Published OnlineFirst July 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0123
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
Synergistic Induction of Apoptosis in Multiple Myeloma Cells
by Bortezomib and Hypoxia-Activated Prodrug TH-302,
In Vivo and In Vitro
Jinsong Hu1,2, Els Van Valckenborgh2, Dehui Xu2, Eline Menu2, Hendrik De Raeve3, Elke De Bryune2,
Song Xu2, Ben Van Camp2, Damian Handisides4, Charles P. Hart4, and Karin Vanderkerken2
Abstract
Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the
hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple
disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we
evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome
inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple
myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase
cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced
cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of
proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression
of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic
BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this
synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the
expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in
the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve
multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies
provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with
multiple myeloma. Mol Cancer Ther; 12(9); 1763–73. 2013 AACR.
Introduction
Multiple myeloma is a malignant plasma-cell disorder
that accounts for 1% of all cancers and 10% to 15% of all
hematologic malignancies (1). In the past decade, there
have been major advances in the treatment of multiple
myeloma. The introduction of novel agents such as bortezomib (proteasome inhibitor) and thalidomide (immunomodulatory drug) has dramatically improved the outcome of patients with multiple myeloma (2–4). Moreover,
Authors' Affiliations: 1Department of Genetics and Molecular Biology,
Xi'an Jiaotong University School of Medicine, Xi'an, China; 2Department of
Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit
Brussel; 3Department of Pathology, Universitair Ziekenhuis Brussel, Brussels, Belgium; and 4Threshold Pharmaceuticals, South San Francisco,
California
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Karin Vanderkerken, Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit
Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. Phone: 32-2477-4418; Fax: 32-2-477-4405; Fax: 0032-2-4774405; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-13-0123
2013 American Association for Cancer Research.
significant advances in both basic and translational
research have enhanced understanding of disease pathogenesis and guided the development of new and more
effective therapies (5). It has been shown previously by us
and other groups that hypoxia is a critical microenvironmental factor in multiple myeloma (6–11). Our data
further support that targeting the hypoxic niche with
hypoxia-activated prodrug (HAP) TH-302 (Fig. 1A) is a
potential new treatment option for multiple myeloma (6).
TH-302 as a 2-nitroimidazole prodrug of the cytotoxic
bromo-isophosphoramide mustard (Br-IPM), exhibits
hypoxia-selective cytotoxicity against a broad spectrum
of human cancer cell lines in vitro, and shows efficacy
across a large panel of human tumor xenografts in vivo.
TH-302 is being evaluated in clinical trials for the treatment of solid tumors as a monotherapy and in combination with chemotherapeutic agents (12).
Bortezomib (PS-341), the first therapeutic proteasome
inhibitor, was approved for refractory, relapsed, and
newly diagnosed multiple myeloma (13). Treatment of
multiple myeloma with bortezomib has dramatically
improved survival for patients with multiple myeloma;
however, some patients do not benefit from this treatment, and those responding ultimately acquire resistance
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Hu et al.
A
N
O
NO2
Br
O
NH
Br
P
N
N
H
CH3
5T33MMvt
Veh
5 µmol/L TH-302
5 µmol/L TH-302
+ 5 nmol/L Btz
5 nmol/L Btz
Figure 1. TH-302 in combination
with bortezomib (Btz) induces
synergistic apoptosis in multiple
myeloma cells. A, the chemical
structure of TH-302. TH-302 is a
nitroimidazole prodrug of
cytotoxin Br-IPM. 5T33MMvt (B),
OPM2 (C), and MMS1 (D) cells were
treated with vehicle (Veh), TH-302,
bortezomib, or combination
therapy at the indicated
concentrations for 16 hours in
hypoxic condition (1% O2),
followed by Annexin V–FITC/7AAD staining and flow cytometry
analysis. Percentage of cells is
shown within each quadrant.
Results are representative of three
independent experiments.
7-AAD
B
Annexin V–FITC
OPM2
Veh
5 µmol/L TH-302
5 µmol/L TH-302
+ 5 nmol/L Btz
5 nmol/L Btz
7-AAD
C
Annexin V–FITC
MMS1
Veh
5 µmol/L TH-302
100 nmol/L Btz
5 µmol/L TH-302
+ 100 nmol/L Btz
7-AAD
D
Annexin V–FITC
(14). On the other hand, severe treatment-related toxicities
such as peripheral neuropathy have been observed in
conjunction with the use of bortezomib (15). Combination
therapy using lower doses of bortezomib is an approach to
increase efficacy and reduce side effects, thereby enhancing the likelihood of survival for patients with multiple
myeloma (16).
In the current study, we investigated the anti–multiple
myeloma effects of TH-302 combined with bortezomib in
hypoxic conditions. Our results show that the combination of TH-302 and bortezomib synergistically induced
apoptosis in multiple myeloma cells in vitro, associated
with the activation of caspases, the decrease of antiapoptotic Bcl-2 family members, and importantly, the increase
of proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA. Further experiments in the 5T33MMvv mouse model in vivo showed that
the combination of TH-302 and bortezomib can improve
multiple disease parameters and result in a significant
prolonged survival. Taken together, these findings provide preclinical rationale for further evaluation of the
combination of TH-302 and bortezomib in clinical trials.
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Mol Cancer Ther; 12(9) September 2013
Materials and Methods
Drugs
TH-302 was provided by Threshold Pharmaceuticals.
TH-302 was dissolved in sterile PBS at 10 mmol/L for in
vitro studies and at 5 mg/mL for in vivo studies. Bortezomib was from LC Laboratories. For in vitro studies,
bortezomib was reconstituted in dimethyl sulfoxide
(DMSO) at a stock concentration of 10 mmol/L and stored
at 20 C until use, and this stock was diluted in medium
just before use so that the concentration of DMSO never
exceeded 0.1%. For in vivo studies, clinical-grade vials of
bortezomib were reconstituted using a sterile NaCl 0.9%
solution. Solutions were sterilized by filtration through a
0.22-mm syringe filter.
Cells and cell culture conditions
The human LP1, OPM2 (both from American Type
Culture Collection), MMS1 (a kind gift from Y. Okuno,
Kyoto University, Kyoto, Japan) and murine myeloma
cell line 5T33MMvt (a kind gift of J. Radl, TNO, Leiden,
the Netherlands) were used in this study. A stock was
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Published OnlineFirst July 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0123
Antimyeloma Activity of TH-302 Combined with Bortezomib
prepared of all authenticated cell lines and experiments
were carried out within 2 months after thawing vials. The
murine cell line was authenticated by its expression of a
specific idiotype. The multiple myeloma cells were cultured in normoxic (20% O2) and hypoxic conditions (1%
O2) as described previously (6).
Quantification of apoptosis
A total of 1 106 cells were washed twice with PBS and
stained with 2 mL 7-amino-actinomycin D (7-AAD; BD
Biosciences) and 5 mL Annexin V–fluorescein isothiocyanate (FITC; BD Biosciences) in 100 mL of binding buffer,
and incubated at 4 C for 15 minutes. Then, cells were
resuspended in 400 mL of binding buffer and immediately
analyzed using a FACSCanto flow cytometer (Becton
Dickinson).
Synergy study of the combination treatment
Multiple myeloma cells were treated simultaneously
with various concentrations of TH-302 and bortezomib for
16 hours in hypoxic conditions. Apoptosis induced by
each agent alone and by the combination treatment was
monitored by flow-cytometric analysis of multiple myeloma cells stained with Annexin V–FITC and 7-AAD as
described earlier. The degree of synergism between the
two compounds was determined using the CompuSyn
Software (ComboSyn Inc.) developed by Chou (17). The
combination index (CI) for each combination was calculated at a nonconstant ratio.
Western blotting
Western blot analysis was conducted as previously
described (6). The primary antibodies against Bcl-2, BclxL, Mcl-1, BAD, BID, BAX, caspase-3, caspase-8, caspase-9, PARP, phospho-p53 (Ser15), and b-actin were
from Cell Signaling Technology and diluted at 1:1,000.
The primary antibodies against PUMA, NOXA, p53,
p21(WAF1/CIP1), ATF4, GRP-78, and CHOP (C/EBP
homologous protein; also known as DNA damage–
inducible gene 153) were from Santa Cruz Biotechnology and diluted at 1:500.
Quantitative real-time PCR (qRT-PCR)
RNA extraction was conducted using the RNeasy Kit
(Qiagen). Total RNA was reverse transcribed using the
Verso cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Real-time PCR
was conducted with Maxima SYBR Green/ROX qPCR
Master Mix (Fermentas) on an ABI Prism 7900 Fast instrument using gene-specific primers. Primer sequences
used for amplifications are as follows: b-actin, forward:
50 -ATCGTGCGTGACATTAAGGAGAAG-30 ; reverse: 50 AGGAAGGAAGGCTGGAAGAGTG-30 . BRCA1, forward: 50 -ACCGTTGCTACCGAGTGTCTG-30 ; reverse:
5 0 -GTGATGTTCCTGAGATGCCTTTGC-3 0 . BRCA2,
forward: 50 -AACCGTGTGGAAGTTGCGTATTG-30 ; reverse: 50 -GGCTCCCGTGGCTGGTAAATC-30 . FANCD2,
forward: 50 -CAGGAGAGCACAGCAGATGAGAG-30 ; re-
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verse: 50 -AGGCAGGAGAATCGCTTGAACC-30 . RAD51,
forward: 50 -TCAAGTGGATGGAGCAGCGATG-30 ; reverse: 50 -GGCAGTCACAACAGGAAGAGG-30 . p53, forward: 5 0 -TTGCGTGTGGAGTATTTGGATGAC-3 0 ; reverse: 5 0 -AGTGTGATGATGGTGAGGATGGG-3 0 .
p21 ( W A F 1 / C I P 1 ) , forward: 50 -CCAGCGACCTTCCTCATCCAC-30 ; reverse: 50 -CCATAGCCTCTACTGCCACCATC-30 . PUMA, forward: 50 -CTGCCGCCCACCACCATC-30 ; reverse: 50 -TGAAGGAGCACCGAGAGGAGAG30 . NOXA, forward: 50 -ACCGTGTGTAGTTGGCATCTCC-30 ; reverse: 50 -AGGTTCCTGAGCAGAAGAGTTTGG-30 . GRP-78, forward: 50 -AGGAGGAGGACAAGAAGGAGGAC-30 ; reverse: 50 -CAGGAGTGAAGGCGACATAGGAC-30 . CHOP, forward: 50 -TGCTTCTCTGGCTTGGCTGAC-30 ; reverse: 50 -CCGTTTCCTGGTTCTCCCTTGG-30 . The thermal cycling conditions included 2
minutes at 50 C and 10 minutes at 95 C followed by 40
cycles of 95 C for 0.15 minutes and 60 C for 1 minute. Ct
values were collected for b-actin and the genes of interest
during the log phase of the cycle. Quantification of given
genes expressed as mRNA level was normalized to b-actin
RNA using the DDCt method.
Animals and 5T33MMvv multiple myeloma model
The 5T33MM model originated spontaneously in
aging C57BL/KaLwRij mice and has since been propagated in vivo by intravenous transfer of the diseased
marrow in young syngeneic mice (18). C57BL/KaLwRijHsd mice were purchased from Harlan CPB. Male
mice were 6 to 10 weeks old when used, and housed and
treated following the conditions approved by the Ethical Committee for Animal Experiments, VUB (license
no. LA1230281).
In vivo analysis of tumor burden
To study the effects of TH-302 alone, bortezomib
alone, and the combination on myeloma progression,
four groups of C57BL/KaLwRij mice (n ¼ 10) were
injected intravenously with 0.5 106 5T33MMvv cells;
one group of 10 na€ve mice was included as negative
control. One week after tumor cell inoculation, mice
were treated with either TH-302 alone [100 mg/kg,
twice weekly, intraperitoneally (i.p.) injection], or bortezomib alone (0.8 mg/kg, twice weekly, s.c. injection),
or bortezomib in combination with TH-302 (twice weekly, TH-302 was injected 4 hours after bortezomib), or
vehicle (0.9% NaCl) until the first mouse showed signs
of morbidity. Serum paraprotein concentration was
assessed using standard electrophoretic techniques.
Bone marrow tumor burden was assessed by determining plasmacytosis on cytosmears. Bone marrow angiogenesis was assessed by determining microvessel density (MVD) as previously described (19).
Survival analysis
Five days after tumor cell inoculation, four groups
(n ¼ 10) of mice injected with 5T33MMvv cells were
treated with either TH-302 alone (100 mg/kg, twice
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Hu et al.
weekly, i.p. injection), or bortezomib alone (0.8 mg/kg,
twice weekly, s.c. injection), or bortezomib in combination
with TH-302 (twice weekly, TH-302 was injected 4 hours
after bortezomib), or vehicle (0.9% NaCl). Kaplan–Meier
analysis was used for analyzing overall survival.
Statistical analysis
For statistical analysis of the in vivo data, differences
between two groups were determined by Mann–Whitney
test. Kaplan–Meier analysis was used to determine the
effects on the survival time of the tumor-bearing mice, the
differences between groups were tested for statistical
significance using the two-tailed log-rank test. For qRTPCR data analysis, a two-tailed paired t test was conducted for the analysis of differences in cells with or
without treatment with a software package GraphPad
Prism (GraphPad software). P < 0.05 was considered
statistically significant.
Results
Combination effects of bortezomib and TH-302 on
multiple myeloma cell apoptosis
In our previous study, we showed that the TH-302
exhibits hypoxia-selective cytotoxicity against multiple
myeloma cells in vitro and shows attractive efficacy in
multiple myeloma in vivo (6). In the present study, we
investigated whether bortezomib can potentiate TH302–induced apoptosis of multiple myeloma cells in
hypoxic conditions. To examine the effects of the combination, three multiple myeloma cell lines were exposed
to increasing concentrations of the two agents. Cell
apoptosis was measured after 16 hours by flow cytometry. As shown in Fig. 1, bortezomib in combination with
TH-302 increased the apoptosis levels in 5T33MMvt,
OPM2, and MMS1 cells. The interaction between
bortezomib and TH-302 was further analyzed using the
Chou–Talalay method (17), to determine whether this
combination exhibited additive or synergistic cytotoxicity. Using CompuSyn software, we calculated the CIs of
varying concentrations of TH-302 with bortezomib. In
5T33MMvt cells, at doses ranging from 2.5 to 10 mmol/L
TH-302 combined with 5 to 20 nmol/L bortezomib, CI
ranged from 0.308 to 0.891, suggesting that this combination was synergistic (Table 1). Similar results were also
observed in OPM2 and MMS1 cells (Supplementary
Tables S1 and S2).
Combination bortezomib and TH-302 induces
apoptosis through activation of the caspase cascade
Caspase activation plays a central role in the execution
of apoptosis. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic
signaling pathway and thus leads to rapid cell death (20).
To determine whether caspase activation is involved in
the augmented apoptosis by the combination treatment
with bortezomib and TH-302, two cell lines 5T33MMvt
and OPM2, exhibiting good and moderate response to the
combination treatment, were chosen for further studies.
1766
Mol Cancer Ther; 12(9) September 2013
Table 1. CI analysis of TH-302 combined with
bortezomib at a nonconstant ratio in 5T33vt
cells
TH-302
(mmol/L)
Bortezomib
(nmol/L)
Fa
CI
Description
2.5
2.5
2.5
5
5
5
10
10
10
5
10
20
5
10
20
5
10
20
0.382
0.556
0.605
0.535
0.603
0.828
0.576
0.777
0.826
0.638
0.569
0.891
0.452
0.550
0.370
0.538
0.308
0.404
Synergism
Synergism
Slight synergism
Synergism
Synergism
Synergism
Synergism
Synergism
Synergism
NOTE: Analysis was conducted using the CompuSyn software (ComboSyn, Inc.). Descriptions are based on CI values
and the recommendations of CompuSyn: <0.1, very strong
synergism; 0.1–0.3, strong synergism; 0.3–0.7, synergism;
0.7–0.85, moderate synergism; 0.85–0.9, slight synergism.
Abbreviation: Fa, fraction affected as tested by the flow
cytometry analysis of 5T33vt cells stained with FITC-labeled
Annexin V and 7-AAD after 16 hours with each indicted
treatment.
We examined the activation of caspase cascades as
defined by their cleavage at specific aspartate residues
by Western blot analysis. As seen in Fig. 2, treating the
cells with the combination induced a greater processing of
caspase-3, -8, and -9 and PARP in both 5T33MMvt and
OPM2 cells, coincided with the appearance of bands
representing cleavage products of caspase-3, -8, and -9
and PARP.
Effects of TH-302 and bortezomib on the expression
of Bcl-2 family proteins
To characterize the mechanism responsible for bortezomib–TH-302–mediated synergism in multiple myeloma, we next investigated the changes of Bcl-2 family
members at the protein level. In Fig. 3A and B, the
changes of antiapoptotic protein Bcl-2, Bcl-xL, and
Mcl-1 in two multiple myeloma cell lines are shown.
TH-302 alone causes a decrease in the expression levels
of all three antiapoptotic proteins, whereas bortezomib
alone does not affect the expression of Bcl-xL, slightly
decreases the expression of Bcl-2, and markedly
increases the expression of Mcl-1. When treated with
the combination, the expression of Bcl-xL is further
decreased as compared with TH-302 alone, whereas
Mcl-1 and Bcl-2 are similarly decreased as compared
with TH-302 alone.
In addition, the effects of TH-302 and bortezomib on
proapoptotic Bcl-2 family proteins were investigated.
As shown in Fig. 3C and D, no significant changes
were observed in BAX proteins. However, among the
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Antimyeloma Activity of TH-302 Combined with Bortezomib
A
Figure 2. Bortezomib/TH-302
induces apoptosis through
activation of the caspase cascade
in multiple myeloma cells.
5T33MMvt (A) and OPM2 (B) cells
were cultured with TH-302 in the
presence or absence of
bortezomib (Btz) for 16 hours under
hypoxic conditions (1% O2). The
concentrations of drugs, either
alone or in combination, were as
follows: 5 nmol/L bortezomib, 5
mmol/L TH-302. Whole-cell lysates
were subjected to immunoblot
analysis with anti–caspase-3, -8,
-9, anti-PARP, and anti–b-actin
antibodies. Blots shown are
representative of three
independent experiments.
B
5T33MMvt
–
+
–
– +
–
+
+
–
– +
+
+
Cleaved caspase-3 (17 kD)
Cleaved caspase-3 (19/17 kD)
Caspase-8 (57 kD)
Cleaved caspase-8 (45 kD)
Caspase-8 (57 kD)
Cleaved caspase-8 (43 kD)
Caspase-9 (49 kD)
Cleaved caspase-9 (39/37 kD)
Cleaved caspase-8 (18 kD)
Cleaved caspase-9 (17 kD)
Caspase-9 (47 kD)
Cleaved caspase-9 (37/35 kD)
Cleaved caspase-9 (17 kD)
Actin
–
–
Btz
TH-302
Caspase-3 (35 kD)
PARP (116 kD)
Cleaved PARP (89 kD)
A
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+
–
Caspase-3 (35 kD)
PARP (116 kD)
Cleaved PARP (89 kD)
Actin
proapoptotic BH3-only members, both TH-302 and bortezomib induced the cleavage of BAD and BID, thereby
consequently producing the truncated BAD and BID
Figure 3. Changes of Bcl-2 family
members induced by TH-302 and
bortezomib (Btz) under hypoxic
conditions by Western blot
analysis. A and B, changes of
antiapoptotic Bcl-2, Bcl-xL, and
Mcl-1 in TH-302- and bortezomibtreated 5T33MMvt (A) and OPM2
(B) cells. Multiple myeloma cells
were treated with vehicle, TH-302
(5 mmol/L), bortezomib (5 nmol/L),
or combination therapy for 16
hours under hypoxic conditions
(1% O2). Whole-cell lysates were
immunoblotted with the indicated
antibodies. C and D, the effects of
TH-302 and bortezomib on the
expression of proapoptotic Bcl-2
family members. 5T33MMvt (C)
and OPM2 (D) multiple myeloma
cells were treated with TH-302
(5 mmol/L), bortezomib (5 nmol/L),
or combination therapy for 16
hours under hypoxic conditions
(1% O2). Whole-cell lysates were
immunoblotted with the indicated
antibodies. The results are
representative of three
independent experiments.
OPM2
–
Btz
TH-302
proteins, which are stronger inducer of apoptosis than
the full-length proteins (21–23). In contrast to BAD and
BID, the expression levels of the proapoptotic BH3-only
5T33MMvt
+ – + Btz (nmol/L)
– + + TH-302 (µmol/L)
B
–
–
OPM2
+ – + Btz (nmol/L)
– + + TH-302 (µmol/L)
Bcl-2 (26 kD)
Bcl-2 (26 kD)
Bcl-xL (30 kD)
Bcl-xL (30 kD)
Mcl-1L (40 kD)
Mcl-1 (35 kD)
Mcl-1s (32 kD)
Cleaved Mcl-1 (19 kD)
Cleaved Mcl-1 (17 kD)
Actin
C
D
5T33MMvt
–
+
–
–
– +
Actin
+ Btz (nmol/L)
+ TH-302 (µmol/L)
OPM2
–
+
–
– +
–
+ Btz (nmol/L)
+ TH-302 (µmol/L)
BAX (20 kD)
BAX (20 kD)
BAD (23 kD)
Cleaved BAD (18 kD)
BID (22 kD)
Cleaved BID (15 kD)
BAD (23 kD)
Cleaved BAD (18 kD)
BID (22 kD)
Cleaved BID (15 kD)
PUMA (23 kD)
PUMA (23 kD)
NOXA (15 kD)
NOXA (15 kD)
Actin
Actin
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p-(s15)-p53
p53
p21
PUMA
NOXA
Actin
C
0
2.5
5 nmol/L Btz
p-(s15)-p53
p53
p21
PUMA
NOXA
Actin
E
0
5
10 µmol/L TH-302
ATF4
GRP-78
CHOP
PUMA
NOXA
Actin
G
0
10
50 nmol/L Btz
ATF4
GRP-78
CHOP
PUMA
NOXA
Actin
B
Relative mRNA expression levels (fold)
10 µmol/L TH-302
D
Relative mRNA expression levels (fold)
5
*
9
8
Veh
5 µmol/L TH-302
7
6
5
4
3
*
**
*
*
*
*
*
2
1
0
p53
p21
PUMA
NOXA BRCA1 BRCA2 FANCD2 RAD51
*
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Veh
5 nmol/L Btz
*
*
*
*
p53
p21
F
Relative mRNA expression levels (fold)
0
PUMA
*
*
NOXA BRCA1 BRCA2 FANCD2 RAD51
*
20.0
Veh
10 µmol/L TH-302
17.5
15.0
12.5
**
10.0
7.5
*
5.0
2.5
0.0
GRP-78
CHOP
NOXA
PUMA
H
Relative mRNA expression levels (fold)
A
*
30
**
25
20
Veh
50 nmol/L Btz
*
15
10
*
5
0
GRP-78
CHOP
PUMA
NOXA
Figure 4. The role of p53 and ER stress in upregulating of proapototic PUMA and NOXA by TH-302 and bortezomib (Btz). A and B, TH-302 upregulates and
activates p53 in OPM2 cells. A, OPM2 cells were cultured with indicated doses of TH-302 for 16 hours under hypoxic conditions (1% O2), then harvested
and probed with the indicated antibodies by Western blot analysis. B, qRT-PCR analysis of the expression p53 and its target genes in TH-302–treated
OPM2 cells. OPM2 cells were cultured with vehicle (Veh) or 5 mmol/L TH-302 for 16 hours in hypoxic condition (1% O2), then harvested for RNA extraction
and cDNA synthesis and PCR analysis. For more details see Materials and Methods. C and D, bortezomib activates p53 in OPM2 cells. C, OPM2 cells
were cultured with indicated doses of bortezomib for 16 hours in hypoxic condition (1% O2), then harvested and probed with the indicated antibodies by
Western blot analysis. D, qRT-PCR analysis of the expression p53 and its target genes in bortezomib-treated OPM2 cells. OPM2 cells were cultured with
vehicle or 5 nmol/L bortezozmib for 16 hours in hypoxic condition (1% O2), then harvested for RNA extraction and cDNA synthesis and PCR analysis.
E and F, TH-302 activates ER stress signaling in LP1 cells. E, LP1 cells were cultured with vehicle or 5 or 10 mmol/L TH-302 for 16 hours in hypoxic
condition (1% O2), then harvested and probed with the indicated antibodies by Western blot analysis. (Continued on the following page.)
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Antimyeloma Activity of TH-302 Combined with Bortezomib
protein PUMA and NOXA were increased by both TH-302
and bortezomib.
p53-dependent and -independent pathways involved
in the induction of PUMA and NOXA
To further address the mechanisms underlying the
induction of PUMA and NOXA, we investigated the roles
of p53-dependent and -independent pathways in
response to TH-302 and bortezomib treatment. When
exposed to hypoxic conditions, TH-302 reduction at the
nitroimadazole site of the prodrug by intracellular reductases leads to the release of the DNA alkylating agent
bromo-isophosphoramidate mustard, which is genotoxic
(24). In response to genotoxic stress, the activation of p53
plays a crucial role in governing cell apoptosis. Because
PUMA and NOXA are direct targets in p53-mediated
apoptosis (25), we then investigated whether p53 is
involved in TH-302- and bortezomib-induced apoptosis.
As shown in Fig. 4A, we confirmed that p53 was upregulated and activated by TH-302 in OPM2 cells, which
express the wild-type p53. In parallel to the activation of
p53, as monitored by the phosphorylation on Ser-15, the
expression level of the target gene p21WAF1/CIP1 was also
increased. Similar results were also observed in bortezomib-treated multiple myeloma cells in hypoxic condition
(Fig. 4C), and the results confirmed the finding that the
induction and activation of p53 is one of the important
molecular mechanisms mediating antimyeloma activity
of bortezomib (26, 27). Moreover, by quantitative measuring of the transcriptional expression levels of the p53related DNA repair genes such as BRCA1, BRCA2, RAD51,
and FANCD2, we found that TH-302 upregulates the
transcription of these DNA repair genes, suggesting the
activation of DNA repair machinery. However, when
treated with bortezomib, the expression levels of DNA
repair genes were decreased (Fig. 4B and D). Interestingly,
in the p53-mutated multiple myeloma cell line LP1, we
still observed the induction of PUMA and NOXA, implicating the role of p53-independent pathways in the regulation of PUMA and NOXA. We then investigated the
contribution of endoplasmic reticulum (ER) stress to the
induction of PUMA and NOXA. As expected, we observed upregulated expression levels of the ER stress markers
GRP-78 and CHOP in response to the treatment by both
bortezomib and TH-302. Moreover, the expression of
ATF4, one major regulator of ER stress signaling pathway,
was also increased (Fig. 4E and G). Furthermore, by using
qRT-PCR, we show that the expression of ER stress markers and PUMA as well as NOXA were all upregulated at
transcription level (Fig. 4F and H).
Combination of bortezomib and TH-302 suppresses
multiple myeloma cell growth in vivo
To test whether the enhanced myeloma cell apoptotic
response is observed in vivo, we examined the efficacy of
the combination of bortezomib and TH-302 in the
5T33MM mouse model. In the first series of experiments,
C57BL/KaLwRijHsd mice inoculated with 5T33MMvv
cells were either assigned to receive TH-302, bortezomib,
or a combination of both starting at day 7 after inoculation
(Fig. 5A). In mice treated with TH-302 alone, bortezomib
alone, or the combination, a significant reduction in serum
paraprotein concentrations and plasmacytosis in the bone
marrow were observed compared with the vehicle-treated mice (Fig. 5B and C). Importantly, the combinationtreated group showed more decreases in the serum paraprotein concentrations and plasmacytosis than in the
single-drug–treated groups. Immunohistochemical staining for CD31 showed significantly decreased MVD in
the treatment groups (Fig. 5D). Similarly, combination
treatment led to more significant decrease of MVD than
each single-drug group. No significant reduction in body
weight was observed in treated mice compared with
vehicle-treated controls (data not shown).
To examine whether the combination of TH-302 and
bortezomib can prolong the survival of multiple myeloma–inoculated mice, a second series of experiments was
carried out. As shown in Fig. 5E, in vivo treatment of
5T33MM mice with a combination of TH-302 and bortezomib resulted in a significantly prolonged survival when
compared with the vehicle group (P < 0.0001), the TH-302alone–treated group (P < 0.0001), and the bortezomibalone–treated group (P < 0.0001). The median survival
time of each group was 25.5 (vehicle), 34 (bortezomib),
41.5 (TH-302), and 53.5 (bortezomib and TH-302) days,
respectively. These data clearly show that the combination of TH-302 and bortezomib results in improvements in
multiple disease parameters and increased overall survival of the mice.
Discussion
Despite advances in chemotherapy and stem-cell transplantation, multiple myeloma remains an incurable disease. As shown in our previous report, multiple myeloma
cells localize at the hypoxic regions in the bone marrow
(6, 10). This finding not only provides us new insights into
the multiple myeloma–bone marrow microenvironment,
but also provides us a new treatment target taking advantage of hypoxia in multiple myeloma. By using a HAP TH302, we previously showed that targeting hypoxia is a
potential new treatment option for multiple myeloma (6).
(Continued.) F, qRT-PCR analysis of the expression of ER stress markers GRP-78 and CHOP and its target genes PUMA and NOXA in TH-302–treated LP1 cells.
LP1 cells were cultured with vehicle or 10 mmol/L TH-302 for 16 hours in hypoxic condition (1% O2), then harvested for RNA extraction and cDNA synthesis
and PCR analysis. G and H, bortezomib activates ER stress in LP1 cells. G, LP1 cells were cultured with vehicle or 10 or 50 nmol/L bortezomib for 16 hours
in hypoxic condition (1% O2), then harvested and probed with the indicated antibodies by Western blot analysis. H, qRT-PCR analysis of the expression
of ER stress markers GRP-78 and CHOP and its target genes PUMA and NOXA in bortezomib-treated LP1 cells. LP1 cells were cultured with vehicle or 50 nmol/L
bortezomib for 16 hours in hypoxic condition (1% O2), then harvested for RNA extraction and cDNA synthesis and PCR analysis. Data represent the mean SD for
three separate experiments. , P < 0.05; , P < 0.01 versus vehicle-treated samples.
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Hu et al.
A
Day 0
7
17
Treatment with Btz, TH-302
Inoculation of 5T33vv cells
**
90
80
70
60
50
40
30
20
10
0
**
**
***
Naïve
Veh
TH-302
**
C
***
Paraprotein (g/dL)
Plasmacytosis (%)
B
Sacrifice
Btz TH-302+Btz
**
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
**
***
Naïve
D
**
Veh
TH-302
Btz TH-302+Btz
**
**
30
**
**
20
MVD
**
10
0
Naïve
110
100
90
80
70
60
50
40
30
20
10
0
TH-302
Btz TH-302+Btz
Veh
Btz
TH-302
TH-302 + Btz
Percentage survival
E
Veh
0
10
20
30
40
50
Days
60
In the present study, we investigated whether the combination of hypoxia-activated treatment with bortezomib
can achieve better therapeutic efficacy, based on a finding
that bortezomib has preferential cytotoxicity toward hypoxic solid tumor cells (28). We showed here that the
combinatorial use of TH-302 and bortezomib can mediate
synergistic antitumor effects in multiple myeloma.
Induction of apoptosis in tumor cells represents a key
strategy of cancer chemotherapy. It has been shown that
Bcl-2 family members are central regulators of apoptosis
(29). All Bcl-2 family members contain at least one of four
conserved Bcl-2 homology (24) domains. According to
their function and structure, the Bcl-2 family can be
divided into two classes: the antiapoptotic and proapoptotic members. The antiapoptotic proteins have all four BH
domains and include Bcl-2, Bcl-xL, Mcl-1, Bcl-w, and A1.
1770
Mol Cancer Ther; 12(9) September 2013
70
80
90
Figure 5. Combination treatment
with TH-302 and bortezomib (Btz)
in the 5T33MM murine multiple
myeloma model. A, a schematic
diagram showing the treatment
process. B, therapeutic effects on
tumor load. Data are expressed as
percentage 5T33MM cells of total
cell number by determining
€nwald–
plasmacytosis on May–Gru
Giemsa–stained cytosmears of
mononuclear bone marrow cells.
C, treatment effects on serum
paraprotein. The concentrations
of serum paraprotein were
determined by serum
electrophoresis. D, MVD analysis.
The number of microvessels in the
femur of the mice were evaluated
by staining and counting CD31
on bone marrow sections. E,
effects of bortezomib and TH-302
on disease-free survival assessed
by a Kaplan–Meier analysis. The
treatment started at day 5 after
tumor inoculation. Mean SD
for groups of 10 mice are
shown. , P < 0.01; , P < 0.001;
Veh, vehicle.
100
The proapoptotic Bcl-2 proteins are further functionally
divided into two subgroups: the effector molecules such as
BAX and BAK, containing BH domains 1–3, and the BH3only proteins such as BAD, BID, BIK, BIM, BMF, BNIP3,
HRK, NOXA, and PUMA, which contain only the BH3
domain (30). In the current study, following the findings
that the combination of bortezomib and TH-302 can synergistically induce apoptosis (Fig. 1 and Table 1), we
further investigated the changes of some important Bcl2 family members in multiple myeloma cells. The changes
of Bcl-2 family members are shown in Fig. 3 and are
summarized in Table 2 based on quantitative analysis.
We found that the protein levels of antiapoptotic Bcl-2,
Bcl-xl, and Mcl-1 were decreased by TH-302 in hypoxic
conditions in all tested multiple myeloma cell lines. Moreover, when treated with bortezomib alone, the expression
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Antimyeloma Activity of TH-302 Combined with Bortezomib
Table 2. The changes of Bcl-2 family proteins by TH-302 and bortezomib in multiple myeloma cells
Member
Pro/antiapoptotic
Bortezomib
TH-302
BortezomibþTH-302
Bcl-2
Bcl-xL
Mcl-1
BAX
BAD
BID
PUMA
NOXA
Antiapoptotic
Antiapoptotic
Antiapoptotic
Proapoptotic
Proapoptotic
Proapoptotic
Proapoptotic
Proapoptotic
Downregulation
No change
Upregulation, cleavage
No change
Cleavage
Cleavage
Upregulation
Upregulation
Downregulation
Downregulation
Downregulation, cleavage
No change
Cleavage
Cleavage
Upregulation
Upregulation
Downregulation
Downregulation
Downregulation, cleavage
No change
Cleavage
Cleavage
Upregulation
Upregulation
of Bcl-2 was decreased, the expression of Bcl-xL was not
disturbed, and the expression of Mcl-1 was increased.
Nevertheless, when the multiple myeloma cells were
treated with the combination of drugs, we observed clearly a decrease of all three antiapoptotic Bcl-2 family proteins. Among these three investigated antiapoptotic Bcl-2
family members, the changes of Mcl-1 are noticeable. Mcl1 is overexpressed in multiple myeloma cells, and plays
essential roles for the survival of multiple myeloma cells
by its ability to oppose to a wide variety of proapoptotic
stimuli (31). Downregulation or reduction of Mcl-1 has
been proposed to play very important roles in response to
drug-induced apoptotic stimuli (32). The expression of
Mcl-1 can be regulated at both transcriptional and posttranslational levels. The accumulation of Mcl-1 upon
bortezomib treatment has been shown to induce multiple
myeloma cell resistance to bortezomib-induced lethality,
and was speculated to be the result of the inhibition of
ubiquitation-mediated degradation (33). More recently,
we showed that the accumulation of Mcl-1 by bortezomib
was transcriptionally upregulated by the selective activation of the ATF4 signaling branch of the unfolded protein
response (UPR; ref. 34). Moreover, Mcl-1 was also shown
to be a substrate for caspases during induction of apoptosis (35–37). In this regard, the cleavage of Mcl-1 by
caspases is related to the decrease of the full length of the
Mcl-1 protein. More importantly, the cleaved Mcl-1 shows
strong proapoptotic activity by enhancing its turnover
and impairing its ability to interact with BH3-only proapoptotic proteins such as NOXA-, PUMA-, and BIMinduced apoptosis (38). In line with the finding of Mcl-1
cleavage, the cleavage of PARP and pro-caspase-3/8/9
shows similar changes when treated with TH-302 and
bortezomib either alone or in combination, suggesting a
tight correlation between caspase activation and Mcl-1
reduction (Figs. 2 and 3A). Similar to the changes of
Mcl-1 antiapoptotic proteins, we also observed that TH302 and bortezomib can result in the cleavage of several
BH3-only proapoptotic proteins such as BAD and BID
(Fig. 3B and Table 2). However, the cleavage of BAD and
BID does not imply the decrease of proapoptotic function, because previous studies have shown that the
cleavage of BAD and BID is a caspase-dependent process, and the cleaved BAD and BID fragments exhibit
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stronger mitochondrial localization and higher apoptotic capability than intact ones, thus promoting apoptosis
(21, 22).
One of the most striking findings of the study was that
both TH-302 and bortezomib can induce the expression of
proapoptotic BH3-only proteins PUMA and NOXA. The
induction of PUMA and NOXA was speculated to be
another crucial event mediating the synergistic apoptosis.
PUMA and NOXA have been shown to be the transcriptional targets of p53 and essential mediators in p53induced apoptosis (39–42). In the current study, we found
that both TH-302 and bortezomib can activate p53 and its
downstream target gene p21WAF1/CIP1 in multiple myeloma cells expressing wild-type p53 (Fig. 4A and C). However, the mechanisms underlying p53 activation by TH302 and bortezomib are different. TH-302 is a hypoxiaactivated DNA cross-linking agent and under hypoxic
condition will lead to genotoxic stress in multiple myeloma cells, inducing p53 signal transduction and apoptosis. In response to the genotoxic stress caused by TH-302,
the activation of p53 may play a direct role in triggering
the onset of DNA repair processes. Accordingly, we found
that the transcription levels of DNA repair genes such as
BRCA1, BRCA2, RAD51, and FANCD2 were significantly
increased by TH-302 (Fig. 4B). However, in multiple
myeloma cells treated with bortezomib, although p53 was
activated, the DNA repair machinery induction was
delayed by downregulating the transcription of these
DNA repair genes (Fig. 4D). Growing evidence has shown
that bortezomib is particularly toxic to multiple myeloma
cells due to an increased susceptibility of multiple myeloma cells to ER stress-induced apoptosis (43). Treatment
of multiple myeloma cells with bortezomib leads to induction of proapoptotic UPR components, including growth
arrest and CHOP. A previous study has indicated that the
major apoptotic regulator p53 was significantly increased
in response to ER stress, and participates in ER stressinduced apoptosis (40, 44). In this regard, our findings are
consistent with the previous studies that the antimyeloma
activity of bortezomib was partly mediated by the induction and activation of p53, and downregulation of
the expression of the DNA repair machinery (26, 27). Very
recently, Lin and colleagues further showed that ER
stress stimulates p53 expression through NF-kB
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Hu et al.
activation (45). Work by Hideshima and colleagues
showed that bortezomib actually activates two upstream
NF-kB–activating kinases (RIP2 and IKKb), promotes
downregulation of NF-kB’s inhibitor (IkBa), and
increases NF-kB DNA binding both in vitro and in vivo
(46). Taken together, it suggests that the induction and
activation of p53 by bortezomib was mediated by ER
stress rather than genotoxic stress.
In multiple myeloma cell lines with p53 mutations,
we still observed the induction of PUMA and NOXA by
TH-302 and bortezomib, strongly implicating the existence of p53-independent mechanisms. We found that
the activation of CHOP by TH-302 and bortezomib
through ER stress may be another upstream regulator.
As shown in Fig. 4E and G, both TH-302 and bortezomib
can trigger ER stress signaling, confirmed by upregulation of ER stress markers GRP-78 and CHOP. As an
important ER stress downstream effector, the transcription factor CHOP works as a critical initiator of ER
stress-induced apoptosis through upregulating the
expression of proapoptotic BH3-only proteins PUMA
and NOXA (47, 48). The mechanism how bortezomib
induces ER stress has been well shown; however, the
reasons why TH-302 can trigger both ER and genotoxic
stresses have not been fully clarified. TH-302 consists of
two distinct parts: a bis-alkylating effector and an oxygen concentration-activating trigger. The hypoxia-selective release of the Br-IPM may lead to protein alkylation
in addition to DNA cross-linking, disturb the homeostasis of the ER, and induce ER stress.
In summary, our findings in the present study show
that the combination of TH-302 and bortezomib has
synergistic cytotoxicity in multiple myeloma, and the
synergy is tightly related to the changes in the balance
between proapoptotic and antiapoptotic Bcl-2 proteins
favoring induction of apoptosis. Importantly, the
experiments carried out in the in vivo murine 5T33MM
model showed impressive improvements in multiple
disease parameters and significantly prolonged survival
after treatment with the combination. Taken together,
our data provide a novel insight into the potential
application of bortezomib and TH-302 for multiple
myeloma, and provide support for further clinical evaluation of the combination of bortezomib and TH-302 for
patients with multiple myeloma.
Disclosure of Potential Conflicts of Interest
D. Handisides has ownership interest (including patents) in Threshold.
No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: J. Hu, D. Handisides, C.P. Hart, K. Vanderkerken
Development of methodology: J. Hu, E. Van Valckenborgh
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J. Hu, H. De Raeve, S. Xu, K. Vanderkerken
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Hu, D. Xu, E. Menu
Writing, review, and/or revision of the manuscript: J. Hu, E. Van Valckenborgh, E. Menu, H. De Raeve, E. De Bryune, B. Van Camp, C.P. Hart,
K. Vanderkerken
Study supervision: K. Vanderkerken
Acknowledgments
The authors thank Prof. Mark De Ridder, Dr. Valeri N. Verovski, and
Heng Jiang (Cancer Research Unit of Oncologic Center, Universitair
Ziekenhuis Brussel, Brussel, Belgium) for their expertise and equipment
used in the hypoxia experiments. The authors also thank A. Willems and
C. Seynaeve for expert technical assistance, Prof. F. Gorus (Universitair
Ziekenhuis Brussel) for analysis of serum electrophoresis.
Grant Support
This work was supported by Vlaamse Kankerliga (to K.Vanderkerken),
FWO-Vlaanderen, Stichting Tegen Kanker (to K. Vanderkerken), Onderzoeksraad VUB (to K. Vanderkerken), and the Fundamental Research
Funds for the Central Universities (No. 2012jdhz59; to J. Hu). E. Van
Valckenborgh, E. Menu, E. De Bryune are post-doctoral fellows of FWOVlaanderen.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received February 21, 2013; revised May 24, 2013; accepted June 10,
2013; published OnlineFirst July 5, 2013.
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Mol Cancer Ther; 12(9) September 2013
Downloaded from mct.aacrjournals.org on June 15, 2017. © 2013 American Association for Cancer Research.
1773
Correction
Correction: Synergistic Induction of
Apoptosis in Multiple Myeloma Cells by
Bortezomib and Hypoxia-Activated
Prodrug TH-302, In Vivo and In Vitro
In this article (Mol Cancer Ther 2013;12:1763–73), which appeared in the September
2013 issue of Molecular Cancer Therapeutics (1), Dr. Elke De Bruyne's name was
misspelled. The correct author listing is below. The authors regret this error.
Jinsong Hu, Els Van Valckenborgh, Dehui Xu, Eline Menu, Hendrik De Raeve, Elke De
Bruyne, Song Xu, Ben Van Camp, Damian Handisides, Charles P. Hart, and Karin
Vanderkerken.
Reference
1. Hu J, Van Valckenborgh E, Xu D, Menu E, De Raeve H, De Bruyne E, et al. Synergistic induction of
apoptosis in multiple myeloma cells by bortezomib and hypoxia-activated prodrug TH-302, in vivo
and in vitro. Mol Cancer Ther 2013;12:1763–73.
Published OnlineFirst June 9, 2015.
doi: 10.1158/1535-7163.MCT-15-0298
Ó2015 American Association for Cancer Research.
1762 Mol Cancer Ther; 14(7) July 2015
Molecular
Cancer
Therapeutics
Published OnlineFirst July 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0123
Synergistic Induction of Apoptosis in Multiple Myeloma Cells by
Bortezomib and Hypoxia-Activated Prodrug TH-302, In Vivo and In
Vitro
Jinsong Hu, Els Van Valckenborgh, Dehui Xu, et al.
Mol Cancer Ther 2013;12:1763-1773. Published OnlineFirst July 5, 2013.
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