) and
diminished trabecular structures (Table 2) in vehicle-treated mice
as compared with GSI XII-treated mice. Quantitative analysis of
bone structural changes showed significant changes between the
two groups, particularly in trabecular bone structures (Table 2). For
example, bone volume fraction (BV/TV), trabecular number (Tb.N)
as well as volumetric bone mineral density were higher in GSI XII-
treated mice as compared with vehicle-treated mice (Table 2),
indicating that Notch inhibition through GSI XII reduces bone loss
and diminishes osteolytic lesions in the MOPC315.BM model.
DISCUSSION
In this study, we used our recently described MOPC315.BM MM
model to investigate whether Notch inhibition through the
g-secretase inhibitor GSI XII has anti-MM activity in vivo.
22
We
showed that MOPC315.BM cells are characterized by aberrant
Notch activity in vitro, which was specifically downregulated
through GSI XII. As a result, viability of murine MM cells was
reduced and apoptosis was induced. Tumour biologic relevance of
these findings was revealed by our in vivo studies: (1) decreased
MOPC315.BM
injection
d0
IgA=10-30 μg/ml
d5 d8 d10 d12 d14 d15 - serum IgA analysis
sacrifice
bone analysis
* p=0.0113
daily treatment
day 0
1.5
3
4.5
6
0
0
25
50
75
100
0
IgA [mg/ml]
day 8 day 10 day 12
vehicle GSI vehicle GSI vehicle GSI vehicle GSI
n. s. * p=0.023 * p=0.036 * p=0.035
51015
GSI
vehicle
days
progression-free [%]
Figure 3. Notch inhibition reduces myeloma-specific paraprotein levels in the MOPC315.BM MM model. (a)Schemeindicatestimepointsof
MOPC315.BM injection, serum collection for measurement of MM-specific M315 IgA levels and GSI XII treatment. GSI XII treatment (10 mg/kg,
daily) for 14 days started after M315 serum levels reached B10–30 mg/ml. (b) M315 serum levels of vehicle-treated (n¼13) and GSI XII-treated
(n ¼9) mice on days 0, 8, 10 and 12 of treatment. Two-tailed t-test was used for statistical analysis. (c) Kaplan–Meier plot for time points when
150 mg/ml M315 serum levels were reached. Logrank test was used for statistical analysis. P-values as indicated.
Notch in myeloma bone disease
R Schwarzer et al
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&2014 Macmillan Publishers Limited Blood Cancer Journal
myeloma-specific IgA serum levels and (2) decreased myeloma
bone disease indicated diminished tumour burden in the
MOPC315.BM MM model. We have previously shown that the
MOPC315.BM model closely resembles human disease, as
mice experience generalized bone loss and focal osteolytic bone
lesions.
22
Our data here showed that Notch inhibition reduced
bone loss, specifically in the trabecular compartment and curtailed
the presence of osteolytic lesions. Particularly, cortical porosity
was less pronounced in GSI XII-treated mice indicative of
diminished changes in cortical bone structures. We suggest that
Notch inhibition efficiently targets MM cells and their
microenvironment in the MOPC315.BM model.
There is increasing evidence that Notch signalling is involved in
differentiation and activation of osteoclasts.
30–33
Activation of
Table 1. mCT analyses of cortical bone of vehicle-treated and GSI XII-treated MOPC315.BM mice (36 days of GSI XII treatment)
Variable GSI XII n¼9 s.d. Vehicle n¼13 s.d. P-value Signif. diff.
Ct.area (mm
2
) 0.78 0.07 0.78 0.04 0.918 NS
Tt.area (mm
2
) 0.95 0.07 0.97 0.04 0.444 NS
Ct.Po.Ar (mm
2
) 0.16 0.02 0.18 0.02 0.018 Yes
Ct.Th (mm) 0.11 0.01 0.11 0.01 0.589 NS
Ct. vBMD (mg HA/cm
3
) 917 30 902 24 0.234 NS
Abbreviations: Ar, area; Ct., cortical; mCT, microcomputed tomography; GSI, g-secretase inhibitor; HA, hydroxylapatite; Po., porosity; Th, thickness; Tt., total;
vBMD, volumetric bone mineral density. Two-tailed t-test was used for statistical analysis. Significant P-value is indicated in bold.
MOPC315.BM
injection
sacrifice
bone analysis
d37
daily treatment
– serum IgA
analysis
0.1
0.15
0.2
0.25
vehicle GSI
Ct.Po.Ar (mm2)
1 mm 1 mm
* p=0.04
vehicle GSI
1
2
3
4
5
0
vehicle GSI
vehicle GSI
day of death
IgA [mg/ml]
* p=0.018
d0
Figure 4. Osteolytic lesions are diminished after treatment of MOPC315.BM mice with GSI XII. (a) Scheme indicates time points of
MOPC315.BM injection, serum collection for measurement of MM-specific M315 IgA levels and GSI XII treatment. After intravenous inoculation
of MOP315.BM cells, mice were randomized into two groups (vehicle-treated versus GSI XII-treated mice). Treatment started immediately
and was discontinued after 36 days. M315 serum levels were measured at the time of death and latest at day 37. (b) M315 serum levels of
vehicle-treated (n¼13) and GSI XII-treated (n¼9) mice on the day of death. One-tailed t-test with Welsh’s correction was used for statistical
analysis. (c) Transversal sections through representative proximal tibiae of vehicle-treated mice and GSI XII-treated mice. Arrows indicate
osteolytic lesions. (d) Three-dimensional reconstruction of proximal tibiae demonstrate reduced wall thickness, diminished trabecular
structures and holes in vehicle-treated mice as compared with GSI XII-treated mice. (e) Box plot for values of the cortical porosity area (in mm
2
)
in vehicle-treated and GSI XII-treated mice. Two-tailed t-test was used for statistical analysis. P-values as indicated.
Table 2. mCT analyses of the trabecular bone of vehicle-treated and GSI XII-treated MOPC315.BM mice (36 days of GSI XII treatment)
Variable GSI XII n¼9 s.d. Vehicle n¼13 s.d. P-value Signif. diff.
Tb. BV/TV (mm
3
/mm
3
) 0.066 0.025 0.044 0.016 0.016 Yes
Tb.Th (mm) 0.046 0.003 0.046 0.004 0.814 NS
Tb.Sp (mm) 0.352 0.079 0.417 0.053 0.032 Yes
Tb.N(1/mm) 3 0.6 2.5 0.3 0.009 Yes
Tb. vBMD (mg HA/cm
3
) 144 31 119 18 0.03 Yes
Abbreviations: BV, bone volume; mCT, microcomputed tomography; GSI, g-secretase inhibitor; HA, hydroxylapatite; Sp, separation; Tb., trabecular; Th, thickness;
TV, total volume; vBMD, volumetric bone mineral density. Two-tailed t-test was used for statistical analysis. Significant P-values are indicated in bold.
Notch in myeloma bone disease
R Schwarzer et al
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Blood Cancer Journal &2014 Macmillan Publishers Limited
Notch through Jagged1 enhances the osteoclast transcription
factor NFATc1 promoter activity and expression, and thereby
promotes osteoclastogenesis.
32,34
Recently, a mechanism
of this regulation has been proposed, demonstrating that
calcium/calmodulin-dependent protein kinase IV (CaMKIV)
bindstoNIC,stabilizestheproteinandinhibitsitsproteasomal
degradation.
31
Thus, promoter activity of the Notch-dependent
target gene NFATc1 is enhanced.
31
In the present study we
provided evidence that Notch inhibition through GSI XII
transcriptionally downregulated NFATc1 expression in RANKL-
induced RAW264.7 cells in vitro. Concomitantly, the
osteoclast differentiation marker TRAP5 was suppressed.
Our current data indicate that Notch inhibition impairs
osteoclast differentiation in vitro. They are in accordance with a
previous study that used small hairpin RNA for Notch2 and
GSI X for inhibition of Notch in osteoclast progenitors and
demonstrated decreased expression of NFATc1 resulting in
inhibition of osteoclastogenesis.
32
We speculate that this
mechanism presumably contributes to the in vivo efficacy of GSI
XII in MOPC315.BM mice with decreased osteolytic lesions and
generalized bone loss. However, we cannot exclude that Notch
inhibition through GSI XII exerts its effects in vivo mainly through
induction of MM cell apoptosis.
Recent evidence from a breast cancer mouse model indicated
that the Jagged1/Notch pathway promotes osteolytic bone
disease of breast cancer through engagement of Notch
signalling in osteoblasts and osteoclasts.
35
Notch inhibition
reverses the Jagged/Notch-mediated bone effects and
reduces development of bone metastasis.
35
In MM, tumour
cells originate from transformed post-germinal center B cells,
which selectively migrate to the bone marrow to establish
bone disease.
11
Our data provided evidence that in mice,
which received GSI XII immediately after MOPC315.BM cell
injection, tumour burden and bone lesions were significantly
reduced as compared with controls (Figure 4). We hypothesize
that Notch inhibition controls MOPC315.BM cell growth and
interferes with MM cell homing and/or interaction with the bone
marrow microenvironment. Supporting this hypothesis it has
most recently been demonstrated that anti-Notch treatment
prevents bone marrow infiltration of human MM cells in a
mouse non-obese diabetic/severe combined immunodeficient
xenograft model by modulation of the chemokine receptor
CXCR4/stromal cell-derived factor-1 system.
21
This is consistent
with the observation that MOPC315.BM cells similar to human
MM cells express CXCR4, which interacts with stromal cell-
derived factor-1 on stromal cells for their recruitment to the
bone marrow.
36
In conclusion, our study provided preclinical evidence
in vivo for Notch inhibition through GSI XII as a valid
therapeutic strategy against myeloma bone disease.
Single use of GSI XII does not eradicate the tumour.
However, it is has been shown that g-secretase inhibitors,
namely GSI XII, increase sensitivity to drugs such as bortezomib,
17
which have recently essentially improved therapeutic outcomes
in MM.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, TRR54,
TPB6 to FJ and BD), the Deutsche Krebshilfe (108658 to FJ and RM); and the
Wilhelm-Sander Stiftung (2011.018.1 to FJ). It was further supported by the
Norwegian Cancer Society (BB) and the Multiple Myeloma Research Foundation
(BB). We thank Katharina Pardon and Alexander Haake for excellent technical
assistance.
AUTHOR CONTRIBUTIONS
RS conceived the hypothesis, designed and performed experiments and
analysed the data. NN, JG, RS and BC performed experiments and analysed the
data. BMW, GND, AL, RM, BB and BD analysed data and contributed to writing
the manuscript. FJ conceived the hypothesis, designed experiments and wrote
the manuscript.
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Notch in myeloma bone disease
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Blood Cancer Journal &2014 Macmillan Publishers Limited
